Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 14 May to 22 June, 2019.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 474 and in compliance with GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
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:
reference to other study
Reason / purpose:
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 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).

Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 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:
reference to same study
Reason / purpose:
reference to other study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Inspected on 2019-04-02 / Signed on 2019-08-01
Limit test:
no
Justification for study design:
- Basis for dose level selection: 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).
- Route of administration: The dietary route of administration was chosen to simulate the conditions of potential human exposure.
- Animal model: The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males 69 to 75 days old. Females: 83 to 89 days old.
- Weight at study initiation: Males: 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
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 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).
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: 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.
Toxicity phase females: At least six weeks.
Recovery phase males: Three weeks before pairing up to necropsy after minimum of six weeks followed by a minimum 14-day recovery.
Recovery phase females: At least six weeks followed by a minimum 14-day recovery.
Animals of the F1 generation received no direct administration of test item; any exposure was in utero or via the milk.
Frequency of treatment:
Continuously
Dose / conc.:
0 ppm
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:
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.
- 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.
Replacement before allocation: Abnormal estrus cycle - 11 females; Body weight range extremes - 3 males
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced, during each week of treatment and recovery, on Days 0, 6, 13 and 20 after mating and on Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before feeding of the treated diets on Day 1).

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Toxicity and Recovery phase males and females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and weekly thereafter, including the recovery phase. On Day 5 of recovery the animals were fed diets prepared for the females in the lactation phase in error (recovery control animals received control diet with the corn oil stabiliser, and recovery animals in Group 4 received treated diet (8000 ppm); On the day of necropsy.
F0 Reproductive phase females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and weekly before pairing; Days 0, 7, 14 and 20 after mating; Day 1, 4, 7 and 13 of lactation; On the day of necropsy.

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

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

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

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

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

Thyroid Hormone Analysis
- Time schedule for examination
At termination: F0 males, All F0 Reproductive phase females
Day 4 of age - F1 offspring, two females per litter (where possible) - no pups were eliminated when litter size dropped below ten/litter
- one for T4 (serum)#
- one for TSH (plasma - optional analysis)
# priority given to serum sample
Day 13 of age: F1 offspring, two males and two females per litter (where possible)
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female
# priority given to serum sample

PARTURITION OBSERVATIONS AND GESTATION LENGTH:
- Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
- Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.
Oestrous cyclicity (parental animals):
Estrous Cycle
Wet smears:
Using pipette lavage during the following phases:
- For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to the Reproductive phase of the study.
- After pairing until mating.
- For four days before scheduled termination (all Reproductive phase, Toxicity phase and Recovery phase females).

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

GROSS NECROPSY
- Necropsy: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Main phase females
The following were recorded:
Each uterine horn: Number of implantation sites was counted and confirmed.

