Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 to 29 September, 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 474 and in compliance with GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19 April to 12 November, 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 422 and in compliance with GLP
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Inspected on 2018-01-23 / Signed on 2018-06-05
Limit test:
no
Justification for study design:
- Basis for dose level selection: Dietary levels were selected following the completion of the preliminary toxicity study (Covance Study number: NN81HP) following consultation with the Sponsor.
- Route of administration: The dietary route of administration was chosen to simulate the conditions of potential human exposure.
- Animal model: The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.
Species:
rat
Strain:
other: Crl:CD(SD)
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 Crl:CD(SD) was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males: 69 to 75 days old; Females: 83 to 89 days old.
- Weight at study initiation: Males: 323 to 392 g; Females: 245 to 299 g
- Housing: 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.
- Number of animals per cage: Pre-pairing: up to four animals of one sex; Pairing: one male and one female; Males after mating: up to four animals; Gestation: one female; Lactation: one female + litter
- Diet: SDS VRF1 Certified powdered diet, ad libitum (removed overnight before blood sampling for hematology and blood chemistry investigations and during urine collection)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (removed overnight during urine collection)
- Acclimation period: Females: 20 days before commencement of treatment; Males: six days prior to the commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 h light : 12 h dark
- 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.
Paper shavings: Two handfuls provided to each Reproductive phase female cage from Day 20 after mating and throught lactation and changed at the same frequency as the cage bedding.

IN-LIFE DATES: from 19 April to 12 November, 2018.
Route of administration:
oral: feed
Vehicle:
other: Feed
Details on exposure:
DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet
- Correction factor: A correction factor was not required.
- Stabilizer: Corn oil (test material to corn oil ratio 5:1).
- Method of preparation: The test item was incorporated into the diet to provide the required concentrations by initial preparation of a premix. The amount of test item and corn oil required for the premix were added to an equal amount of plain (basal) diet and stirred. An amount of plain diet equal to the weight of
the mixture was added and the mixture was stirred again until visibly homogenous. The doubling up process was repeated until approximately half the premix diet was added. At this stage the mixture was ground with a mechanical grinder. The mixture was made up to the weight of the premix with plain diet. The premix was then mixed using a turbula mixer for 100 cycles. This premix was diluted with further quantities of plain diet using the doubling up process to prepare the required concentration test mixes. Each formulation was mixed using a turbula mixer for 100 cycles.
- Frequency of preparation: Weekly.
- Storage of formulation: Frozen (-10 to -30°C). Diet was allowed to thaw before feeding commenced.

Details on mating procedure:
- Animals: Toxicity phase and Recovery phase males with Reproductive phase females (Toxicity and Recovery phase females were not paired for mating).
- M/F ratio per cage: 1:1 from within the same treatment groups
- Pairing commenced: After a minimum of three weeks of treatment.
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Presence of sperm within the vaginal smear and/or ejected copulation plugs referred to as Day 0 of gestation.
- 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 800 and 20000 ppm were analyzed to assess the stability and homogeneity of the test item in the diet matrix. Formulations were found to be homogenous and stable for 15 days when stored frozen (-10 to -30°C) and for four days when stored at ambient temperature (15 to 25°C).
- Achieved concentration: Samples of each formulation prepared for administration in Week 1 and in the final week of treatment were analyzed for achieved concentration of the test item
Duration of treatment / exposure:
Reproductive phase females: Three weeks before pairing, then throughout pairing and gestation until Day 12 of lactation (necropsy on Day 13 of lactation (the diet was available to the animals until the morning of necropsy)).
Toxicity phase males: Three weeks before pairing up to necropsy after minimum of six weeks.
Toxicity phase females: At least six weeks.
Recovery phase males: Three weeks before pairing up to necropsy after minimum of six weeks followed by a minimum 14-day recovery.
Recovery phase females: At least six weeks followed by a minimum 14-day recovery.
Animals of the F1 generation received no direct administration of test item; any exposure was in utero or via the milk.
Frequency of treatment:
Continuously
Dose / conc.:
0 ppm (nominal)
Remarks:
Basal diet + corn oil / Group 1 (Control)
Dose / conc.:
1 500 ppm
Remarks:
Group 2 (Low dose)
Dose / conc.:
3 500 ppm
Remarks:
Group 3 (mid dose)
Dose / conc.:
7 500 ppm
Remarks:
Group 4 (High dose)
No. of animals per sex per dose:
Reproductive phase females: 10 animals/dose
Toxicity phase females: 5 females/dose in all groups; 5 males/dose in control and high dose groups; 10 males/dose in low and mid dose groups
Recovery phase animals: 5 animals/sex/dose in control and high dose groups
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: the dose levels selected for investigation in this OECD 422 combined repeated dose toxicity study and reproductive/developmental toxicity screening study (0, 1500, 3500 and 7500 ppm) were chosen in conjunction with the Sponsor and were based on the results of a 14-day preliminary study conducted at these laboratories (Covance Study No. NN81HP).
- Rationale for animal assignment: On arrival and non-selective allocation to cages.
Estrous cycles were evaluated prior to treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the reproductive phase of the study.
On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets by Study Management 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.
- Post-exposure recovery period in satellite groups: 14 days
- Other: 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.
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.
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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). During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced, during each week of treatment and recovery, on Days 0, 6, 13 and 20 after mating and on 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 feeding of the treated diets on Day 1).

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Toxicity and Recovery phase males and females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and weekly thereafter, including the recovery phase. On Day 5 of recovery the animals were fed diets prepared for the females in the lactation phase in error (recovery control animals received control diet with the corn oil stabiliser, and recovery animals in Group 4 received treated diet (8000 ppm); On the day of necropsy.
F0 Reproductive phase females: Weekly during acclimatization; Before feeding of the treated diets 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 AND COMPOUND INTAKE (if feeding study):
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals: Daily, including the recovery phase. On Day 5 of recovery the animals were fed diets prepared for the females in the lactation phase in error (recovery control animals received control diet with the corn oil stabiliser, and recovery animals in Group 4 received treated diet (8000 ppm). Food consumption was not recorded for Toxicity phase males and Reproductive phase females during the period when paired for mating (Week 3), but recommenced for males in Week 4. Food consumption was recorded continuously for Toxicity and Recovery phase females. For Reproductive phase females after mating food consumption was performed daily throughout gestation and lactation (until Day 12).
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

OTHER:
NEUROBEHAVIOURAL EXAMINATION:
- Time schedule:
Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery animals in Groups 1 and 4 and on the lowest numbered toxicity phase males and females in Groups 2 and 3 during Week 6 of treatment.
Motor activity: During Week 6 of treatment, the motor activity of all recovery animals in Groups 1 and 4 and on the lowest numbered toxicity phase males and females in Groups 2 and 3 was measured.

OPHTHALMOLOGY
- Time schedule:
Pre-treatment: All Toxicity and Recovery phase animals and spare animals; Week 6: All Toxicity phase females and the first five Toxicity phase males of Groups 1 and 4
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope. Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY AND CLINICAL CHEMISTRY:
- Time schedule for collection of blood:
Week 6: Five lowest numbered surviving Toxicity phase males and females in each group; Recovery: All male Recovery animals
- Animals fasted: Yes, blood samples were collected after overnight withdrawal of food; animals were also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
- Animals were held under light general anaesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein. Sampling was performed on the morning after overnight collection of urine.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count, Differential leucocyte count: Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count, Prothrombin time and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin, Bile acids, Urea, Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus, Total protein, Albumin and Albumin/globulin ratio (A/G Ratio).

Urinalysis
- Time schedule for collection of urine:
Week 6: Five lowest numbered surviving Toxicity phase males and females in each group; Recovery: All Recovery animals
Metabolism cages used for collection of urine: Yes; animals were placed in an individual metabolism cage, without access to food or water. Urine samples were collected overnight.
- Parameters:
Using manual methods: Clarity and Color/Appearance (App) - by visual assessment; Volume (Vol) - using a measuring cylinder; pH - using a pH meter; Specific gravity (SG) - by direct refractometry using a SG meter
Using Multistix reagent strips interpreted using the Clinitek®500 instrument: Ketones, Bile pigments, Urobilinogen, Blood pigments
Using a Roche P Modular Analyzer: Protein, Creatinine, Glucose, Sodium, Potassium, Chloride
A microscopic examination of the urine sediment was performed: Epithelial cells, Leucocytes (WBC), Erythrocytes (RBC), Casts and Other abnormal components (A)
The slide was also examined for abnormalities in spermatozoa and crystals.

