Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 January 2017 - 26 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 422 without any deviation.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
09 May 2017 - 23 June 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Preliminary study performed similarly to OECD Guideline 407, 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:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
test item was administered for 14 days only; 4/sex/dose only used; test item formulation analysis, neurobehavioral examination, haematology, clinical biochemistry and histopathology not reported.
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor - 1002888716
- Appearance: Paste, pale yellow
- Expiration date of the lot/batch: 17 May 2020

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Dry area, closed containers, room temperature 15 to 25°C
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: Groups 1 and 2 - Males: 71 to 78 days; Females: 85 to 92 days / Group 3 - Males: 83 to 90 days; Females: 97 to 104 days / Group 4 - Males: 93 to 100 days; Females: 107 to 114 days
- Weight at study initiation: Males - Group 1: 344-367 g; Group 2: 350-370 g; Group 3: 432-462 g; Group 4: 422-466 g / Females - Group 1: 239-259 g; Group 2: 244-255 g; Group 3: 235-268 g; Group 4: 257-295 g
- Housing: Animals were housed in groups of 4 (same sex) in polycarbonate cages. Cages comprised of a polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
- Diet: SDS VRF1 Certified powdered diet, ad libitum
- Water: Potable water from public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: Groups 1 and 2: Eight days before commencement of treatment
Group 3: 20 days before commencement of treatment
Group 4: 30 days before commencement of treatment.

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

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

IN-LIFE DATES: 10 May to 23 June 2017
Route of administration:
oral: feed
Details on route of administration:
The oral (dietary), route of administration was chosen as it is a possible route of human exposure.
Vehicle:
other: feed
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: None.
- Method of preparation: The required amount of test item was weighed out into a suitable container. With magnetic stirring, the required amount of solvent was added until the test item was fully dispersed. The dispersion was added to the appropriate amount of plain diet, followed by the addition of a further appropriate amount of plain diet.
The bulk was stirred well and then the solvent evaporated off in a fume hood, until constant weight was achieved. The required amount of corn oil was then added to the mixture and the bulk mixed firstly using a mechanical grinder, and then with a Turbula mixer. Appropriate amounts of plain diet was then added to aliquots of this pre-mix, with mixing, until the requisite test item concentration was achieved.
- Frequency of preparation: Weekly
- Storage of formulation: Eight days ambient (15 to 25°C) and 15 days frozen (-10 to -30°C) stability confirmed at 500 ppm to 20000 ppm.

STABILITY AND HOMOGENEITY
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the diet matrix as part of the main OECD 422 study (Envigo Study Number: WX59DP). The specimen formulations were analyzed to assess the stability and homogeneity of the test item in the diet matrix at concentrations of 500 ppm and 20000 ppm.
Stability and homogeneity of the formulations was confirmed for15 days when stored frozen (-10 to -30°C) and for eight days at ambient temperature (15°C to 25°C).
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No formulation analysis was performed in this study. However, 200 g samples of first preparations per group were stored at (-10 to 30°C).
Duration of treatment / exposure:
14 days
Frequency of treatment:
Continuously
Dose / conc.:
0 ppm
Remarks:
Group 1 (control)
Dose / conc.:
7 500 ppm
Remarks:
Group 2
Dose / conc.:
10 000 ppm
Remarks:
Group 3
Dose / conc.:
5 000 ppm
Remarks:
Group 4
No. of animals per sex per dose:
4
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: In the absence of toxicity and palatability data on this test item, a staggered approach, starting with 7500 ppm was decided. In discussion with the Sponsor and following the review of the Group 2 data (7500 ppm), where there was a slight effect on body weight and food intake (especially males), a dose level of 10000 ppm was selected as the dose level for Group 3. In discussion with the Sponsor and following the review of the Group 3 data (10000 ppm), where there was body weight loss in males and stasis in the females and a decrease in food intake, a dose level of 5000 ppm was selected as the dose level for Group 4.
- 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. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before treatment commenced: Body weight range extremes - one males.
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 acclimatization 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 to monitor general health on Days 1, 4, 8, 11 and 15.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded for 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 from Day -3 up to termination.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; All animals were killed by carbon dioxide asphyxiation with subsequent exsanguination and subjected to detailed necropsy. Animals in Groups 2, 3 and 4 were killed following 14 days of treatment. Animals in Group 1 were killed following 36 days of treatment. Only the thoracic and abdominal cavities were opened. The cranial cavity was not opened as there were no observations during the study to indicate a possible neurotoxic action. 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 together and presentated in the table 7.5.1/1. Requisite organs were weighed for all animals.

