Concrete of Cistus ladaniferus (Cistaceae) obtained from stems and leaves by organic solvents extraction

Toxicity to reproduction

Administrative data

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

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. QA statement)

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 or test system 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.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 30 May to 22 June, 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

Preliminary study, used as range-finder experiment for OECD 422 screening test performed in GLP laboratory.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline required

Principles of method if other than guideline:

The purpose of this study was to assess the systemic toxic potential of Cistus Concrete in a 14-day oral dietary study in Crl:CD(SD) rats, to select a suitable high dose for a subsequent OECD 422 combined repeated dose toxicity study and reproductive/developmental screening study (Covance Study Number: KL09NM).

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Males: 72 to 78 days; Females: 86 to 92 days
- Weight at study initiation: Males: 367-435 g; Females: 262-328 g
- Housing: Animals were housed in groups of 4/sex in polycarbonate cages
- Diet: SDS VRF1 Certified powdered diet, ad libitum (non-restricted)
- Water: Potable water from public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70%
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light
- Environmental Enrichment
Aspen chew block: Provided to each cage throughout the study and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study and replaced when necessary.

IN-LIFE DATES: From 30 May To 22 June 2018.

Route of administration:

oral: feed

Details on route of administration:

The dietary route of administration was chosen to simulate the conditions of possible human exposure.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet.
- Stabiliser: Corn oil (test material to corn oil ratio 5:1).
- Correction factor: A correction factor was not required.
- Method of preparation:
On each occasion of the preparation of the premix the required amount of test item and solvent was weighed out and magnetically stirred until the test item was fully dispersed. The mixture was added to the appropriate amount of plain diet. A further approximately 100g of plain diet to clean the weighing container was used and then added to the mixture. This mixture was stirred well, then left for the solvent to evaporate for at least 45 minutes while stirred frequently,
until a constant weight was achieved and at least 90% of the initial weight of added solvent was removed. The required amount of corn oil was added to the mixture. Additional diet was used to rinse the corn oil weighing container which was then added to the mixture. This was ground using a mechanical grinder then made up to the correct weight with plain diet. It was transferred to a plastic container and mixed using a Turbula mixer for 100 cycles at 16 rpm to ensure all the test item 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, it was treated the same except that no test item was used.
- Frequency of preparation: Weekly
- Storage of formulation: Deep-frozen (-10 to -30 °C) for 15 days

STABILITY AND HOMOGENEITY
Before the commencement of treatment, the suitability of the proposed mixing procedure and the homogeneity and stability of the test material in the carrier diet, was determined as part of the main OECD 422 study (Study Number: KL09NM).
In that study, dietary formulations in the range 800 to 20000 ppm were analyzed to assess the stability and homogeneity of the test item in the diet matrix. The
formulations were confirmed homogenous and stable for up to four days when stored at ambient temperature (15 to 20°C) or 15 days frozen (-10 to -30°C).
No formulation analysis was performed on this study. However, 200 g samples of the first preparations per group were retained in Pharmacy at -10 to -30°C.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No analysis of the formulated diets was performed on this study, however, samples of the first preparations were stored at - 10 to - 30 °C.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Continuously

Dose / conc.:

0 ppm

Remarks:

Untreated diet of the same batch with corn oil / Group 1 (control)

Dose / conc.:

3 500 ppm

Remarks:

Group 2 (Low dose)

Dose / conc.:

7 500 ppm

Remarks:

Group 3 (mid dose)

Dose / conc.:

15 000 ppm

Remarks:

Group 4 (High dose)

No. of animals per sex per dose:

4 animals/sex/dose.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary concentrations used in this study (0, 3500, 7500 and 15000 ppm) were selected in conjunction with the Sponsor. In a recent acute oral toxicity study conducted according to OECD guideline 423 in rats, the LD50 of the test item was found to be higher than 2000 mg/kg bw. Based on these results, 15000 ppm (equivalent to 1000 mg/kg bw/day) was selected as the highest dietary concentration in order to assess the palatability and the systemic toxicity of this test item.
- Rationale for animal assignment: Randomly allocated on arrival. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
- Animal Replacement
On Day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed ±20% of the mean for the appropriate sex. No replacements were necessary.

