Gum of Cistus ladaniferus (Cistaceae) obtained from stems and leaves by extraction with alkaline solution

Endpoint summary

Description of key information

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD Guideline 422, GLP, Rel.1), the NOAEL for systemic toxicity was7500 ppm (mean achieved doses of 322 mg/kg bw/day for males, 331 mg/kg bw/day for toxicity phase females, 342 mg/kg bw/day for females during gestation and 731 mg/kg bw/day for females during lactation).The mean of the mean achieved doses for males and females toxicity, obtained during the OECD 422 study corresponds to 326 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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 27 February to 12 July, 2019

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 2019-04-02 / Signed on 2019-08-01

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.

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: 322-400 g; Females: 238 to 311 g
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization and pre-treatment periods.
- Number of animals per cage:
* Pre-pairing, toxicity phase females and recovery animals: 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
- Acclimatization and pre-treatment: Males: six days before commencement of treatment.Females: 20 days before commencement of treatment.
- 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:

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.

IN-LIFE DATES: from 13 March to 12 July 2019.

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:

other: Feed

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 substance was incorporated into the diet to provide the required concentrations by initial preparation of a premix. The test item was melted at 70°C before weighing. On each occasion of the preparation of the premix the required amount of test item and solvent (acetone) were 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 amount (approximately 100 g) of plain diet was used to clean the weighing container and then added to the mixture. This mixture was stirred well, then left for the solvent (acetone) 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. The formulation 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.
- Storage of formulation: Frozen (-10 to -30°C).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Stability and homogeneity: The homogeneity and stability of Labdanum Gum in the concentration range of 800 ppm to 20000 ppm in SDS VRF1 diet was confirmed as part of another study, Covance study number GS09VH.
- Achieved concentration: Samples of each formulation prepared for administration 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 of 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 target dietary concentrations 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 (Envigo Study No. HX59HH).
- 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.

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.
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.

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.

Other examinations:

Thyroid Hormone Analysis:
At termination: F0 males, All F0 Reproductive phase females

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 or for Reproductive phase females prior to pairing, during gestation or during lactation that were considered related to dietary administration of Labdanum Gum. There were no signs observed during the recovery period that were considered to be associated with previous treatment with Labdanum Gum.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male that received 1500 ppm (No. 7) was killed for welfare reasons on Day 29 of study due to a damaged right eye. Macroscopic examination revealed a protruding and abnormal dark coloured right eye. This death was considered not to be related to treatment with Labdanum Gum.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the first week of treatment there was an initial slight reduction in group mean body weight gain for Toxicity and Recovery phase males that received 3500 or 7500 ppm compared to Control. Overall group mean body weight gains for the treatment period (Days 1 to 43) were slightly lower than Control for all groups of treated males (97%, 83% and 93% of Control at 1500, 3500 and 7500 ppm respectively), however, no dose-response and no statistical significance were apparent. There was an initial group mean body weight loss, between Days 1 to 4 of treatment, for all groups of treated females, this minor loss was greatest at 7500 ppm. Thereafter, group mean body weight gain was observed for all groups of treated females, with the exception of Days 29 to 36 where a group mean body weight loss was apparent for females that received1500 or 3500 ppm and weight gain was low at 7500 ppm. The overall group mean body weight gains (Days 1 to 43) for Toxicity and Recovery phase females treated at 3500 or 7500 ppm were statistically significantly lower than Control (57% and 47% of Control at 3500 and 7500 ppm respectively).
During gestation, the group mean body weight gain for all groups of treated Reproductive phase females was slightly higher than Control (114%, 111% and 106% of Control at 1500, 3500 and 7500 ppm respectively). Overall body weight gain throughout lactation (Days 1 to 13) was slightly lower than Control for females that received 1500 or 7500 ppm and comparable to Control for females that received 3500 ppm (81%, 107% and 78% of Control at 1500, 3500 and 7500 ppm respectively), although statistically significant lower values than Control were observed in all treated groups between Days 7-13 (30%, 45% and 70% of Control at 1500, 3500 and 7500 ppm respectively).
During the recovery phase (R1 to R15), group mean body weight gain for males that previously received 7500 ppm was slightly lower than Control (80% of Control). Group mean body weight gain during the recovery phase for females that previously received 7500 ppm was much higher than Control although not statistically significant (2000% of Control).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