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

HISTOPATHOLOGY
- Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: Initially in modified Davidson’s fluid; Eyes: In Davidson’s fluid.
- Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: All F0 animals killed or dying prematurely; Toxicity phase males and females in Groups 1 and 4 at scheduled termination.
Abnormalities: All remaining adult animals.
Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
Postmortem examinations (offspring):
SACRIFICE
Time of necropsy:
Selected offspring for Day 4 thyroid hormone analysis - Day 4 of age.
Scheduled kill - Day 13 of age.
Method of sacrifice:
- Offspring- selected for thyroid hormone sampling on Day 4 or Day 13 of age: Decapitation
- Offspring - not selected for thyroid hormone sampling: Intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed. Abnormal tissues retained.
- F1 offspring on Day 4 of age:
Blood sampling required
Externally normal offspring discarded without examination.
Externally abnormal offspring identified on despatch to necropsy, examined externally, and retained pending possible future examination.
- F1 offspring on Day 13 of age
Blood sampling required
All animals (but not including those selected for thyroid hormone analysis) were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Thyroid glands were preserved from one male and one female in each litter.
Animals selected for thyroid hormone analysis: externally normal offspring discarded without examination; externally abnormal offspring examined.
Statistics:
See "Any other information on materials and methods incl. tables"
Reproductive indices:
Mating Performance and Fertility:
- Percentage mating = (Number animals mating / Animals paired) x 100
- Conception rate (%) = (Number animals achieving pregnancy / Animals mated) x 100
- Fertility index (%) = (Number animals achieving pregnancy / Animals pairing) x 100
Gestation Length and Index: Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day.
Gestation index was calculated for each group as:
- Gestation index (%) = (Number of live litters born / Number pregnant ) x 100
Offspring viability indices:
Survival indices:
- Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100
- Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
- Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering) x 100
- Lactation index (%) = (Number of live offspring on Day 13 of lactation / Number live offspring on Day 4 (after blood sampling)) x 100
Sex ratio: The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 and 13 of age.
- Percentage males = (Number of males in litter / Total number of offspring in litter) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed during the treatment period for Toxicity and Recovery phase animals 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 received 1500 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 or7500 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):
The overall food intake of all groups of treated Toxicity and Recovery phase males was generally marginally lower than Control throughout the first three weeks of treatment (Days 1 to 22).
There was an initial statistically significant reduction in food intake, between Days 1 to 5 and 6-7, for females that received 7500 ppm following the commencement of treatment. Thereafter, the food intake of females that received 7500 ppm remained marginally lower than Control. The food intake of females that received 1500 or 3500 ppm was generally marginally lower than or comparable to Control throughout the treatment period.
The food intake during early gestation (Days 0 to 8) for Reproductive phase females that received 7500 ppm was marginally lower than Control, with a statistical significance on Days 0-1 (78% of Control), but comparable to Control thereafter. The food intake during gestation for Reproductive phase females that received 1500 or 3500 ppm was comparable to Control.
The food intake during lactation (Day 1 to 13) for Reproductive phase females that received 7500 ppm was generally marginally lower than Control. The food intake during lactation for Reproductive phase females that received 1500 or 3500 ppm was generally slightly higher than Control.
The food intake during recovery for males and females that had previously received 7500 ppm was comparable to 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
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.
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
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 treatment. Females allocated to the Toxicity phase all showed regular 4 to 5 day estrous cycles prior to treatment with the exception of one Control female with an irregular cycle. Females allocated to the Recovery phase all showed regular 4 to 5 day cycles prior to treatment with the exception of one Control female with an irregular cycle.
There was no effect of treatment on estrous cycle regularity, however, it was noted that during the treatment period a high number of Reproductive phase females were observed to be acyclic, this included four females in the Control group. One Control female and one female that received 3500 ppm were observed to have irregular cycles and one female that received 1500 ppm was observed to have extended estrus. However, given that all females mated within 1-4 days, with the exception of one Control female which mated within 5-8 days, these acyclic and irregular cycles are considered to be incidental. At termination,
all Toxicity phase females and a majority of Recovery phase females were cycling and all Reproductive phase females were in diestrus.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
- Pre-coital interval, mating performance and fertility were considered unaffected by treatment with Labdanum Gum, when compared to Control.
- Gestation length and gestation index were considered unaffected by treatment at any dose level.
Key result
Dose descriptor:
NOAEL
Effect level:
7 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed.
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment related clinical signs observed amongst offspring following parental dietary administration of Labdanum Gum.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no effect of treatment with Labdanum Gum on the mean number of implantations,
litter size, post implantation survival index, live birth index, viability index or lactation index.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of treatment on offspring body weights on Day 1 of age. The overall body weight gain (Day 1 to 13 of age) of male and female offspring derived from parents that received 7500 ppm was marginally lower than Control particularly in males.
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):
There was no effect of treatment with Labdanum Gum on the mean number of implantations, litter size, post implantation survival index, live birth index, viability index or lactation index.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
There was no effect of treatment on ano-genital distance in the male and female offspring at any dietary concentration investigated.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipples were observed in male offspring of parents treated with Labdanum Gum.
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:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
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:
not specified
Key result
Reproductive effects observed:
not specified
Conclusions:
Under the test conditions 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. It was also concluded the NOAEL for reproductive/developmental toxicity was the high dose (target dietary concentration of 7500 ppm) with mean achieved doses of 342 mg/kg bw/day during gestation and 731 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 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).