Thyroid Hormone Analysis
- Time schedule for examination
At termination: F0 males, All F0 Reproductive phase females
Day 4 of age - F1 offspring, two females per litter (where possible) - no pups were eliminated when litter size dropped below ten/litter
- one for T4 (serum)#
- one for TSH (plasma - optional analysis)
# priority 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

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.
Oestrous cyclicity (parental animals):
Estrous Cycle
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 the Reproductive phase of the study.
- After pairing until mating.
- For four days before scheduled termination (all Reproductive phase, Toxicity phase and Recovery phase females).

Dry smears:
Reproductive phase females: from beginning of treatment until animals were paired for mating, using cotton swabs (approximately three weeks).
Litter observations:
Clinical observations: Examined at approximately 24 h after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights: Days 1, 4, 7 and 13 of age.
Ano-genital distance: Day 1 - all F1 offspring.
Nipple/areolae count: Day 13 of age - male offspring.
Postmortem examinations (parental animals):
SACRIFICE
Time of necropsy
Toxicity phase:
F0 males and females: After Week 6 investigations were completed.
Reproductive phase females:
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 13 of lactation.
Recovery phase
F0 Males and females: After at least 14 days without treatment.
- Method of sacrifice: All adult animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination. (No animal was exposed to carbon dioxide until after completion of thyroid hormone assays).

GROSS NECROPSY
- 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.
Main phase females
The following were recorded:
Each uterine horn: Number of implantation sites was counted and confirmed.

ORGAN WEIGHTS
- For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

HISTOPATHOLOGY
- 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 F0 animals killed or dying prematurely; Toxicity phase males and females in Groups 1 and 4 at scheduled termination.
Abnormalities: All remaining adult 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.
Postmortem examinations (offspring):
SACRIFICE
Time of necropsy:
Selected offspring for Day 4 thyroid hormone analysis - Day 4 of age.
Scheduled kill - Day 13 of age.
Method of sacrifice:
- Offspring- selected for thyroid hormone sampling on Day 4 or Day 13 of age: Decapitation
- Offspring - not selected for thyroid hormone sampling: Intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed. Abnormal tissues retained.
- F1 offspring on Day 4 of age:
Blood sampling required
Externally normal offspring discarded without examination.
Externally abnormal offspring identified on despatch to necropsy, examined externally, and retained pending possible future examination.
- F1 offspring on Day 13 of age
Blood sampling required
All animals (but not including those selected for thyroid hormone analysis) 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.
Animals selected for thyroid hormone analysis: externally normal offspring discarded without examination; externally abnormal offspring examined.
Statistics:
See "Any other information on materials and methods incl. tables"
Reproductive indices:
Mating Performance and Fertility:
- Percentage mating = (Number animals mating / Animals paired) x 100
- Conception rate (%) = (Number animals achieving pregnancy / Animals mated) x 100
- Fertility index (%) = (Number 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:
- Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100
- 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 of lactation / Number live offspring on Day 4 (after blood sampling)) x 100
Sex ratio: The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 and 13 of age.
- Percentage males = (Number of males in litter / Total number of offspring in litter) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed during the treatment period for toxicity and recovery phase animals, reproductive females prior to pairing, during gestation and lactation that were considered related to dietary administration of Cistus Concrete. There were no signs observed during the recovery period that were considered to be associated with the previous treatment with Cistus Concrete.
Mortality:
no mortality observed
Description (incidence):
- On Day 2 of lactation, a female receiving 7500 ppm was terminated early due to total litter loss. Several signs including cold to touch, dull eyes, whole-body pallor, brown aqueous discharge from the vaginal area and a thin build were observed for this female prior to the female’s dispatch. Macropathology findings comprised pale kidneys, pale liver, inactive and pale mammary glands and fetal placental material in the stomach.
- On Day 8 of lactation, a second female receiving 7500 ppm was dispatched early due to total litter loss. Macropathology findings comprised pale and inactive mammary glands. Microscopic findings for both these females included decreased secretory activity of the mammary tissue.
- On Day 25 of gestation, one female receiving 1500 ppm was killed due to having surpassed the designated gestational time. Macroscopic examination of thisfemale revealed no abnormalities and the animal was confirmed to be not pregnant.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Group mean body weight gains for the treatment period (Days 1 to 43) were slightly lower for toxicity and recovery phase males receiving 3500 or 7500 ppm (88 and 86% of Control, respectively) but was mainly due to the initial decrease during Days 1-4 of treatment. Body weight gain for males receiving 1500 ppmduring this period was generally similar to Controls.
- Group mean body weight gains for the treatment period (Days 1 to 43) for toxicity and recovery females were moderately low for females receiving 1500 ppm (62% of Control) and lower for females receiving 3500 or 7500 ppm (38 and 24% of Control), all differences attained statistical significance. The lower body weight gain for toxicity and recovery phase females receiving 7500 ppm was predominately due to the mean weight loss of 17 g during Days 1 to 4 of treatment. Although the reduction in overall body weight gain from Days 1 to 43 of treatment when compared with Controls was indicative of a clear dose
dependent effect, the body weight gain from Days 4 to 43 of treatment did not show a dose-dependent relationship and therefore body weight gain for females receiving 7500 ppm showed recovery after Day 4 of dosing. Mean body weight values recorded for reproductive phase females only from Day 4 of treatment were a maximum of 10% lower when compared to mean Control body weight values.
- During gestation, absolute body weights at 7500 ppm were statistically significantly lower than in Controls; the overall mean body weight gain of females receiving 7500 ppm was statistically significantly lower than Control (-16% of Control), with statistical significance attained during Days 0 to 7 and 14 to 20 of gestation. Following parturition, the mean body weight gain for females at all dose levels was essentially similar to Control, although females receiving 3500 or 7500 ppm had lower absolute body weights.
- During the recovery phase (R0-R15) the mean absolute body weights for the males that received 7500 ppm were higher compared to Controls although the gains during the recovery period were similar to Controls. The absolute body weights for females that received 7500 ppm were statistically significantly lower compared to Controls, however mean body weight gain was statistically significantly higher for females previously receiving 7500 ppm Cistus concrete (almost 2.5-fold the Control value).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Group mean food consumption for toxicity and recovery phase males during the treatment period was similar to Controls for males receiving 1500 and 3500 ppm of Cistus Concrete administered via the diet. A reduction in food consumption (-33% of Control) was observed in males receiving 7500 ppm on Day 1 of treatment, however subsequent food consumption values were similar to Controls and slightly lower values compared to Controls could occur in all treated groups during the treatment period.
- Food consumption was generally lower for toxicity, recovery and reproductive phase females receiving 3500 ppm and 7500 ppm compared to Controls during the pre-pairing period and for the remainder of the treatment period for toxicity and recovery phase females, with values still attaining statistical significance several times after Day 5 of treatment at 7500 ppm.
- Toxicity, recovery and reproductive phase females receiving 3500 ppm and 7500 ppm also showed a marked reduction in food intake during the first four days of Cistus Concrete administered via the diet. Food consumption for females receiving 1500 ppm tended to be similar to the Controls during the treatment period with the exception of Day 1 of treatment.
- A general trend to a slight reduction in food consumption was observed for females receiving 7500 ppm during the gestation period, the reduction was greater during the first week of gestation although intake on Day 0-1 was the same as the Controls. Group mean food consumption for reproductive phase females receiving 1500 or 3500 ppm during gestation was similar to Controls.
- Following parturition, daily food intake was generally statistically significantly lower for females receiving 7500 ppm, leading to a mean overall decrease of 29% compared to Controls. The food intake of females receiving 1500 or 3500 ppm was similar to that of Controls throughout the lactation period.
- Food consumption was similar to the Controls through Days R1-R14 in the recovery phase for females. Overall food consumption for males during the recovery period was higher than the Controls (17% increase compared to Control), with food intake values particularly high during the first two days of recovery.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No effects of treatment were observed during ophthalmic examinations in Week 6 of treatment for male and female animals treated at all dose levels.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Haematology investigations after six weeks of dietary administration of the test substance revealed a statistically significant reduction in reticulocyte counts at all dose levels for males, however a dose dependent relationship was not apparent, and all values were within Historical Control Data (HCD) range. Following a two-week recovery period, reticulocyte counts remained slightly low compared to concurrent controls however statistical significance was not attained.
- Red cell mass (haematocrit, haemoglobin and red blood cell counts) were statistically low for females at 7500 ppm compared to Controls however all values for these parameters were within the HCD range. Following a two-week recovery period, haematocrit, haemoglobin and red blood cell counts for females that received 7500 ppm were similar to Controls.
- Activated partial thromboplastin time was slightly shorter for females receiving 7500 ppm compared to Controls and this difference attained statistical significance, as this change was confined to one sex, not associated with any changes to other clotting factors and within the HCD range it is considered this difference represents normal biological variation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- A statistically significant reduction in bile acids was apparent amongst males and females receiving 3500 and 7500 ppm, however mean values for both sexes were within the HCD range. Following the recovery period bile acids were still lower for males that received 7500 ppm compared to concurrent Controls however bile acids for females was slightly higher than Controls.
- Creatinine levels were statistically significantly higher for females receiving 7500 ppm compared to Controls, but the mean value was within the HCD range and following a two-week recovery period, females previously receiving 7500 ppm of Cistus Concrete creatinine levels were similar to Controls.
- All other differences from Controls, including those that attained statistical significance, were generally small, confined to one sex, or the magnitudes were not dose-related and, consequently, were considered to represent normal biological variation.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Urinalysis investigations performed in Week 6 of treatment indicated the total amount of glucose was low in males and females at all dose levels, although a dose relationship was apparent for females only with statistical significance confined to females receiving 7500 ppm. The total amount of protein in the urine was low in females at 3500 or 7500 ppm and total creatinine output was also low in females at these levels, with the differences from Controls attaining statistical significance for both parameters. The means for the aforementioned parameters were all within the reported HCD range.
- There was a decrease of sodium output in males and females at all dose levels when compared with that of the Controls, a dose relationship was evident in males only and all mean values were with the HCD range. Potassium and chloride output were also reduced for females at all dose levels when compared to Controls, statistical significance was attained for chloride output only but there was no dose response and all mean values were within the HCD range.
- All other differences from Controls, including those that attained statistical significance, were generally small, confined to one sex, or the magnitudes were not dose-related and, consequently, were considered to represent normal biological variation

Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- No treatment-related effects were observed for sensory reactivity and grip strength assessment during Week 6 of treatment for males and females at all dose levels.
- Motor activity assessment of males during Week 6 of treatment revealed no treatment-related effects in males at all dose levels. Motor activity assessment of females during Week 6 of treatment revealed an increase in the overall scores for both high and low beam break activity at all dose levels. The higher incidence of overall low beam break scores attained statistical significance at all dose levels. A dose dependent response was not evident in the total scores and when compared with Historical Control Data (HCD), all statistically significant values and total beam scores were within the normal range. The total low beam score for the Control female was below the range documented in the HCD which led to the incidental increases observed in the treated groups. The increased overall high beam scores for females at all dose levels showed a dose-dependent response however statistical significance was not attained. In addition, the control value was below the range documented in the HCD and the values of the treated groups were within the HCD ranges. Statistical significance was attained for the 30-minute interval at 3500 and 7500 ppm and at the 60-minute interval at 7500 ppm, and these values were also within the normal range documented in the HCD. The increase in motor activity observed in the females was not associated with any clinical signs or signs recorded during the arena observation and all total scores were within the HCD range; therefore, these differences were considered fortuitous in nature and were unrelated to treatment with Cistus Concrete.
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):
The microscopic examination performed after 6 weeks treatment revealed no test item-related lesions. All other histological changes (incidental findings) were considered to be unrelated to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age. Slightly lower mean T4 concentrations were observed in the adult males, however there was no dose relationship observed and the historical control data (HCD) showed that the Control values for this study were high. There was no consistent effect on T4 levels in the offspring. Consequently, there was no requirement to measure T4 in the samples obtained from offspring on Day 4 of age or from the adult females and none of the TSH (thyroid stimulating hormone) samples required analysis.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
- All females allocated to the reproductive phase of the study showed regular 4 to 5 day estrous cycles prior to the start of and during treatment. Females allocated to the toxicity phase all showed regular 4 to 5 day estrous cycles prior to treatment with the exception of one female in each dose group including the control.
- For females allocated to the recovery phase all control females and three females in the 7500 ppm group showed regular 4 or 5 day estrous cycles, with the remaining two females in the 7500 ppm groups showing an irregular cycle or were acyclic.
- During the treatment period the majority of reproductive phase females had regular estrous cycles of 4 to 5 days, with the exception of one control female which had an irregular cycle, two acyclic females receiving 1500 ppm, two acyclic females and one female showing an irregular cycle at 3500 ppm and two females receiving 7500 ppm which had irregular cycles.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
- All females had a pre-coital interval of 1 to 4 days, with the exception of one female receiving 1500 ppm and one female receiving 3500 ppm, which had pre-coital intervals of 5 to 8 or 13 to 14 days, respectively. At termination, the majority of toxicity phase females and all recovery phase females were cycling and all reproductive phase females were in diestrus.
- Gestation length and gestation index were considered unaffected by treatment at any dose level.
Key result
Dose descriptor:
NOAEL
Effect level:
7 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed.
Key result
Critical effects observed:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- At 7500 ppm, pups in two litters (litters 149 and 150) were abnormally cold to touch on Day 1 of age; this was followed by a high rate of pup mortality in both litters such that all pups in litter 150 were dead by Day 2 of age while in litter 149 all but one pup were dead by Day 4 of age, with the single remaining pup dead on Day 8 of age.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- At 7500 ppm, pups in two litters (litters 149 and 150) were abnormally cold to touch on Day 1 of age; this was followed by a high rate of pup mortality in both litters such that all pups in litter 150 were dead by Day 2 of age while in litter 149 all but one pup were dead by Day 4 of age, with the single remaining pup dead on Day 8 of age.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no effect of treatment on mean offspring body weights on Day 1 of age. On Day 13 of age the mean body weights of male and female offspring of parents receiving 7500 ppm Cistus Concrete were slightly lower than that of the Control; body weight gain of the offspring to Day 13 of age in the 7500 ppm group was statistically significantly reduced by 16% in male and female offspring when compared to Control.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
Two females (4F No’s. 149 and 150) receiving 7500 ppm had total litter losses. The mean numbers of implantation sites for females at 7500 ppm were slightly lower compared to Controls resulting in a slightly lower mean total litter size. The mean implantation value at 7500 ppm was just outside the Historical Control Data (HCD) range however the mean litter size value was within the normal range documented in the HCD. The mean number of implantation sites and litter size were generally unaffected at 1500 and 3500 ppm. Group mean post implantation survival index, mean live birth index, viability index and lactation index were unaffected by the dietary administration of Cistus Concrete.
There were no effects on sex ratio that were associated to treatment with Cistus Concrete.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
Ano-genital distances for male offspring were slightly but statistically significantly longer for male offspring of parents treated with 7500 ppm Cistus Concrete; however, values were within the HCD ranges. There was no effect on ano-genital distance in the female offspring and the remaining dose levels for male offspring.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipples were observed in male offspring of parents treated with Cistus Concrete.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of offspring that either died prematurely or at scheduled termination on Day 13 of age did not reveal any findings that could be attributed to treatment.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
Screening for development (foetal and pup growth survival until day 4)
Generation:
F1
Effect level:
3 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Two total litter losses, clinical signs, low pup body weights in conjunction with the general reduction in food consumption and bodyweight gain observed in the reproductive females at 7500 ppm.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Under the test conditions of this study it was concluded that the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was 7500 ppm (mean achieved doses: 422 mg/kg bw/day in males and 411 mg/kg bw/day in females). The exact relationship of the two total litter losses in females receiving 7500 ppm to test-item administration is undetermined however, as both incidents have comparable timings, clinical signs, low pup body weights in conjunction with the general reduction in food consumption and bodyweight gain observed in the reproductive females of this group, a relationship to Cistus Concrete cannot be completely discounted and therefore this finding is considered adverse. It was therefore concluded that within the context of this study, the NOAEL for reproductive/developmental toxicity was 3500 ppm (mean achieved doses; 193 mg/kg bw/day before pairing, 208 mg/kg bw/day during gestation and 487 mg/kg bw/day during lactation).
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, the test item was administered to groups of Crl:CD(SD) rats at dietary concentrations of 1500, 3500 and 7500 ppm. An additional subgroup was used to assess reversibility, persistence or delayed occurrence of systemic effects for 14 days post treatment. A similarly constituted control group was assigned to each phase, and received the vehicle, powdered SDS VRF1 Certified diet with corn oil, throughout the same relative treatment period.