7500 ppm were statistically significantly lower than Control (57% and 47% of Control at 3500 and 7500 ppm respectively). During gestation, the group mean body weight gain for all groups of treated Reproductive phase females was slightly higher than Control (114%, 111% and 106% of Control at 1500, 3500 and 7500 ppm respectively). Overall body weight gain throughout lactation (Days 1 to 13) was slightly lower than Control for females that received 1500 or 7500 ppm and
comparable to Control for females that received 3500 ppm (81%, 107% and 78% of Control at 1500, 3500 and 7500 ppm respectively), although statistically significant lower values than Control were observed in all treated groups between Days 7-13 (30%, 45% and 70% of Control at 1500, 3500 and 7500 ppm respectively). During the recovery phase (R1 to R15), group mean body weight gain for males that previously received 7500 ppm was slightly lower than Control (80% of Control). Group mean body weight gain during the recovery phase for females that previously received 7500 ppm was much higher than Control although not statistically significant (2000% of Control).

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 examination in Week 6 of treatment for males and female animals treated at all dose levels.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematology investigations for Toxicity phase animals during Week 6 of treatment with Labdanum Gum revealed no treatment related changes in either sex.
Activated partial thromboplastin time was statistically significantly higher than Control for males that received 3500 ppm, however, a dose response was not apparent.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood chemistry investigations during Week 6 of treatment revealed that creatinine levels for all groups of treated males were statistically significantly higher than Control but within the HCD range. Following a two-week recovery period, creatinine levels for males that had previously received 7500 ppm were comparable to Control.
During Week 6, a statistically significant decrease in urea concentration was apparent in females that received 7500 ppm but the value was within the HCD range. Following a two-week recovery period, urea concentration in females that had previously received 7500 ppm was comparable to Control.
During Week 6, a statistically significant increase in phosphorus levels was apparent amongst males and females that received 7500 ppm but all values were within the HCD range. Following a two-week recovery period, phosphorus levels in males and females that had previously received 7500 ppm were comparable to Control.
During Week 6, globulin levels for females that received 7500 ppm were statistically significantly higher than Control and slightly outside of the HCD range. Following a
two-week recovery period, globulin levels in females that had previously received 7500 ppm were comparable to Control. All other differences from Control, including those that attained statistical significance, were generally small, confined to one sex, or the magnitudes were not dose-relatded and,consequently, were considered to represent normal biological variation.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Total creatinine output was higher than Control for males treated at 3500 or 7500 ppm, however, all values were within the HCD range and no such trend was apparent in the females. Sodium concentration was higher than Control for all groups of treated females, however, all values were within the HCD range and no such trend was apparent in the males.
All other differences from Control 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):

Sensory Reactivity Observations and Grip Strength:
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:
Motor activity assessment of males and females during Week 6 of treatment revealed no treatment-related effects at any dose level investigated.
The total low beam scores for all groups of treated males were slightly lower than Control, however, the individual data revealed some inter-group variability with no common trends. The total high beam score for females that received 7500 ppm were slightly higher than Control, however, there was one atypical female (No.170) which had consistently high high beam scores throughout the 1-hour testing period compared to the other females in this group. This female showed a normal pattern of lower activity during the second half of the recording period so this data is deemed to be genuine.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Following six weeks of treatment, terminal body weights for Toxicity phase females that received 7500 ppm were statistically significantly lower than Control.
A dosage related decrease in group mean body weight adjusted adrenal weights was observed in Toxicity phase males, with statistical significance attained at 7500 ppm. All values were within the HCD range. Following the two-week recovery period, group mean body weight adjusted adrenal weight for males that previously received 7500 ppm were comparable to Control.
Group mean body weight adjusted thymus weights were statistically significantly lower than Control in Toxicity phase males at the end of the treatment period, however, no dose response was apparent. All values were within the HCD range. Following the two-week recovery period, group mean body weight adjusted thymus weights for males that previously received 7500 ppm were comparable to Control.
Group mean body weight adjusted ovary weights were slightly low at 7500 ppm for the Toxicity phase females but the difference did not attain statistical significance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related macroscopic findings. All findings were considered incidental and 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 of exposure revealed no test item-related lesions. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within their stages. No cell or stage specific abnormalities were noted.