HISTOPATHOLOGY: No; but tissues were routinely preserved for microscopic examinations in 10% neutral buffered formalin (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.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed that were considered to be related to treatment with the test item.
Mortality:
no mortality observed
Description (incidence):
No mortality occured during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the first week of treatment males receiving 7500 ppm had significantly low overall weight gain compared with Controls. Mean bodyweight loss was recorded during Days 1-2 and 4-5. Bodyweight gain during Days 8-15 was similar to that of the Control. Following commencement of treatment for females receiving 7500 ppm, group mean bodyweight and bodyweight gain was similar to or higher than Controls, and was considered unaffected by treatment.

Bodyweight loss was evident during Days 1-4 of treatment for males and females, and slightly lowers weight gain was evident during Days 4-8 for males receiving 10000 ppm when compared to Controls. Thereafter bodyweight gain was similar to or exceeded that of the Controls.

Bodyweight loss was evident during Days 1-4 of treatment for males receiving 5000 ppm when compared to Controls. Thereafter bodyweight gain was similar to that of the Controls. Females receiving 5000 ppm had a similar bodyweight pattern to that of the Controls, during the first week of treatment. Thereafter bodyweight gain exceeded that of the Controls.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Overall food consumption was considered unaffected by treatment with the test item.

Food consumption during Days 1-11 of treatment for males receiving 7500 ppm was lower than that of Controls and intake of females on Day 1 was slightly low when compared with Controls.

Food intake was significantly lower on Day 1 of treatment for animals receiving 10000 ppm when compared with Controls. Thereafter, food consumption for males was similar to that of the Controls but intake generally remained low during the first week of treatment for females.

Food intake was slightly lower on Day 1 of treatment for animals receiving 5000 ppm when compared with Controls. Thereafter consumption was similar to that of the Controls.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were no visual water consumption effects.
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):
Absolute and bodyweight relative spleen weights and bodyweight relative liver weights were slightly higher for animals that received the test item when compared to Controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of animals on Day 15 (Day 37 for Controls) of study did not reveal any findings that were considered related to treatment with the test item.
Histopathological findings: non-neoplastic:
not examined
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
> 7 500 - < 10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Critical effects observed:
no

Achieved dose:

The overall mean achieved doses in animals receiving 5000, 7500 and 10000 ppm were 283, 465 and 669 mg/kg/day in males and 307, 506 and 631 mg/kg/day in females, respectively.

Conclusions:
The effects on bodyweight and food consumption at the start of treatment, from which the animals quickly recovered, were considered likely to be related to the palatability of the test item. The high dietary concentration of 8000 ppm was expected to elicit initial mean body weight loss or stasis and initial low food consumption and potential adverse effects in target organs, especially in males.
It is considered that 8000 ppm should be a suitable high dose to be tested in the subsequent OECD 422 study, Envigo Study No. WX59DP.
Executive summary:

In a repeated dose toxicity range-finding study, groups of Crl:CD(SD) rats (4/sex/dose) received test item orally, via the diet at concentrations of 7500, 10000 or 5000 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, 7500 or 10000 ppm were 283, 465 and 669 mg/kg/day in males and 307, 506 and 631 mg/kg/day in females, respectively.

All animals survived the dosing period, with no adverse clinical signs or macroscopic changes in organ appearance at any dose level. There were no visual water consumption effects.