Toxicity phase males were treated for three weeks prior to pairing up to necropsy after a minimum of six weeks. Toxicity phase females were treated for at least six weeks. Recovery phase males were treated for three weeks before pairing up to necropsy after a minimum of six weeks followed by a 14-day recovery period. Recovery phase females were treated for six weeks followed by a 14-day recovery period. Reproductive phase females were treated for three weeks before pairing, throughout pairing and gestation until Day 12 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 13 of lactation (the treated diet was made available until the morning of necropsy). Selected F1 offspring were killed on Day 4 of age for blood sampling collection for thyroid hormone analysis. The remaining F1 offspring were killed on Day 13 of age. The offspring received no direct administration to the test item; any exposure was in utero or via the milk.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, ophthalmic examinations, hematology (peripheral blood), blood chemistry, urinalysis, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight, bone marrow micronucleus tests, 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.

The mean concentrations of Cistus Concrete in test formulations analyzed for the study were within ±10% of nominal concentrations, confirming accurate formulation. The difference from the mean value remained within 5%, confirming precise analysis.

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 male and female offspring. The overall mean achieved dosages for toxicity phase animals during the treatment period were 85, 186 and 422 mg/kg bw/day for males and 86, 194 and 411 mg/kg bw/day for females receiving 1500, 3500 or 7500 ppm, respectively. The overall mean achieved dosages for reproductive phase females receiving 1500, 3500 or 7500 ppm were 92, 208 and 439 mg/kg bw/day and 207, 487 and 904 mg/kg bw/day during gestation and lactation, respectively.

Dietary administration of Cistus Concrete to parental Sprague Dawley (Crl:CD(SD)) rats at all dose levels for three weeks prior to pairing, during pairing and then up to termination of the toxicity phase males and females after 6 weeks of treatment and reproductive phase females on Day 13 of lactation was generally well tolerated. Two females receiving 7500 ppm were prematurely killed as a result of a total litter loss and a relationship to treatment could not be completely discounted. In addition, one female receiving 1500 ppm was killed prematurely as this female failed to litter by Day 25 of gestation, because this female was not pregnant. There were no test-item related signs observed during the detailed physical examination and arena observations and no effects on sensory reactivity, grip strength or motor activity.

Overall body weight gains during the treatment period for toxicity and recovery phase males given 3500 or 7500 ppm were slightly low, body weight gains for males receiving 1500 ppm were similar to Controls. Overall group mean body weight gains for toxicity and recovery phase females were moderately low for females receiving 1500 ppm and significantly lower for females receiving 3500 or 7500 ppm. During the gestation period overall body weight gain for females receiving 7500 ppm was low (-16% of Control) and during the lactation period body weight gains of treated groups were similar to Controls. During the recovery period the mean body weight gains for males that received 7500 ppm was similar to Controls whereas mean body weight gain was statistically significantly higher for females previously receiving 7500 ppm Cistus Concrete (almost 2.5-fold the control value).

Mean food intake for toxicity and recovery phase males at all dose levels was generally similar to Controls throughout the treatment period, with the exception of males receiving 7500 ppm on Day 1 of treatment (-33% of Control). At 7500 ppm, subsequent food consumption values for males were similar to Controls and slightly lower values compared to Controls could occur in all treated groups during the treatment period. Food consumption was generally lower for toxicity, recovery and reproductive phase females receiving 3500 ppm and 7500 ppm compared to Controls during the pre-pairing period and for the remainder of the treatment period for toxicity and recovery phase females, with values still attaining statistical significance several times after Day 5 of treatment at 7500 ppm. Food consumption for females receiving 1500 ppm tended to be similar to the Controls during the treatment period with the exception of Day 1 of treatment. Mean food consumption during gestation and lactation tended to be low for females receiving 7500 ppm, especially during lactation (mean overall food intake was 29% lower compared to Control). Mean food consumption for females receiving 1500 or 3500 ppm during gestation or lactation was similar to Controls. Food consumption was similar to the Controls through Days R1-R14 in the recovery phase for females. Overall food consumption for males during the recovery period was higher than the Controls (17% increase compared to Control), with food intake values particularly high during the first two days of recovery.

There was no treatment-related ophthalmoscopic finding. The haematological examination of the peripheral blood and biochemical assessment of the

plasma did not reveal any toxicologically significant differences from Controls. The analysis of the urine in Week 6 of treatment revealed low glucose output in both sexes at all dose levels, low protein and creatinine output for females receiving 3500 or 7500 ppm. In addition, sodium output was low in both sexes at all dose levels and potassium and chloride output was reduced for females at all dose levels.

There were no treatment-related effects on estrous cycles, pre-coital interval, mating performance, fertility and gestation length Terminal body weights for toxicity phase males and females at 7500 ppm were statistically significantly lower compared to Controls. Changes in organ weights consisted of slightly higher body weight adjusted mean liver weights for males and females at 7500 ppm when compared to Control. Following the recovery period, mean liver weights remained slightly higher in the males previously treated at 7500 ppm but the females were similar to Controls.

The macroscopic and microscopic examination of the adult males and females revealed no test-item related lesions.

A reduction in mean number of implantation sites of females receiving 7500 ppm resulted in slightly lower mean litter size when compared with Control; the mean implantation was just outside the Historical Control Data (HCD) range however the mean litter size value was

within the normal range documented in the HCD. The mean number of implantation sites and litter size were generally unaffected at 1500 and 3500 ppm. Group mean post implantation survival index, mean live birth index, viability index and lactation index were unaffected by the dietary administration of Cistus Concrete. There were no effects on sex ratio that were associated to treatment with Cistus Concrete.

Offspring body weight gain at 7500 ppm was 16% lower than in Controls but ano-genital distance was unaffected, and no nipples were seen in male pups. There were no findings associated with treatment at macroscopic examination.

Based on the results of this study it was concluded that the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was 7500 ppm (mean achieved doses: 422 mg/kg bw/day in males and 411 mg/kg bw/day in females). The exact relationship of the two total litter losses in females receiving 7500 ppm to test-item administration is undetermined however, as both incidents have comparable timings, clinical signs, low pup body weights in conjunction with the general reduction in food consumption and bodyweight gain observed in the reproductive females of this group, a relationship to Cistus Concrete cannot be completely discounted and therefore this finding is considered adverse. It was therefore concluded that within the context of this study, the NOAEL for reproductive/developmental toxicity was 3500 ppm (mean achieved doses; 193 mg/kg bw/day before pairing, 208 mg/kg bw/day during gestation and 487 mg/kg bw/day during lactation).

This study is considered as acceptable and satisfies the requirement for toxicity to reproduction endpoint.

Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19 April to 12 November, 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 422 and in compliance with GLP
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Inspected on 2018-01-23 / Signed on 2018-06-05
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
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 Crl:CD(SD) was used because of the historical control data available at this laboratory (including thyroid hormone data).
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males: 69 to 75 days old; Females: 83 to 89 days old.
- Weight at study initiation: Males: 323 to 392 g; Females: 245 to 299 g
- Housing: 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.
- Number of animals per cage: Pre-pairing: up to four animals of one sex; Pairing: one male and one female; Males after mating: up to four animals; Gestation: one female; Lactation: one female + litter
- Diet: SDS VRF1 Certified powdered diet, ad libitum (removed overnight before blood sampling for hematology and blood chemistry investigations and during urine collection)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (removed overnight during urine collection)
- Acclimation period: Females: 20 days before commencement of treatment; Males: six days prior to the commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 h light : 12 h dark
- 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.
Paper shavings: Two handfuls provided to each Reproductive phase female cage from Day 20 after mating and throught lactation and changed at the same frequency as the cage bedding.

IN-LIFE DATES: from 19 April to 12 November, 2018.
Route of administration:
oral: feed
Details on route of administration:
The dietary route of administration was chosen to simulate the conditions of potential human exposure.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet
- Correction factor: A correction factor was not required.
- Stabilizer: Corn oil (test material to corn oil ratio 5:1).
- Method of preparation: The test item was incorporated into the diet to provide the required concentrations by initial preparation of a premix. The amount of test item and corn oil required for the premix were added to an equal amount of plain (basal) diet and stirred. An amount of plain diet equal to the weight of
the mixture was added and the mixture was stirred again until visibly homogenous. The doubling up process was repeated until approximately half the premix diet was added. At this stage the mixture was ground with a mechanical grinder. The mixture was made up to the weight of the premix with plain diet. The premix was then mixed using a turbula mixer for 100 cycles. This premix was diluted with further quantities of plain diet using the doubling up process to prepare the required concentration test mixes. Each formulation was mixed using a turbula mixer for 100 cycles.
- Frequency of preparation: Weekly.
- Storage of formulation: Frozen (-10 to -30°C). Diet was allowed to thaw before feeding commenced.

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 800 and 20000 ppm were analyzed to assess the stability and homogeneity of the test item in the diet matrix. Formulations were found to be homogenous and stable for 15 days when stored frozen (-10 to -30°C) and for four days when stored at ambient temperature (15 to 25°C).
- Achieved concentration: Samples of each formulation prepared for administration in Week 1 and in the final week of treatment were analyzed for achieved concentration of the test item.