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. 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.

Key result

Critical effects observed:

no

Formulation Analysis:

On this study each diet preparation was analysed for achieved concentration and homogeneity. The mean concentrations were within -16.0% to -33.6% of the nominal concentration. The coefficient of variation values remained within 5%, with the exception of Week 1 Group 3 and Week 7 Group 3 and 4, which were 5.46%, 5.87% and 6.94%, respectively.

The recoveries were prepared at each occasion as a quality control measure. In Week 2, all three recoveries were outside of the acceptance limits determined during the validation of the analytical method (83.7% - 103.7%). The recoveries were within 105.4% to 108.5% from nominal concentration. This was within the acceptance limits for a validation run (90 to

110%). The corrected concentrations for Week 2 formulations were comparable with all other occasion on the study, therefore this was considered to be an accurate reflection of the diet concentrations.

For Week 6 re-preparation, the procedural recovery prepared at 3500 ppm was outside of the acceptance limits determined during the validation of the analytical method (108.0%). This was excluded from the mean percentage recovery. As two out of three recoveries were within acceptance limits, the results were considered acceptable.

Achieved Dose:

The overall mean achieved doses for Toxicity, Recovery and Reproductive phase animals during treatment that received Labdanum Gum at target dietary concentrations of 1500, 3500 or 7500 ppm were 65, 140 and 322 mg/kg bw/day in males and 68, 155 and

331 mg/kg bw/day in females, respectively.

The overall mean achieved doses for Reproductive phase females during gestation that received target dietary concentrations of 1500, 3500 or 7500 ppm were 70, 175 and 342 mg/kg bw/day respectively.

The overall mean achieved doses for Reproductive phase females during lactation that received target dietary concentrations of 1500, 3500 or 7500 pm were 163, 376 and 731 mg/kg bw/day respectively.

Conclusions:

Under the test conditions of thi study, the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was the high dose 7500 ppm (mean achieved doses of 322 mg/kg bw/day in males and 331 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 before 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 minimum 14-day recovery. Recovery phase females were treated for six weeks followed by a minimum 14-day recovery.

Reproductive phase females were treated for three weeks before pairing, then 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, macropathology and histopathology investigations were undertaken.

Each diet preparation was analysed for achieved concentration and homogeneity. The mean concentrations were within -16.0% to -33.6% of the nominal concentration. The coefficient of variation values remained within 5%, with the exception of Week 1 Group 3 and Week 7

Group 3 and 4, which were 5.46%, 5.87% and 6.94%, respectively. On this study, target dietary concentrations were set in the study plan and referenced throughout the report, however, each diet preparation was analysed for achieved concentration prior to being fed to the animals.

The overall mean achieved doses for Toxicity, Recovery and Reproductive phase animals during treatment that received Labdanum Gum at target dietary concentrations of 1500, 3500 or 7500 ppm were 65, 140 and 322 mg/kg bw/day in males and 68, 155 and 331 mg/kg bw/day in females, respectively. The overall mean achieved doses for Reproductive phase females that received target dietary concentrations of 1500, 3500 or 7500 ppm were 70, 175 and 342 mg/kg bw/day and 163, 376 and 731 mg/kg bw/day during gestation and lactation respectively.