5000 ppm

Following the administration of test diet, bodyweight loss was recorded in males, but weight gain was recorded from Day 4 of treatment, bodyweight gain in females was similar to the Control group. Food intake was slightly lower on Day 1, but there was no overall effect on food consumption for either sex at this level when compared with Controls.

Absolute and bodyweight relative spleen weights and bodyweight relative liver weights were slightly higher when compared to Controls.

7500 ppm

Following the administration of test diet, bodyweight loss or stasis was recorded in males, but sustained weight gain was recorded from Day 7 of treatment. In females, bodyweight gain was similar to Control group.

Food intake was reduced in both sexes on Day 1 when compared with Controls. However, food intake remained low until Day 11 for males when compared with Controls.

Absolute and bodyweight relative spleen weights and bodyweight relative liver weights were slightly higher when compared to Controls.

10000 ppm

Following the administration of test diet, bodyweight loss was recorded, but slight gain or stasis was recorded from Day 4 of treatment. After the first week of treatment weight gains were similar to or exceeded Controls. Food intake was reduced in both sexes on Day 1 when compared with Controls. Thereafter, food consumption in males was generally similar to Controls, but female intake remained low during the first week of treatment; thereafter differences to the Control were small.

Absolute and bodyweight relative spleen weights and bodyweight relative liver weights were slightly higher when compared to Controls

Conclusion

The effects on bodyweight and food consumption at the start of treatment, from which the animals quickly recovered, were considered likely to be related to the palatability of the test item. The high dietary concentration of 8000 ppm was expected to elicit initial mean body weight loss or stasis and initial low food consumption and potential adverse effects in target organs, especially in males.

It is considered that 8000 ppm should be a suitable high dose to be tested in the subsequent OECD 422 study, Envigo Study No. WX59DP.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
OECD 422
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 January 2017 - 26 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 422 without any deviation.
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:
Adopted 29 July 2016
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor - 1002888716
- Appearance: Paste, pale yellow
- Expiration date of the lot/batch: 15 December 2017

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Dry area, closed containers, room temperature 15 to 25°C
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) 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.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males: 69 to 76 days old; Females: 83 to 90 days old.
- Weight at study initiation: Males: 339-391 g; Females: 237-305 g
- Housing: Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing; Cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Number of animals per cage: Pre-pairing: up to five animals of one sex; Pairing: one male and one female; Males after mating: up to five animals; Gestation: one female; Lactation: one female + litter
- Diet: SDS VRF1 Certified powdered diet, ad libitum (removed overnight before blood sampling for thyroid hormone, hematology and blood chemistry investigations)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (removed overnight during urine collection)
- Acclimation period: Females: 20 days before the commencement of treatment; Males: six days before the commencement of treatment.

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: 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 (returned on Day 13 of lactation, after dispatch of the litter)] and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study [except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced at the same time as the cages.