The offspring received no direct administration of the test item; any exposure was in utero or via the milk. Selected F1 offspring were sampled and killed on Day 4 (not analyzed) or Day 13 of age for analysis of blood thyroid hormone levels. The remaining F1 offspring were killed on Day 13 of age.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (by visual assessment), ophthalmic examination, hematology (peripheral blood),

blood chemistry, urinalysis, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, body weight, nipple counts (males only), ano-genital distance and macropathology for all offspring were also assessed.

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 or in offspring on Day 13 of age.

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. There were no treatment-related effects on oestrous cycles, pre-coital interval, mating

performance, fertility and gestation length.

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.

There were no treatment related clinical signs observed amongst offspring following parental dietary administration of Labdanum Gum.

There was no effect of treatment with Labdanum Gum on the mean number of implantations, litter size, post implantation survival index, live birth index, viability index or lactation index. There was no effect of treatment on offspring body weights on Day 1 of age. The overall body weight gain of male and female offspring derived from parents that received 7500 ppm was marginally lower than Control. There was no effect of treatment on ano-genital distance in the male and female offspring and no nipples were seen in male offspring. There were no findings associated with treatment at macroscopic examination.

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.

It was also concluded the NOAEL for reproductive/developmental toxicity was the high dose (target dietary concentration of 7500 ppm) with mean achieved doses of 342 mg/kg bw/day during gestation and 731 mg/kg bw/day during lactation.

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. certificate)
Remarks:
Inspected on 2019-04-02 / Signed on 2019-08-01
Type of assay:
mammalian erythrocyte micronucleus test