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males.

One male that received 1500 ppm was killed for welfare reasons on Day 29 of study due to a damaged right eye. Macroscopic examination revealed a protruding and abnormal dark coloured right eye. This death was not considered to be related to treatment with Labdanum Gum. There were no test-item related signs observed during detailed physical examination and arena observation and no effects on sensory reactivity, grip strength or motor activity.

Effects on body weight performance and food consumption (comprising periods of low mean weight gain and reduced food consumption) were evident throughout the treatment period for all groups of treated Toxicity and Recovery phase males and females. Following the commencement of treatment, an initial reduction in body weight gain was evident for males that received 3500 or 7500 ppm as well as minor mean weight loss in all groups of treated females. In addition, an initial reduction in food intake was evident following the commencement of treatment for females that received 7500 ppm. These effects were considered to be related to the palatability of the test item. Thereafter, the food intake of females that received 7500 ppm remained marginally low. Overall group mean body weight gain throughout the treatment period (Days 1 to 43) was slightly low for all groups of treated males and females that received 3500 or 7500 ppm. The food intake of all groups of treated males was generally marginally lower than Control for the first three weeks of treatment (Days 1 to 22) and the food intake of females that received 1500 or 3500 ppm was generally slightly lower than or comparable to Control. Following six weeks of treatment, the terminal body weights for Toxicity phase females that received 7500 ppm were statistically significantly lower than Control.

Food intake during early gestation (Days 0 to 8) for Reproductive phase females that received 7500 ppm was marginally lower than Control but comparable to Control thereafter. Overall body weight gain throughout lactation (Days 1 to 13) was slightly lower than Control for females that received 1500 or 7500 ppm (but not at 3500 ppm) and the food intake during lactation for females that received 7500 ppm was generally marginally lower than Control.

During the recovery period, group mean body weight gain for males that previously received 7500 ppm was slightly lower than Control, however, group mean body weight gain during the recovery phase for females that previously received 7500 ppm was higher than Control.

There was no treatment-related ophthalmoscopic finding.

Haematological examination of the peripheral blood during Week 6 of treatment did not reveal any toxicologically significant differences from Control.

Biochemical assessment of the plasma during Week 6 of treatment revealed that creatinine levels for all groups of treated males were statistically significantly higher than Control. Following a two-week recovery period, creatinine levels for males that had previously received 7500 ppm were comparable to Control. A statistically significant decrease in urea concentration was apparent in females that received 7500 ppm. Following a two-week recovery period, urea concentration in females that had previously received 7500 ppm was comparable to Control. In addition, a statistically significant increase in phosphorus levels was apparent amongst males and females that received 7500 ppm. Following a two-week recovery period, phosphorus levels in males and females that had previously received 7500 ppm were comparable to Control. Globulin levels for females that received 7500 ppm were statistically significantly higher than Control. Following a two-week recovery period, globulin levels in females that had previously received 7500 ppm were comparable to Control.

The analysis of urine during Week 6 of treatment revealed that total creatinine output was higher than Control for males treated at 3500 or 7500 ppm, however, all values were within the HCD range and no such trend was apparent in the females. In addition, sodium concentration was higher than Control for all groups of treated females, however, all values were also within the HCD range and no such trend was apparent in the males.

Following six weeks of treatment, terminal body weights for Toxicity phase females that received 7500 ppm were statistically significantly lower than Control. Changes in organ weights consisted of a dosage related decrease in group mean body weight adjusted adrenal weights in Toxicity phase males, with statistical significance attained at 7500 ppm. Following the two-week recovery period, group mean body weight adjusted adrenal weight for males that previously received 7500 ppm were comparable to Control. Group mean body weight adjusted thymus weights were statistically significantly lower than Control in all groups of treated Toxicity phase males, however, no dose response was apparent. Following the two-week recovery period, group mean body weight adjusted thymus weights for males that previously received 7500 ppm were comparable to Control. Group mean body weight adjusted ovary weights were slightly low at 7500 ppm for the Toxicity phase females but the difference did not attain statistical significance.