Duration of treatment / exposure:
Reproductive phase females: Three weeks before pairing, then throughout pairing and gestation until Day 12 of lactation (necropsy on Day 13 of lactation (the diet was available to the animals until the morning of necropsy)).
Toxicity phase males: Three weeks before pairing up to necropsy after minimum of six weeksof treatment.
Toxicity phase females: At least six weeks.
Recovery phase males: Three weeks before pairing up to necropsy after minimum of six weeks followed by a minimum 14-day recovery.
Recovery phase females: At least six weeks followed by a minimum 14-day recovery.
Frequency of treatment:
Continuously
Dose / conc.:
0 ppm
Remarks:
Control group (Basal diet + corn oil) / Group 1
Dose / conc.:
1 500 ppm
Remarks:
Group 2 (Low dose)
Dose / conc.:
3 500 ppm
Remarks:
Group 3 (mid dose)
Dose / conc.:
7 500 ppm
Remarks:
Group 4 (High dose)
No. of animals per sex per dose:
Reproductive phase females: 10 animals/dose
Toxicity phase females: 5 females/dose in all groups; 5 males/dose in control and high dose groups; 10 males/dose in low and mid dose groups
Recovery phase animals: 5 animals/sex/dose in control and high dose groups
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: the dose levels selected for investigation in this OECD 422 combined repeated dose toxicity study and reproductive/developmental toxicity screening study (0, 1500, 3500 and 7500 ppm) were chosen in conjunction with the Sponsor and were based on the results of a 14-day preliminary study conducted at these laboratories (Covance Study No. NN81HP).
- Rationale for animal assignment: On arrival and non-selective allocation to cages.
Estrous cycles were evaluated prior to treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the reproductive phase of the study.
On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets by Study Management 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.
- Post-exposure recovery period in satellite groups: 14 days
- Other: 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.
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.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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). During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced, during each week of treatment and recovery, on Days 0, 6, 13 and 20 after mating and on Days 1, 6 and 12 of lactation and each week during recovery, 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 feeding of the treated diets on Day 1).

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Toxicity and Recovery phase males and females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and twice weekly thereafter (including recovery phase), and on the day of necropsy.
F0 Reproductive phase females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and twice weekly before pairing. Days 0, 7, 14 and 20 after mating and Days 1, 4, 7 and 13 of lactation. On the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals: Daily, including the recovery phase. Food consumption was not recorded for males and females during the period when paired for mating (Days 22 to 29) but recommenced for males on Day 30. For Reproductive females after mating food consumption was recorded daily until Day 12 of lactation.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

WATER CONSUMPTION:
Fluid intake was assessed by daily visual observation. No significant effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups: Pre-treatment: All Toxicity and Recovery phase animals and spare animals; Week 6: All Toxicity phase females, toxicity phase males and recovery animals of Groups 1 and 4
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope. Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 6: Five lowest numbered Toxicity phase males per group and all Toxicity phase females; Recovery Week 2: all Recovery phase animals.
- Anaesthetic used for blood collection: Yes, Animals were held under light general anaesthesia induced by isoflurane.
- Animals fasted: Yes, blood samples were collected after overnight with drawal of food; animals were also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
- Blood samples were withdrawn from the sublingual vein. Sampling was performed on the morning after overnight collection of urine.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count, Differential leucocyte count: Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count, Prothrombin time and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin, Bile acids, Urea, Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus, Total protein, Albumin and Albumin/globulin ratio (A/G Ratio).

URINALYSIS: Yes
- Time schedule for collection of urine: Week 6: Five lowest numbered surviving Toxicity phase males and females in each group.
Metabolism cages used for collection of urine: Yes; animals were placed in an individual metabolism cage,with food and water deprivation. Urine samples were collected overnight.
- Parameters:
Using manual methods: Clarity and Color/Appearance (App) - by visual assessment; Volume (Vol) - using a measuring cylinder; pH - using a pH meter; Specific gravity (SG) - by direct refractometry using a SG meter
Using Multistix reagent strips interpreted using the Clinitek®500 instrument: Ketones, Bile pigments, Urobilinogen, Blood pigments
Using a Roche P Modular Analyzer: Protein, Creatinine, Glucose, Sodium, Potassium, Chloride
A microscopic examination of the urine sediment was performed: Epithelial cells, Leucocytes (WBC), Erythrocytes (RBC), Casts and Other abnormal components (A)
The slide was also examined for abnormalities in spermatozoa and crystals.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and dose groups:
Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery animals in Groups 1 and 4 and on the lowest numbered toxicity phase males and females in Groups 2 and 3 during Week 6 of treatment.
Motor activity: During Week 6 of treatment, the motor activity of all recovery animals in Groups 1 and 4 and on the lowest numbered toxicity phase males and females in Groups 2 and 3 was measured.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
SACRIFICE
Time of necropsy
Toxicity phase:
F0 males and females: After Week 6 investigations were completed.
Reproductive phase females:
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 13 of lactation.
Reproductive phase females whose litters die before Day 13: On or after day last offspring dies.
F1 Offspring Selected offspring for thyroid hormone analysis – Day 4 of age. Scheduled kill - Day 13 of age.
Recovery phase
F0 Males and females: After at least 14 days without treatment.
- Method of sacrifice: All adult animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination. Decapitation for Offspring - selected for
thyroid hormone sampling on Day 4 and 13 of age and intraperitoneal injection of sodium pentobarbitone for Offspring - not selected for thyroid hormone sampling.

GROSS NECROPSY
- 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.

ORGAN WEIGHTS
- For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

HISTOPATHOLOGY
- 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 F0 animals killed prematurely. The following animals from Groups 1 and 4 at scheduled termination:
- Five lowest numbered surviving Toxicity phase males
- Toxicity phase females
Abnormalities: All remaining Toxicity phase, Recovery phase and Reproductive phase adult 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.
Other examinations:
Thyroid Hormone Analysis:
Day 4 of age: Offspring: up to two females selected for sampling per litter (if possible, male pups reserved for nipple evaluation):
- one for T4 (serum)#
- one for TSH (plasma)
No females were selected for sampling if:
- The resultant live litter size would fall below ten offspring.
- The resultant number of live female offspring would fall to less than three.
- If only four female offspring were available within a litter but the overall litter size would fall to less than ten, one female was selected with priority given to the serum sample.
Day 13 of age: 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.
At termination: All F0 Toxicity phase males. F0 Reproductive phase females, no samples were taken from females which failed to litter or with total litter loss.
All Recovery phase animals.