Test material

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

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males 69 to 75 days old. Females: 83 to 89 days old.
- Weight at study initiation: Males: 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.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
None.
Details on exposure:
DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet
- Correction factor: A correction factor was not required.
- Stabilizer: Corn oil (test material to corn oil ratio 5:1).
- Method of preparation: The test substance was incorporated into the diet to provide the required concentrations by initial preparation of a premix. 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).
Duration of treatment / exposure:
Toxicity phase males: Three weeks before pairing up to necropsy after minimum of six weeks of treatment.
Toxicity phase females: At least six weeks.
Frequency of treatment:
Continuously
Post exposure period:
None
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Control group (Basal diet + corn oil) / Group 1
Dose / conc.:
15 000 ppm
Remarks:
Group 2 (Low dose)
Dose / conc.:
3 500 ppm
Remarks:
Group 3 (Mid dose)
Dose / conc.:
7 500 ppm
Remarks:
Group 4 (High dose)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Positive control(s):
Five bone marrow smears previously prepared from rats administered cyclophosphamide (Envigo Study Number GY05QJ) will be stained and coded along with the bone marrow smears prepared in this study.
- Route of administration: diet
- Doses / concentrations: 20 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow smears
Details of tissue and slide preparation:
- Preparation of bone marrow smears
Animals were killed on the day after receiving their last daily dose. One femur was dissected from the first five male and female animals per group (surviving to necropsy) and the proximal head removed. Using a syringe and needle, bone marrow was flushed from the marrow cavity with approximately 3 mL pre filtered foetal calf serum into appropriately labelled centrifuge tubes (1 per animal). The resulting cell suspensions were centrifuged at 1000 rpm for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal bovine calf serum to facilitate smearing in the conventional manner on glass microscope slides (Schmid 1976). At least 4 smears/slides were prepared from each animal. The bone marrow smears were air-dried and then fixed for a minimum of 10 minutes in absolute methanol.
- Fixation and staining of slides
The slides were rinsed in purified water and stained using an acridine orange solution at 0.0125 mg/mL and stored at room temperature in the dark until required. The remaining unstained slides were kept in reserve.
- Microscopic examination
At least 3 slides (prepared in a separate study [GY05QJ]) from animals treated with Cyclophosphamide (CPA), a well characterized clastogen, were stained and coded along with the bone marrow smears prepared from this study.
Prior to scoring the slides were wet mounted with coverslips using purified water. Coded slides were examined by fluorescence microscopy and 4000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. At least one smear was examined per animal, any remaining smears being held temporarily in reserve in case of technical problems with the first smear.
The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, the test article is considered clearly negative if, in all experimental conditions examined:
a) None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control,
b) There is no dose-related increase when evaluated by an appropriate trend test,
c) All results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits),
and d) Bone marrow exposure to the test substance(s) occurred.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly positive if:
a) At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control,
b) This increase is dose-related when evaluated with an appropriate trend test,
and c) Any of these results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
When conducting a dose-response analysis, at least three treated dose groups should be analysed. Statistical tests should use the animal as the experimental unit. Positive results in the micronucleus test indicate that a test chemical induces micronuclei, which are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species.
Statistics:
Analysis of data: The results obtained for each treatment group will be compared with the results obtained for the vehicle control group using non-parametric statistical methods. Analysis will be performed for the study using data from all animals that survive to scheduled termination to maximise the power of statistical analysis. If less than five animals per group survive to scheduled termination the study may be repeated.
For the proportion of polychromatic erythrocytes at 24 hours, an asymptotic one-tailed Jonckheere’s test for trend (Jonckheere 1954) with “step-down” will be used on Groups 1 to 4 for a decrease from control. Exact one-tailed Wilcoxon pairwise tests (Wilcoxon 1945), for a decrease from control, will also be carried out on Group 1 (control) versus Groups 2, 3, 4 and 5 (positive control slides).
For incidences of micronucleated polychromatic erythrocytes at 24 hours, an exact one-tailed Linear-by-Linear association test (Cytel 1995) with “step-down” will be used on Groups 1 to 4 for an increase from control. Also, exact one-tailed pairwise Permutation tests (Cytel 1995), for an increase from control, will be carried out on Group 1 (control) versus Groups 2, 3, 4 and 5 (positive control slides). Statistical significance will be declared at the 5% level for all tests.
Quasar (version 1.4) and/or SAS (version 9.1.3) and/or StatXact 3 in-house statistical analysis packages may be used for statistical analysis.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:
The 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).
- Solubility: no data
- Clinical signs of toxicity in test animals:
There were no clinical signs observed during the treatment period for Toxicity and Recovery phase animals 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.
- Rationale for exposure:
Continuously (OECD 422)
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
* Percentage of Micronucleated Polychromatic Erythrocyte Counts (%MPCE):
The data for the concurrent vehicle control were within the ranges determined by the laboratory historical control data, therefore, the performance of the vehicle was consistent with a valid assay. The coded positive control slides prepared from study GY05QJ demonstrated the ability of the analyst to detect increases in micronucleated polychromatic erythrocytes.
There were no statistically significant increases in %MPCE observed in male or female Crl:CD(SD) rats administered Labdanum Gum at any dose level compared to vehicle control values. All individual and group mean values were within the current vehicle historical control range (95% confidence limits).
* Micronucleated Normochromatic Erythrocytes (MNCE):
Labdanum Gum did not cause any substantial increases in the incidence of micronucleated normochromatic erythrocytes in male or female Crl:CD(SD) rats.
* Proportion of Polychromatic Erythrocytes (%PCE):
The data for the concurrent vehicle control %PCE were within or close to the ranges determined by the laboratory historical control data (95% confidence limits), therefore, the performance of the vehicle was consistent with a valid assay.
A statistically significant decrease in %PCE (p<0.01) was observed in male Crl:CD(SD) rats administered Labdanum Gum at 3500 and 7500 ppm compared to vehicle control values. A statistically significant trend was also observed from Group 1 to 4 (p<0.001), upon exclusion of Group 4, the trend was again statistically significant (p<0.001). However, individual and group mean values from all treatment groups did not exceed the lower 95% confidence limit of the current vehicle historical control range. In additional to this the group mean vehicle control was marginally higher than the upper 95% confidence limit. Therefore, the statistically significant decreases observed are considered to be of questionable biological relevance.
There were no statistically significant decreases in %PCE observed in female Crl:CD1(SD) rats administered Labdanum Gum at any dose level compared to vehicle control values. All individual and group mean values were within or close to the current vehicle historical control range (95% confidence limits).