Macroscopic and microscopic examination of a full range of organs/tissues from the adult males and females revealed no test-item related lesions.

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was the high dose (target dietary concentration of 7500 ppm) with mean achieved doses of 322 mg/kg bw/day in males and 331 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 UN GHS.

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

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 18 April to 18 July, 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 Labdanum G um, in a 14-day oral dietary study in Crl:CD(SD)rats, and to aid in the selection of a suitable high dose for a subsequent OECD 422 combined toxicity and reproductive/developmental screening study (Envigo Study No. NR09JX)

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: 70 to 77 days; Females: 84 to 91 days
- Weight at study initiation: Males: 341-407 g; Females: 258-354 g
- Housing: Animals were housed in groups of 4/sex in polycarbonate cages
- Diet: SDS VRF1 Certified powdered diet, ad libitum (removed overnight before blood chemistry collection)
- Water: Potable water from public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 7 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 DATE: From 18 April To 18 July, 2018.VFGC

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: the test substance was incorporated into the diet to provide the required concentrations by initial preparation of a premix. the test item was melted at 70°C before weighing. On each occasion of the preparation of the premix the required amount of test item and solvent were weighed out and magnetically stirred until the test item was fully dispersed. The mixture was added to the appropriate amount (approximately 100 g) of plain diet was used to clean the weighing container and then added to the mixture. The mixture was stirred well, then left for the solvent to evaportae 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. The formulation 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.
- Frequency of preparation: Weekly
- Storage of formulation: frozen (- 10 to - 30°C)
STABILITY AND HOMOGENEITY
Before the commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations analyzed to assess homogeneity and stability in Envigo Study Number: GS09VH.The formulations were confirmed homogenous and stable for up to 28 hours when stored at ambient temperature (15 to 20°C) or 22 days frozen (-10 to -30°C).
No formulation analysis was performed on this study. However, 200 g of samples of the first preparation 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.:

5 000 ppm

Remarks:

Group 2 (Low dose)

Dose / conc.:

10 000 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, 5000, 10000 and 15000 ppm) were selected in conjunction with the Sponsor. In two recent acute oral toxicity studies conducted according to OECD guidelines 420 and 423 in rats on two different extracts of the same botanical origin as the test item, the LD50 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 Day 1, 4, 8, 11 and 15 to monitor general health.

BODY WEIGHT: Yes
- The weight of each animal was recorded for the 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. 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. The retained tissues were checked before disposal of the carcass.

ORGAN WEIGHTS: For bilateral organs, left and right organs were weighed individually and presentated in the table 7.5.1/1. Requisite organs were weighed for animals killed at the scheduled interval.

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).

Other examinations:

None.

Statistics:

Summary statistics (e.g. means and standard deviations) presented in this report were calculated from computer-stored individual raw data. Group mean values and standard deviations were frequently calculated using a greater number of decimal places than presented in the appendices. It is, therefore, not always possible to derive exact group values from the data presented in the appendices.
For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no changes in clinical condition that were considered to be related to treatment with Labdanum Gum.

Mortality:

no mortality observed

Description (incidence):