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days on Days -3, 1, 4, 8, 11 and 15 on each animal
to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: the weight of each animal was recorded three days before treatment commenced, on the day that treatment commenced (Day 1), daily throughout the study and before 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 daily throughout the study from Day -3.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. No significant effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; Animals were killed following 14 days of treatment by carbon dioxide asphyxiation and subjected to detailed necropsy. 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, unless specified above. Requisite organs were weighed for animals killed at scheduled intervals.
HISTOPATHOLOGY: No; but tissues with macroscopic abnormalities were preserved for microscopic examinations in 10% neutral buffered formalin for microscopic examination (except testes: In modified Davidson’s fluid).

Statistics:

Summary statistics (e.g. means and standard deviations) presented in this report were calculated from computer-stored individual raw data.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed that were considered to be related with Cistus Concrete.

Mortality:

no mortality observed

Description (incidence):

There were two premature deaths in this study, two females receiving 15000 ppm were dispatched to necropsy on Day 7 of treatment for welfare reasons, due to progressive body weight loss from the commencement of treatment. There were no findings observed at macroscopic examination for both females.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight of males and females given Cistus Concrete by dietary administration was affected at all dose levels for males and females, the effect was particularly evident at the commencement of treatment (Days 1 to 4).
Group mean body weight loss or reduced body weight gain was evident during Days 1 to 4 of treatment at all dose levels for males, thereafter a period of good weight gain was recorded and weight change was generally similar to Controls. As a result, group mean body weight gains from Days 1 to 15 of treatment were markedly lower for males receiving 3500, 7500 or 15000 ppm (62, 60 and 65% of Control, respectively), although without a clear dose-dependent effect. Group mean body weight losses were particular evident for females receiving 3500, 7500 and 15000 ppm of Cistus Concrete during Days 1 to 4 of treatment, with a mean loss of 3, 12 and 28 g, respectively. In general, an improvement in body weight gain for females at all dose levels was observed during the second week of treatment. Consquently, overall body weight gains during the treatment period were markedly lower for females receiving 7500 ppm (63% of Controls) and 15000 ppm (a group mean body weight stasis of 0 g was recorded from Days 1 to 15 of treatment).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Overall food consumption was slightly low for males at all dose levels and for females receiving 3500 ppm and moderately lower for females receiving 7500 or 15000 ppm compared with Controls. A dose response was evident for females only.
Food consumption for males at 7500 or 15000 ppm was significantly lower on Day 1 of treatment and remained slightly lower on Day 2 of treatment when compared with Controls. Food consumption of males receiving 3500 ppm was slightly low on Day 1 of treatment. Overall food consumption from Days 1 to 15 of treatment among males receiving 3500, 7500 or 15000 ppm was 92, 88 and 92% of Controls, respectively.
Food consumption for females receiving 7500 or 15000 ppm was significantly lower during the first week of treatment. Food intake improved during the second week of treatment. Food consumption for females receiving 3500 ppm was slightly low on Days 1 and 2 of treatment. Overall food consumption for females receiving 3500, 7500 or 15000 ppm was 89, 74 and 63% of Controls, respectively and therefore indicative of a dose-dependent effect.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

There was no effect of treatment on water consumption observed at all dose levels. On one occasion (Day 4 of treatment) increased water consumption was observed for females that receivied 15000 ppm.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean absolute and body weight relative liver weights were higher (+13% and +19%, respectively) for males that received 15000 ppm when compared with Controls. Mean absolute and body weight relative liver weights were slightly higher for females at all dose levels when compared with Controls, with both changes showing a dose-dependent response.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination of animals on Day 15 of study did not reveal any findings that were considered related to treatment with Cistus Concrete.
At scheduled necropsy, one male that received 7500 ppm had an abnormal shape and absent caput of the right epididymis, the right testes was described as soft, small and had pale areas. In addition, this male had clear cysts observed on both kidneys. Additionally, pelvic dilatation of the right kidney was observed in one male that received 15000 ppm.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Key result

Dose descriptor:

other: 7500 ppm is considered as the high dose level in the OECD 422 screening study.