Any other information on results incl. tables

Table7.6.2/1: Summary of results and statistical analysis

Male data

Treatment

Treatment
(ppm)

Proportion of PCE % # (SD)

Incidence MPCE mean # (SD)

Group mean % MPCE #

Control

0

55.5 (4.4)

4.0 (3.1)

0.10

Labdanum Gum

1500

52.1 (3.9)

3.4 (1.5)

0.09

Labdanum Gum

3500

46.7**^^^ (2.1)

3.6 (1.8)

0.09

Labdanum Gum

7500

45.8**^^^ (3.6)

4.2 (0.8)

0.11

Cyclophosphamidea

20 mg/kg

45.4* (2.0)

60.0* (9.6)

1.50

Female data

Treatment

Treatment
(ppm)

Proportion of PCE % # (SD)

Incidence MPCE mean # (SD)

Group mean % MPCE #

Control

0

51.8 (0.8)

4.0 (2.3)

0.10

Labdanum Gum

1500

51.0 (0.7)

4.4 (0.5)

0.11

Labdanum Gum

3500

57.9 (3.3)

3.8 (1.3)

0.10

Labdanum Gum

7500

56.5 (4.2)

4.0 (1.9)

0.10

Cyclophosphamidea

20 mg/kg

45.6* (1.9)

73.3* (6.8)

1.83

Control

Basal diet + corn oil

 

PCE

Polychromatic erythrocytes

 

MPCE

Number of micronucleated polychromatic erythrocytes observed per 4000 polychromatic erythrocytes examined

 

SD

Standard deviation

 

a

Positive control slides from study GY05QJ

 

 

 

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

 

 

 

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):

 

 

 

 

 

 

Pairwise

*

p< 0.05

(significant)

 

 

**

p< 0.01

(significant)

 

Trend

^^^

p< 0.001

(significant)

 

 

otherwise

p> 0.05

(not significant)

 

Applicant's summary and conclusion

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

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

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

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

Bone marrow smears were obtained from the first 5 male and female animals,surviving to scheduled necropsy,from the vehicle control group and each of the test item groups on the day after administration of the final dose.

One smear from each animal was examined for the presence of micronuclei in 4000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes was also kept.

The data for the concurrent vehicle control (group mean % polychromatic erythrocytes [%PCE] and % micronucleated polychromatic erythrocytes [%MPCE]) were within or close to the ranges determined by the laboratory historical control data (95% confidence limits), therefore, the performance of the vehicle was consistent with a valid assay. The coded positive control slides prepared from study GY05QJ demonstrated the ability of the analyst to detect increases in micronucleated polychromatic erythrocytes.

There were no statistically significant increases in %MPCE observed in male Crl:CD(SD) rats administered Labdanum Gum at any dose level compared to vehicle control values. All individual and group mean values were within the current vehicle historical control range (95% confidence limits).

A statistically significant decrease in %PCE (p<0.01) was observed in male Crl:CD(SD) rats administered Labdanum Gum at 3500 and 7500 ppm compared to vehicle control values. A statistically significant trend was also observed from Group 1 to 4 (p<0.001), upon exclusion of Group 4, the trend was again statistically significant (p<0.001). However, individual and group mean values from all treatment groups did not exceed the lower 95% confidence limit of the current vehicle historical control range. In additional to this the group mean vehicle control was marginally higher than the upper 95% confidence limit. Therefore, the statistically significant decreases observed are considered to be of questionable biological relevance.

There were no statistically significant increases in %MPCE and no statistically significant decreases in %PCE observed in female Crl:CD1(SD) rats administered Labdanum Gum at any dose level compared to vehicle control values. The group mean values were within or close to the current vehicle historical control range (95% confidence limits).

Under the test conditions of this study, Labdanum Gum did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes in male and female Crl:CD(SD) rats following 6 weeks of dietary administration in this in vivo test procedure.

This study is considered as acceptable and satisfies the requirement for genetic toxicity in vivo endpoint.