There were no deaths during this study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Throughout Days 1 to 11 of treatment, group mean body weight gain for males that received Labdanum Gum at 5000 or 10000 ppm was higher than Control. Body weight loss was evident for one Control male (1M 18) during the first week of treatment and this contributed to a lower overall mean body weight gain for the Control. Overall mean body weight gain of males that received Labdanum Gum at 15000 ppm was lower than Control (81% of Control) and body weight stasis was evident during Days 1 to 4 of treatment.
A group mean body weight loss was evident for females that received Labdanum Gum at 5000 ppm during Days 1 to 8 of treatment, with a mean loss of 16 g. Group mean body weight losses were also evident for females that received 10000 or 15000 ppm during Days 1 to 4 of treatment, with mean losses of 19 and 23 grespectively. In general, a slight improvement in body weight gain for all treated female groups was observed during thesecond week of treatment. However, mean body weight gain during the treatment period wasmarkedly lower than Control for females that received 5000 ppm (26% of Control) and
10000 ppm (22% of Control). Females that received Labdanum Gum at 15000 ppm showed a group mean body weight loss of 10 g from Days 1 to 15 of treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The overall food intake of males that received Labdanum Gum at all dietary concentrations investigated was comparable to Control during Days 1 to 15 of treatment. The food intake of males that received 15000 ppm was slightly low on the first day of treatment.
The food intake of females that received Labdanum Gum at 5000, 10000 or 15000 ppm showed a dosage-related decrease during Days 1 to 15 of treatment (79%, 74% and 68% of Control respectively), with a marked reduction seen on Days 1 to 4 for females that received 10000 or 15000 ppm, with reduced intake persisting until Day 8 at 15000 ppm.

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 intake at any dietary concentration investigated.

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):

Group mean absolute and body weight relative liver weights were higher than Control for males that received 5000, 10000 or 15000 ppm, there was a dose relationship for relativeweights. Seminal vesicle and prostate weights were marginally low at 15000 ppm (11% and 13% of Control, respectively).
Group mean absolute and body weight relative liver weights were higher than Control for females that received Labdanum Gum at dietary concentrations of 5000, 10000 or
15000 ppm, showing no dose relationship.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of the animals on Day 15 of study did not reveal any test item related findings

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

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

Key result

Critical effects observed:

not specified

None.

Conclusions:

Based on the results obtained in this 14-day toxicity study, dose levels of 15000 or 10000 ppm are considered unsuitable for use on the main OECD 422 study due to the body weight and food consumption effects observed in the females. It was concluded that a dose level of 7500 ppm Labdanum Gum is considered suitable for use as the high dose level in the OECD 422 study (Envigo Study No. NR09JX).

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 5000, 10000 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 5000, 10000 or 15000 ppm were 283, 583 and 853 mg/kg bw/day in males and 259, 510 and 715 mg/kg bw/day in females, respectively.

There were no deaths and no changes in clinical condition that were considered to be related to treatment with Labdanum Gum.Throughout Days 1 to 11 of treatment, group mean body weight gain for males that received Labdanum Gum at 5000 or 10000 ppm was higher than Control. Overall mean body weight gain of males that received Labdanum Gum at 15000 ppm was lower than Control (81% of Control). A group mean body weight loss (-16 g) was evident for females that received Labdanum Gum at 5000 ppm during Days 1 to 8 of treatment. Group mean body weight losses were also evident for females that received 10000 or 15000 ppm during Days 1 to 4 of treatment (-19 and -23 g respectively). In general, a slight improvement in body weight gain for all groups of treated females was observed during the second week of treatment. However, mean body weight gain during the treatment period was markedly lower than Control for females that received 5000 ppm and 10000 ppm (26% and 22% of Control, respectively).

Females that received Labdanum Gum at 15000 ppm showed a group mean body weight loss (-10 g) throughout the treatment period.

The overall food intake of males that received Labdanum Gum at all dietary concentrations investigated was comparable to Control during Days 1 to 15 of treatment. The food intake of females that received Labdanum Gum at 5000, 10000 or 15000 ppm showed a dose-related decrease during Days 1 to 15 of treatment, with a marked reduction seen on Days 1 to 4 for females that received 10000 or 15000 ppm, with reduced intake persisting until Day 8 at 15000 ppm. There was no effect of treatment on water intake at any dietary concentration investigated. High absolute and body weight adjusted liver weights were observed in all groups of treated animals, especially in males (18% of Control at 15000 ppm). Seminal vesicle and prostate weights were marginally low at 15000 ppm (11% and 13% of Control, respectively). However, macroscopic examination of the animals on Day 15 of study did not reveal any test item related findings.