Statistics:
See "Any other information on materials and methods incl. tables".
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed during the treatment period for toxicity and recovery phase animals, reproductive females prior to pairing, during gestation and lactation that were considered related to dietary administration of Cistus Concrete. There were no signs observed during the recovery period that were considered to be associated with the previous treatment with Cistus Concrete.
Mortality:
no mortality observed
Description (incidence):
- On Day 2 of lactation, a female receiving 7500 ppm was terminated early due to total litter loss. Several signs including cold to touch, dull eyes, whole-body pallor, brown aqueous discharge from the vaginal area and a thin build were observed for this female prior to the female’s dispatch. Macropathology findings comprised pale kidneys, pale liver, inactive and pale mammary glands and fetal placental material in the stomach.
- On Day 8 of lactation, a second female receiving 7500 ppm was dispatched early due to total litter loss. Macropathology findings comprised pale and inactive mammary glands. Microscopic findings for both these females included decreased secretory activity of the mammary tissue.
- On Day 25 of gestation, one female receiving 1500 ppm was killed due to having surpassed the designated gestational time. Macroscopic examination of thisfemale revealed no abnormalities and the animal was confirmed to be not pregnant.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean body weight gains for the treatment period (Days 1 to 43) were slightly lower for toxicity and recovery phase males receiving 3500 or 7500 ppm (88 and 86% of Control, respectively) but was mainly due to the initial decrease during Days 1-4 of treatment. Body weight gain for males receiving 1500 ppmduring this period was generally similar to Controls.
Group mean body weight gains for the treatment period (Days 1 to 43) for toxicity and recovery females were moderately low for females receiving 1500 ppm (62% of Control) and lower for females receiving 3500 or 7500 ppm (38 and 24% of Control), all differences attained statistical significance. The lower body weight gain for toxicity and recovery phase females receiving 7500 ppm was predominately due to the mean weight loss of 17 g during Days 1 to 4 of treatment. Although the reduction in overall body weight gain from Days 1 to 43 of treatment when compared with Controls was indicative of a clear dose
dependent effect, the body weight gain from Days 4 to 43 of treatment did not show a dose-dependent relationship and therefore body weight gain for females receiving 7500 ppm showed recovery after Day 4 of dosing. Mean body weight values recorded for reproductive phase females only from Day 4 of treatment were a maximum of 10% lower when compared to mean Control body weight values.
During gestation, absolute body weights at 7500 ppm were statistically significantly lower than in Controls; the overall mean body weight gain of females receiving 7500 ppm was statistically significantly lower than Control (-16% of Control), with statistical significance attained during Days 0 to 7 and 14 to 20 of gestation. Following parturition, the mean body weight gain for females at all dose levels was essentially similar to Control, although females receiving 3500 or 7500 ppm had lower absolute body weights.
During the recovery phase (R0-R15) the mean absolute body weights for the males that received 7500 ppm were higher compared to Controls although the gains during the recovery period were similar to Controls. The absolute body weights for females that received 7500 ppm were statistically significantly lower compared to Controls, however mean body weight gain was statistically significantly higher for females previously receiving 7500 ppm Cistus concrete (almost 2.5-fold the Control value).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Group mean food consumption for toxicity and recovery phase males during the treatment period was similar to Controls for males receiving 1500 and 3500 ppm of Cistus Concrete administered via the diet. A reduction in food consumption (-33% of Control) was observed in males receiving 7500 ppm on Day 1 of treatment, however subsequent food consumption values were similar to Controls and slightly lower values compared to Controls could occur in all treated groups during the treatment period.
- Food consumption was generally lower for toxicity, recovery and reproductive phase females receiving 3500 ppm and 7500 ppm compared to Controls during the pre-pairing period and for the remainder of the treatment period for toxicity and recovery phase females, with values still attaining statistical significance several times after Day 5 of treatment at 7500 ppm.
- Toxicity, recovery and reproductive phase females receiving 3500 ppm and 7500 ppm also showed a marked reduction in food intake during the first four days of Cistus Concrete administered via the diet. Food consumption for females receiving 1500 ppm tended to be similar to the Controls during the treatment period with the exception of Day 1 of treatment.
- A general trend to a slight reduction in food consumption was observed for females receiving 7500 ppm during the gestation period, the reduction was greater during the first week of gestation although intake on Day 0-1 was the same as the Controls. Group mean food consumption for reproductive phase females receiving 1500 or 3500 ppm during gestation was similar to Controls.
- Following parturition, daily food intake was generally statistically significantly lower for females receiving 7500 ppm, leading to a mean overall decrease of 29% compared to Controls. The food intake of females receiving 1500 or 3500 ppm was similar to that of Controls throughout the lactation period.
- Food consumption was similar to the Controls through Days R1-R14 in the recovery phase for females. Overall food consumption for males during the recovery period was higher than the Controls (17% increase compared to Control), with food intake values particularly high during the first two days of recovery.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No effects of treatment were observed during ophthalmic examinations in Week 6 of treatment for male and female animals treated at all dose levels.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematology investigations after six weeks of dietary administration of the test substance revealed a statistically significant reduction in reticulocyte counts at all dose levels for males, however a dose dependent relationship was not apparent, and all values were within Historical Control Data (HCD) range. Following a two-week recovery period, reticulocyte counts remained slightly low compared to concurrent controls however statistical significance was not attained.
Red cell mass (haematocrit, haemoglobin and red blood cell counts) were statistically low for females at 7500 ppm compared to Controls however all values for these parameters were within the HCD range. Following a two-week recovery period, haematocrit, haemoglobin and red blood cell counts for females that received 7500 ppm were similar to Controls.
Activated partial thromboplastin time was slightly shorter for females receiving 7500 ppm compared to Controls and this difference attained statistical significance, as this change was confined to one sex, not associated with any changes to other clotting factors and within the HCD range it is considered this difference represents normal biological variation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant reduction in bile acids was apparent amongst males and females receiving 3500 and 7500 ppm, however mean values for both sexes were within the HCD range. Following the recovery period bile acids were still lower for males that received 7500 ppm compared to concurrent Controls however bile acids for females was slightly higher than Controls.
Creatinine levels were statistically significantly higher for females receiving 7500 ppm compared to Controls, but the mean value was within the HCD range and following a two-week recovery period, females previously receiving 7500 ppm of Cistus Concrete creatinine levels were similar to Controls.
All other differences from Controls, including those that attained statistical significance, were generally small, confined to one sex, or the magnitudes were not dose-related and,consequently, were considered to represent normal biological variation.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urinalysis investigations performed in Week 6 of treatment indicated the total amount of glucose was low in males and females at all dose levels, although a dose relationship was apparent for females only with statistical significance confined to females receiving 7500 ppm. The total amount of protein in the urine was low in females at 3500 or 7500 ppm and total creatinine output was also low in females at these levels, with the differences from Controls attaining statistical significance for both parameters. The means for the aforementioned parameters were all within the reported HCD range.
There was a decrease of sodium output in males and females at all dose levels when compared with that of the Controls, a dose relationship was evident in males only and all mean values were with the HCD range. Potassium and chloride output were also reduced for females at all dose levels when compared to Controls, statistical significance was attained for chloride output only but there was no dose response and all mean values were within the HCD range.
All other differences from Controls, including those that attained statistical significance, were generally small, confined to one sex, or the magnitudes were not dose-related and, consequently, were considered to represent normal biological variation.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- No treatment-related effects were observed for sensory reactivity and grip strength assessment during Week 6 of treatment for males and females at all dose levels.
- Motor activity assessment of males during Week 6 of treatment revealed no treatment-related effects in males at all dose levels. Motor activity assessment of females during Week 6 of treatment revealed an increase in the overall scores for both high and low beam break activity at all dose levels. The higher incidence of overall low beam break scores attained statistical significance at all dose levels. A dose dependent response was not evident in the total scores and when compared with Historical Control Data (HCD), all statistically significant values and total beam scores were within the normal range. The total low beam score for the Control female was below the range documented in the HCD which led to the incidental increases observed in the treated groups. The increased overall high beam scores for females at all dose levels showed a dose-dependent response however statistical significance was not attained. In addition, the control value was below the range documented in the HCD and the values of the treated groups were within the HCD ranges. Statistical significance was attained for the 30-minute interval at 3500 and 7500 ppm and at the 60-minute interval at 7500 ppm, and these values were also within the normal range documented in the HCD. The increase in motor activity observed in the females was not associated with any clinical signs or signs recorded during the arena observation and all total scores were within the HCD range; therefore, these differences were considered fortuitous in nature and were unrelated to treatment with Cistus Concrete.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Following six weeks of treatment, terminal body weights for toxicity phase males and females at 7500 ppm were statistically significantly lower compared to Controls. Group mean body weight adjusted liver weights were increased in both sexes given 7500 ppm. The greatest magnitude of change (1.13X) from controls was observed in females given 7500 ppm. Absolute liver weights were only slightly increased for females at 7500 ppm with no apparent dose relationship and for males at 7500 ppm absolute weights were decreased, in both sexes absolute liver weights were within the HCD range.
Group mean absolute heart weights and body weight adjusted thymus weights were low amongst males given 7500 ppm Cistus Concrete (0.85 and 0.61X Control, respectively), and attained statistical significance in each instance. In addition, adjusted levator ani plus bulbocavernosus muscle complex (LABC) weights were high for males given 7500 ppm. However, a dose-dependent relationship was not apparent for body weight adjusted thymus or LABC weights and the absolute values for thymus, LABC and heart weights were all within the historical control data range.
For the reproductive phase females that received 7500 ppm, the body weight adjusted combined weight of the uterus, cervix and oviducts was high, when compared to the Controls (1.32X Control). Absolute weights were however comparable in all groups and therefore this change was attributed to the lower bodyweight of females in Group 4. In addition, the mean absolute weight of the uterus, cervix and oviducts for females at 7500 ppm was within the HCD range.
In the recovery phase, mean adjusted adrenal weights were low compared to the Controls for females previously treated at 7500 ppm. The mean liver weights remained slightly but not statistically significantly higher in the males previously treated at 7500 ppm but the females were similar to Controls, following the 14-day recovery period. Mean absolute heart weights, adjusted thymus and LABC weights were similar to Controls for males previously treated at 7500 ppm.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination performed after 6-weeks of treatment or 2-weeks of recovery 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:
effects observed, non-treatment-related
Description (incidence and severity):
The microscopic examination performed after 6 weeks treatment revealed no test item-related lesions. All other histological changes (incidental findings) were considered to be unrelated to treatment.
Histopathological findings: neoplastic:
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 adult males or in offspring on Day 13 of age. Slightly lower mean T4 concentrations were observed in the adult males, however there was no dose relationship observed and the historical control data (HCD) showed that the Control values for this study were high.There was no consistent effect on T4 levels in the offspring. Consequently, there was no requirement to measure T4 in the samples obtained from offspring on Day 4 of age or from the adult females and none of the TSH (thyroid stimulating hormone) samples required analysis.
Key result
Dose descriptor:
NOAEL
Effect level:
7 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed.
Remarks on result:
other: mean achieved doses: 422 mg/kg bw/day in males and 411 mg/kg bw/day in females
Key result
Critical effects observed:
no

Formulation Analysis:

The homogeneity and stability were confirmed for Cistus Concrete in SDS VRF1 Certified with corn oil stabilizer at a ratio of test item to corn oil of 5 to 1 formulations at nominal concentrations of 800 ppm and 20000 ppm for ambient temperature storage (15 to 25ºC) for

8 days for 800 ppm and 15 days for 20000 ppm and frozen storage (-10 to -30ºC) for up to 15 days for both 800 ppm and 20000 ppm.