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

mortality

organ weights and organ / body weight ratios

Remarks on result:

other: 7500 ppm Cistus Concrete is considered suitable for use as the high dose level in the reproduction/developmental toxicity screening study.

Key result

Critical effects observed:

no

The overall mean achieved doses in animals receiving 3500, 7500 or 15000 ppm were 193, 415 and 841 mg/kg bw/day in males and 200, 391 and 660 mg/kg bw/day in females, respectively.

Conclusions:

Based on the results of this dose range-finding study, dose levels of 1500, 3500 and 7500 ppm were considered applicable for the subsequent dose toxicity study with reproduction/ developmental toxicity screening test (OECD 422, Covance study KL09NM).

Executive summary:

In a 14- day toxicity range-finding study, groups of Crl:CD(SD) rats (4/sex/dose) received test item orally, via the diet at concentrations of 3500, 7500 or 15000 ppm. A similarly constituted control group received the basal diet with a corn oil stabilizer. During the study, clinical condition, body weight, food consumption, visual water consumption, organ weight and macropathology investigations were undertaken.

The overall mean achieved doses in animals receiving 3500, 7500 or 15000 ppm were 193, 415 and 841 mg/kg bw/day in males and 200, 391 and 660 mg/kg bw/day in females, respectively.

There were no treatment-related changes in clinical condition at all dose levels. There were two premature deaths; on Day 7 of treatment two females at 15000 ppm were dispatched to necropsy for welfare reasons due to progressive body weight loss. There were no findings observed at macroscopic examination for both females. Some short periods of reduced body weight gain or minor weight loss were apparent for males and females at all dose levels. These reduced gains or losses predominately occurred during the initial period of treatment. Subsequently, overall body weight gain and/or stasis for the treatment period was still lower for males at all dose levels and for females at 7500 and 15000 ppm when compared to Controls. Overall food consumption was slightly lower for males at all dose levels and for females receiving 3500 ppm and moderately lower for females receiving 7500 or 15000 ppm when compared with Controls. Visual water consumption at all dose levels was unaffected by treatment. There was an indication that absolute and body weight relative liver weights were slightly higher in males that received 15000 ppm and in females at all dose levels when compared with Controls, with an apparent dose-related response in females only. There were no test item-related macroscopic changes observed at necropsy.

Based on the results obtained in this 14-day toxicity study and due largely to the two premature decedents in females receiving 15000 ppm, it was concluded that a dose level of 7500 ppm Cistus Concrete is considered suitable for use as the high dose level in the reproduction/developmental toxicity screening study (OECD 422, Covance study KL09NM).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

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.

Test material

Test material information

Constituent 1

Reference substance name:

Concrete of Cistus ladaniferus (Cistaceae) obtained from stems and leaves by organic solvents extraction

EC Number:

947-090-6

Molecular formula:

not applicable for UVCB

IUPAC Name:

Concrete of Cistus ladaniferus (Cistaceae) obtained from stems and leaves by organic solvents extraction

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 or test system 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:

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Remarks:

Basal diet + corn oil / Group 1 (Control)

Doses / concentrations 2

Dose / conc.:

1 500 ppm

Remarks:

Group 2 (Low dose)

Doses / concentrations 3

Dose / conc.:

3 500 ppm

Remarks:

Group 3 (mid dose)

Doses / concentrations 4

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

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.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

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.