Based on the results obtained in this 14-day toxicity study, dose levels of 15000 or 10000 ppm are considered unsuitable for use on the main OECD 422 study due to the body weight and food consumption effects observed in the females. It was concluded that a dose level of 7500 ppm Labdanum Gum is considered suitable for use as the high dose level in the  OECD 422 study (Envigo Study No. NR09JX).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

326 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Recent GLP study conducted according to OECD Guideline No 422 without any deviation (Klimisch score = 1).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated oral toxicity by oral route:

- In the dose range-finding study for the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (Envigo, 2019, rel.1, SS),groups of Crl:CD(SD) rats (4/sex/dose) received test item orally, via the diet at concentrations of 5000, 10000 or 15000 ppm. A similarly constituted control group received the basal diet with a corn oil stabilizer.

There were no deaths and no changes in clinical condition that were considered to be related to treatment with Labdanum Gum.Throughout Days 1 to 11 of treatment, group mean body weight gain for males that received Labdanum Gum at 5000 or 10000 ppm was higher than Control. Overall mean body weight gain of males that received Labdanum Gum at 15000 ppm was lower than Control (81% of Control). A group mean body weight loss (-16 g) was evident for females that received Labdanum Gum at 5000 ppm during Days 1 to 8 of treatment. Group mean body weight losses were also evident for females that received 10000 or 15000 ppm during Days 1 to 4 of treatment (-19 and -23 g respectively). In general, a slight improvement in body weight gain for all groups of treated females was observed during the second week of treatment. However, mean body weight gain during the treatment period was markedly lower than Control for females that received 5000 ppm and 10000 ppm (26% and 22% of Control, respectively).

Females that received Labdanum Gum at 15000 ppm showed a group mean body weight loss (-10 g) throughout the treatment period.

The overall food intake of males that received Labdanum Gum at all dietary concentrations investigated was comparable to Control during Days 1 to 15 of treatment. The food intake of females that received Labdanum Gum at 5000, 10000 or 15000 ppm showed a dose-related decrease during Days 1 to 15 of treatment, with a marked reduction seen on Days 1 to 4 for females that received 10000 or 15000 ppm, with reduced intake persisting until Day 8 at 15000 ppm. There was no effect of treatment on water intake at any dietary concentration investigated. High absolute and body weight adjusted liver weights were observed in all groups of treated animals, especially in males (18% of Control at 15000 ppm). Seminal vesicle and prostate weights were marginally low at 15000 ppm (11% and 13% of Control, respectively). However, macroscopic examination of the animals on Day 15 of study did not reveal any test item related findings.

Based on the results obtained in this 14-day toxicity study, dose levels of 15000 or 10000 ppm are considered unsuitable for use on the main OECD 422 study (Envigo Study No. NR09JX).

- In the main study, considered as the key study (OECD 422, Covance, 2019, Rel.1), 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.

The overall mean achieved doses for Toxicity, Recovery and Reproductive phase animals during treatment that received Labdanum Gum at target dietary concentrations of 1500, 3500 or 7500 ppm were 65, 140 and 322 mg/kg bw/day in males and 68, 155 and 331 mg/kg bw/day in females, respectively. The overall mean achieved doses for Reproductive phase females that received target dietary concentrations of 1500, 3500 or 7500 ppm were 70, 175 and 342 mg/kg bw/day and 163, 376 and 731 mg/kg bw/day during gestation and lactation respectively.

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males.

One male that received 1500 ppm was killed for welfare reasons on Day 29 of study due to a damaged right eye. Macroscopic examination revealed a protruding and abnormal dark coloured right eye. This death was not considered to be related to treatment with Labdanum Gum. There were no test-item related signs observed during detailed physical examination and arena observation and no effects on sensory reactivity, grip strength or motor activity.