The mean concentrations were within the applied limits of +10/-15%, confirming the accuracy of formulation. The difference from mean remained within 5%, confirming precise analysis. The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure.

Conclusions:
Under the test condition, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 7500 ppm (mean achieved doses: 422 mg/kg bw/day in males and 411 mg/kg bw/day in females).
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, the test item was administered to groups of Crl:CD(SD) rats at dietary concentrations of 1500, 3500 and 7500 ppm. An additional subgroup was used to assess reversibility, persistence or delayed occurrence of systemic effects for 14 days post treatment. A similarly constituted control group was assigned to each phase, and received the vehicle, powdered SDS VRF1 Certified diet with corn oil, throughout the same relative treatment period.

Toxicity phase males were treated for three weeks prior to pairing up to necropsy after a minimum of six weeks. Toxicity phase females were treated for at least six weeks. Recovery phase males were treated for three weeks before pairing up to necropsy after a minimum of six weeks followed by a 14-day recovery period. Recovery phase females were treated for six weeks followed by a 14-day recovery period. Reproductive phase females were treated for three weeks before pairing, throughout pairing and gestation until Day 12 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 13 of lactation (the treated diet was made available until the morning of necropsy)

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, ophthalmic examinations, hematology (peripheral blood), blood chemistry, urinalysis, thyroid hormone analysis, organ weight, macroscopic pathology and histopathology investigations were undertaken.

The mean concentrations of Cistus Concrete in test formulations analyzed for the study were within ±10% of nominal concentrations, confirming accurate formulation. The difference from the mean value remained within 5%, confirming precise analysis.

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

The overall mean achieved dosages for toxicity phase animals during the treatment period were 85, 186 and 422 mg/kg bw/day for males and 86, 194 and 411 mg/kg bw/day for females receiving 1500, 3500 or 7500 ppm, respectively. The overall mean achieved dosages for reproductive phase females receiving 1500, 3500 or 7500 ppm were 92, 208 and 439 mg/kg bw/day and 207, 487 and 904 mg/kg bw/day during gestation and lactation, respectively.

Two females receiving 7500 ppm were prematurely killed as a result of a total litter loss and a relationship to treatment could not be completely discounted. In addition, one female receiving 1500 ppm was killed prematurely as this female failed to litter by Day 25 of gestation, because this female was not pregnant. There were no test-item related signs observed during the detailed physical examination and arena observations and no effects on sensory reactivity, grip strength or motor activity.

Overall body weight gains during the treatment period for toxicity and recovery phase males given 3500 or 7500 ppm were slightly low, body weight gains for males receiving 1500 ppm were similar to Controls. Overall group mean body weight gains for toxicity and recovery phase females were moderately low for females receiving 1500 ppm and significantly lower for females receiving 3500 or 7500 ppm. During the gestation period overall body weight gain for females receiving 7500 ppm was low (-16% of Control) and during the lactation period body weight gains of treated groups were similar to Controls. During the recovery period the mean body weight gains for males that received 7500 ppm was similar to Controls whereas mean body weight gain was statistically significantly higher for females previously receiving 7500 ppm Cistus Concrete (almost 2.5-fold the control value).

Mean food intake for toxicity and recovery phase males at all dose levels was generally similar to Controls throughout the treatment period, with the exception of males receiving 7500 ppm on Day 1 of treatment (-33% of Control). At 7500 ppm, subsequent food consumption values for males were similar to Controls and slightly lower values compared to Controls could occur in all treated groups during the treatment period. Food consumption was generally lower for toxicity, recovery and reproductive phase females receiving 3500 ppm and 7500 ppm compared to Controls during the pre-pairing period and for the remainder of the treatment period for toxicity and recovery phase females, with values still attaining statistical significance several times after Day 5 of treatment at 7500 ppm. Food consumption for females receiving 1500 ppm tended to be similar to the Controls during the treatment period with the exception of Day 1 of treatment. Mean food consumption during gestation and lactation tended to be low for females receiving 7500 ppm, especially during lactation (mean overall food intake was 29% lower compared to Control). Mean food consumption for females receiving 1500 or 3500 ppm during gestation or lactation was similar to Controls. Food consumption was similar to the Controls through Days R1-R14 in the recovery phase for females. Overall food consumption for males during the recovery period was higher than the Controls (17% increase compared to Control), with food intake values particularly high during the first two days of recovery.

There was no treatment-related ophthalmoscopic finding. The haematological examination of the peripheral blood and biochemical assessment of the

plasma did not reveal any toxicologically significant differences from Controls. The analysis of the urine in Week 6 of treatment revealed low glucose output in both sexes at all dose levels, low protein and creatinine output for females receiving 3500 or 7500 ppm. In addition, sodium output was low in both sexes at all dose levels and potassium and chloride output was reduced for females at all dose levels.

Terminal body weights for toxicity phase males and females at 7500 ppm were statistically significantly lower compared to Controls. Changes in organ weights consisted of slightly higher body weight adjusted mean liver weights for males and females at 7500 ppm when compared to Control. Following the recovery period, mean liver weights remained slightly higher in the males previously treated at 7500 ppm but the females were similar to Controls.

The macroscopic and microscopic examination of the adult males and females revealed no test-item related lesions.

Based on the results of this study it was concluded that the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was 7500 ppm (mean achieved doses: 422 mg/kg bw/day in males and 411 mg/kg bw/day in females). Therefore, the test substance is not classified for damage to organs through prolonged oral repeated exposure according to the criteria of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for sub-acute oral toxicity endpoint.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Remarks:
Inspected on 2018-01-16 / Signed on 2018-06-05.
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Physical state: Brownish-green, pasty mass.
- Stability under test conditions: stability of the test item is the supplier's responsability.

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
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 Crl:CD(SD) was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males: 69 to 75 days old; Females: 83 to 89 days old.
- Weight at study initiation: Males: 323 to 392 g; Females: 245 to 299 g
- Housing: 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.
- Number of animals per cage: Pre-pairing: up to four animals of one sex; Pairing: one male and one female; Males after mating: up to four animals; Gestation: one female; Lactation: one female + litter
- Diet: SDS VRF1 Certified powdered diet, ad libitum (removed overnight before blood sampling for hematology and blood chemistry investigations and during urine collection)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (removed overnight during urine collection)
- Acclimation period: Females: 20 days before commencement of treatment; Males: six days prior to the commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 h light : 12 h dark
- 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.
Paper shavings: Two handfuls provided to each Reproductive phase female cage from Day 20 after mating and throught lactation and changed at the same frequency as the cage bedding.

IN-LIFE DATES: from 19 April to 12 November, 2018.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
None.
Details on exposure:
DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet
- Correction factor: A correction factor was not required.
- Stabilizer: Corn oil (test material to corn oil ratio 5:1).
- Method of preparation: The test substance was incorporated into the diet to provide the required concentrations by initial preparation of a premix. On each occasion of the preparation of the premix, the required amount of test substance and corn oil were weighed into a suitable container. An amount of sieved diet that approximately equalled the weight of test substance was added and the mixture stirred together. A further amount of sieved diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
For the control diet, an amount of diet was added directly to the corn oil and then prepared as indicated for the premix.
- Frequency of preparation: Weekly.
- Storage of formulation: Deep-frozen (nominally -30 to -10 °C) until the day before use. Formulations were used within 28 h of removal from the freezer.
Duration of treatment / exposure:
Toxicity phase males: Three weeks before pairing up to necropsy after minimum of six weeks of treatment.
Toxicity phase females: At least six weeks.
Frequency of treatment:
Continuously
Post exposure period:
None
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Control group (Basal diet + corn oil)/Group 1
Dose / conc.:
1 500 ppm
Remarks:
Group 2 (Low dose)
Dose / conc.:
3 500 ppm
Remarks:
Group 3 (Mid dose)
Dose / conc.:
7 500 ppm
Remarks:
Group 4 (High dose)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Positive control(s):
- Positive control: Cyclophosphamide
- Justification for choice of positive control(s): Positive control from Envigo study GY05QJ
- Route of administration: feed
- Doses / concentrations: 20 mg/kg

Examinations

Tissues and cell types examined:
Femora bones were removed for marrow extraction and the prepared slides were examined for polychromatic erythrocytes (PCEs), normochromatic erythrocytes (NCEs) and total erythrocytes.
Details of tissue and slide preparation:
- Preparation of bone marrow smears
Animals were killed on the day after receiving their last daily dose. One femur was dissected from the first five male and female animals per group (surviving to necropsy) and the proximal head removed. Using a syringe and needle, the bone marrow was flushed from the marrow cavity with approximately 3 mL pre-filtered foetal calf serum into appropriately labelled centrifuge tubes (1 per animal). The resulting cell suspensions were centrifuged at 1000 rpm for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides (Schmid 1976). At least 4 smears/slides were prepared from each animal. The bone marrow smears were air-dried
- Fixation and staining of slides
The slides were fixed in methanol and allowed to air dry. Slides were rinsed in purified water and stained using an acridine orange solution at 0.0125 mg/mL and stored at room temperature in the dark until required. The remaining unstained slides were kept in reserve.
- Microscopic examination
At least 3 slides (prepared in a separate Envigo study [GY05QJ]) from animals treated with Cyclophosphamide (CPA), a well characterized clastogen, were stained and coded along with the bone marrow smears prepared from this study. Prior to scoring the slides were wet mounted with coverslips using purified water. Coded
slides were examined by fluorescence microscopy and 4000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. At least one smear was examined per animal, any remaining smears being held temporarily in reserve in case of technical problems with the first smear.
The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.
Evaluation criteria:
A test chemical is considered clearly negative if, in all experimental conditions examined:
a) None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control, b) There is no dose-related increase at any sampling time when evaluated by an appropriate trend test, c) All results are inside the distribution of the historical negative control data (95% confidence limits), and d) Bone marrow exposure to the test item(s) occurred.

A test chemical is considered clearly positive if:
a) At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control, b) This increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and c) Any of these results are outside the distribution of the historical negative control data (95% confidence limits).
Statistics:
Analysis of data: The results obtained for each treatment group will be compared with the results obtained for the control group using non-parametric statistical methods.
The computer systems used on this study to acquire and/or quantify data were Statistics SAS (StatXact).

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: the dose levels selected for investigation in this OECD 422 combined repeated dose toxicity study and reproductive/developmental toxicity screening study (0, 1500, 3500 and 7500 ppm) were chosen in conjunction with the Sponsor and were based on the results of a 14-day preliminary study conducted at these laboratories (Covance Study No. NN81HP).
- Solubility: no data
- Clinical signs of toxicity in test animals: There were no clinical signs observed during the treatment period for toxicity and recovery phase animals, reproductive females prior to pairing, during gestation and lactation that were considered related to dietary administration of Cistus Concrete. There were no signs observed during the recovery period that were considered to be associated with the previous treatment with Cistus Concrete.
- Rationale for exposure: Continuously (OECD 422)


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
* The data for the concurrent vehicle control %MPCE were within the ranges determined by the laboratory historical control data (95% confidence limits), therefore, the performance of the vehicle was consistent with a valid assay.
Cistus Concrete did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes in male or female Crl:CD(SD) rats. All group mean values for male and female Crl:CD(SD) rats treated with Cistus Concrete were within the current vehicle historical control range (95% confidence limits). The coded positive control slides prepared from Envigo study GY05QJ demonstrated the ability of the scorer to detect increases in micronucleated polychromatic erythrocytes.
* Cistus Concrete did not cause any significant increases in the incidence of micronucleated normochromatic erythrocytes in male or female Crl:CD(SD) rats.
* The data for the concurrent vehicle control %PCE were within the ranges determined by the laboratory historical control data (95% confidence limits), therefore, the performance of the vehicle was consistent with a valid assay. Cistus Concrete did not cause any statistically significant decreases in the proportion of polychromatic erythrocytes in male or female Crl:CD(SD) rats. All group mean values for male and female Crl:CD(SD) rats treated with Cistus Concrete were within the current vehicle historical control range (95% confidence limits).

Any other information on results incl. tables

Table 7.6.2/2: Summary of results and statistical analysis (Males)

Sampling time

after final dose

Treatment

(ppm)

Proportion of

PCE, Group mean

% # (SD)

Group mean

MPCE/ 4000

PCE # (SD)

Group mean %

MPCE #

 24 hours

Basal Diet + Corn Oil

(-)

50.0 (6.4)

6.4 (3.4)

0.16

Cistus Concrete

(1500)

45.2 (3.2)

5.8 (2.7)

0.15

Cistus Concrete

(3500)

44.7 (1.4)

6.8 (2.6)

0.17

Cistus Concrete

(7500)

45.8 (3.8)

6.0 (2.1)

0.15

Cyclophosphamide

(20 mg/kg)

45.3 (0.8)

72.7* (6.7)

1.82

Table 7.6.2/2: Summary of results and statistical analysis (Females)

Sampling time

after final dose

Treatment

(ppm)

Proportion of

PCE, Group mean

% # (SD)

Group mean

MPCE/ 4000

PCE # (SD)

Group mean %

MPCE #

 24 hours

Basal Diet + Corn Oil

(-)

46.0 (2.9)

5.8 (4.0)

0.15

Cistus Concrete

(1500)

46.0 (3.3)

6.2 (2.5)

0.16

Cistus Concrete

(3500)

48.2 (1.9)

6.8 (2.9)

0.17

Cistus Concrete

(7500)

46.6 (2.2)

8.8 (2.5)

0.22

Cyclophosphamide

(20 mg/kg)

44.0 (8.3)

60.0* (14.7)

1.50

 

PCE: Polychromatic erythrocytes

MPCE: Number of micronucleated polychromatic erythrocytes observed per 4000 polychromatic erythrocytes examined

a: Positive control slides from Envigo study GY05QJ

SD: Standard deviation

#: Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table.

Applicant's summary and conclusion

Conclusions:
Under the test conditions of this study, Cistus Concrete did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male or female Crl:CD(SD) rats when administered orally by diet in this in vivo test procedure.
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, the test item was administered to groups of Crl:CD(SD) rats at dietary concentrations of 1500, 3500 and 7500 ppm. An additional subgroup was used to assess reversibility, persistence or delayed occurrence of systemic effects for 14 days post treatment. A similarly constituted control group was assigned to each phase, and received the vehicle, powdered SDS VRF1 Certified diet with corn oil, throughout the same relative treatment period.

Toxicity phase males were treated for three weeks prior to pairing up to necropsy after aminimum of six weeks. Toxicity phase females were treated for at least six weeks.

During the study, bone marrow micronucleus test (OECD 474) was undertaken.This phase of the study was designed to assess the potential induction of micronuclei by Cistus Concrete in bone marrow cells of male and female Crl:CD(SD) rats following 6 weeks of dietary administration.

Bone marrow smears were obtained from the first 5 male and female animals, surviving to scheduled necropsy, from the vehicle control group and each of the test item groups on the day after administration of the final dose. At least one smear from each animal was examined for the presence of micronuclei in 4000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes was also kept.

The data for the concurrent vehicle control (group mean % polychromatic erythrocytes [%PCE] and % micronucleated polychromatic erythrocytes [%MPCE]) were within the ranges determined by the laboratory historical control data (95% confidence limits), therefore, the performance of the vehicle was consistent with a valid assay. No statistically significant increases in the frequency of micronucleated polychromatic erythrocytes (%MPCE) and no statistically significant decreases in the proportion of polychromatic erythrocytes (%PCE) were observed in male or female Crl:CD(SD) rats administered Cistus Concrete at any dose level, compared to vehicle control values. All group mean values (%PCE and %MPCE) were within the current vehicle historical control range (95% confidence limits). The coded positive control slides prepared from Envigo study GY05QJ demonstrated the ability of the scorer to detect increases in micronucleated polychromatic erythrocytes.

It is concluded that Cistus Concrete did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male or female Crl:CD(SD) rats when administered orally by diet in this in vivo test procedure.