Effects on body weight performance and food consumption (comprising periods of low mean weight gain and reduced food consumption) were evident throughout the treatment period for all groups of treated Toxicity and Recovery phase males and females. Following the commencement of treatment, an initial reduction in body weight gain was evident for males that received 3500 or 7500 ppm as well as minor mean weight loss in all groups of treated females. In addition, an initial reduction in food intake was evident following the commencement of treatment for females that received 7500 ppm. These effects were considered to be related to the palatability of the test item. Thereafter, the food intake of females that received 7500 ppm remained marginally low. Overall group mean body weight gain throughout the treatment period (Days 1 to 43) was slightly low for all groups of treated males and females that received 3500 or 7500 ppm. The food intake of all groups of treated males was generally marginally lower than Control for the first three weeks of treatment (Days 1 to 22) and the food intake of females that received 1500 or 3500 ppm was generally slightly lower than or comparable to Control. Following six weeks of treatment, the terminal body weights for Toxicity phase females that received 7500 ppm were statistically significantly lower than Control.

Food intake during early gestation (Days 0 to 8) for Reproductive phase females that received 7500 ppm was marginally lower than Control but comparable to Control thereafter. Overall body weight gain throughout lactation (Days 1 to 13) was slightly lower than Control for females that received 1500 or 7500 ppm (but not at 3500 ppm) and the food intake during lactation for females that received 7500 ppm was generally marginally lower than Control.

During the recovery period, group mean body weight gain for males that previously received 7500 ppm was slightly lower than Control, however, group mean body weight gain during the recovery phase for females that previously received 7500 ppm was higher than Control.

There was no treatment-related ophthalmoscopic finding. Haematological examination of the peripheral blood during Week 6 of treatment did not reveal any toxicologically significant differences from Control.

Biochemical assessment of the plasma during Week 6 of treatment revealed that creatinine levels for all groups of treated males were statistically significantly higher than Control. Following a two-week recovery period, creatinine levels for males that had previously received 7500 ppm were comparable to Control. A statistically significant decrease in urea concentration was apparent in females that received 7500 ppm. Following a two-week recovery period, urea concentration in females that had previously received 7500 ppm was comparable to Control. In addition, a statistically significant increase in phosphorus levels was apparent amongst males and females that received 7500 ppm. Following a two-week recovery period, phosphorus levels in males and females that had previously received 7500 ppm were comparable to Control. Globulin levels for females that received 7500 ppm were statistically significantly higher than Control. Following a two-week recovery period, globulin levels in females that had previously received 7500 ppm were comparable to Control.

The analysis of urine during Week 6 of treatment revealed that total creatinine output was higher than Control for males treated at 3500 or 7500 ppm, however, all values were within the HCD range and no such trend was apparent in the females. In addition, sodium concentration was higher than Control for all groups of treated females, however, all values were also within the HCD range and no such trend was apparent in the males.

Following six weeks of treatment, terminal body weights for Toxicity phase females that received 7500 ppm were statistically significantly lower than Control. Changes in organ weights consisted of a dosage related decrease in group mean body weight adjusted adrenal weights in Toxicity phase males, with statistical significance attained at 7500 ppm. Following the two-week recovery period, group mean body weight adjusted adrenal weight for males that previously received 7500 ppm were comparable to Control. Group mean body weight adjusted thymus weights were statistically significantly lower than Control in all groups of treated Toxicity phase males, however, no dose response was apparent. Following the two-week recovery period, group mean body weight adjusted thymus weights for males that previously received 7500 ppm were comparable to Control. Group mean body weight adjusted ovary weights were slightly low at 7500 ppm for the Toxicity phase females but the difference did not attain statistical significance.

Macroscopic and microscopic examination of a full range of organs/tissues from the adult males and females revealed no test-item related lesions.

Based on the results of this study it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity is 7500 ppm. 

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding the specific target organ toxicity after oral dose-repeated exposure acording to the Annex I of the regulation (EC) No. 1272/2008 (CLP) and UN GHS.