IN-LIFE DATES: 19 July 2017 to 10 October 2017
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
- Stabilizer: Corn oil (test material to corn oil ratio 5:1).
- Correction factor: A correction factor was not required.
- Method of preparation: The required amount of test item was weighed out into a suitable container. With magnetic stirring, the required amount of solvent was added until the test item was fully dispersed. The dispersion was added to the appropriate amount of plain diet, followed by the addition of a further appropriate amount of plain diet.
The bulk was stirred well and then the solvent evaporated off in a fume hood, until constant weight was achieved.
The required amount of corn oil was then added to the mixture and the bulk mixed firstly using a mechanical grinder, and then with a Turbula mixer after making to the required weight with plain diet.
Appropriate amounts of plain diet was then added to aliquots of this pre-mix, with mixing, until the requisite test item concentration was achieved.
- Frequency of preparation: Weekly.
- Storage of formulation: Frozen (-10 to -30 °C). Stability and homogeneity of the formulations was confirmed for 15 days when stored frozen (-10 to -30°C) and for eight days at ambient temperature (15°C to 25°C).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the diet matrix at concentrations of 500 ppm and 20000 ppm.
- Achieved concentration: Samples of each formulation prepared for administration in Week 1 and the final week of treatment were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
- Reproductive phase (females): Three weeks before pairing, then throughout pairing and gestation until Day 12 of lactation (necropsy on Day 13 of lactation (the treated diet was available to the animals until the morning of necropsy)).
- Toxicity phase (males): Three weeks before pairing up to necropsy after a minimum of six weeks.
- Toxicity phase (females): A minimum of 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:
Group 1 (control)
Dose / conc.:
2 000 ppm
Remarks:
Group 2 (low dose)
Dose / conc.:
4 000 ppm
Remarks:
Group 3 (mid dose)
Dose / conc.:
8 000 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: Dietary levels were selected following the completion of the preliminary toxicity study (Envigo Study number: SN57HH) following consultation with the Sponsor.
Effects on
body weight and food consumption were seen at 7500 ppm in males at the start of treatment, from which they did not completely recover. The same trend was observed at 10000 ppm in males and females, with slight recovery at the end of treatment. These effects were considered likely to be related to the palatability of test item. Also, high kidneys and liver weights (mean increase of 9 and 28% from controls, respectively) were observed in males at 10000 ppm and even in liver weights at 5000 ppm (mean increase of 15% from controls).
The high dietary concentration of 8000 ppm was expected to elicit initial mean body weight loss or stasis and initial low food consumption and potential adverse effects in target organs, especially in males. The lowest dietary concentration of 2000 ppm was expected to be a no effect level for effects on body weight and food consumption. The intermediate dietary concentration of 4000 ppm allowed evaluation of any concentration related trends and provides a geometric progression of dietary concentrations.

- Rationale for animal assignment: On arrival and non-selective allocation to cages. Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study. On Day 1 of study all animals were weighed and body weights were reviewed 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.

- Rationale for selecting satellite groups: Females within the toxicity and recovery groups were not paired.

- Post-exposure recovery period in satellite groups: 14 days

- Identification of animals: Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment.

- 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: Body weight range extreme Four males and two females
Irregular estrous cycles Five females
Acyclic One female
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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day, by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

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 the recovery phase), and 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 (Week 3), but recommenced for males on Week 4.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each relevant phase.

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 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: Yes
- Time schedule for collection of blood: Week 6: Five lowest numbered surviving Toxicity phase males and females in each group; Recovery: 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 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.
- Blood samples were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant.
- Haematology parameters: Haematocrit, Haemoglobin concentration (Females only in Recovery phase), Erythrocyte count (RBC), Absolute reticulocyte count, Reticulocyte %, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width (Males and Females in recovery phase), Total leucocyte count (Females only in recovery phase), Differential leucocyte count (Males and Females in recovery phase): Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count, Prothrombin time (Males only in recovery time) and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST) (Males only in recovery phase), Gamma-glutamyl transpeptidase (gGT), Total bilirubin, Bile acids, Urea (Males only in recovery phase), Creatinine (Females only in recovery phase), Glucose (Males and Females in recovery phase), Total cholesterol (Males and Females in recovery phase), Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca) (Males only in recovery phase), Inorganic phosphorus, Total protein (Males only in recovery phase), Albumin (Males only in recovery phase) and Albumin/globulin ratio (A/G Ratio) (Females only in recovery phase).

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, 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 (T-Prot and U-Prot), Creatinine (T-Creat and U-Creat), Glucose (T-Gluc and U-Gluc), Sodium, Potassium, Chloride
A microscopic examination of the urine sediment was performed: Epithelial cells, Leucocytes (WBC), Erythrocytes (RBC), Casts and Other abnormal components (A)
The slide was also examined for abnormalities in spermatozoa and crystals.

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

IMMUNOLOGY: No
Sacrifice and pathology:
SACRIFICE
Time of necropsy
- Toxicity phase: F0 males and females - after Week 6 investigations were completed.
- 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, unless specified below. 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 only: All remaining adult animals.
°Processing - Adrenals: Toxicity phase males and females in Group 2 and 3 and all Recovery phase males and females.
°Processing - Kidneys: Toxicity phase males in Group 2 and 3 and all Recovery phase males.
°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:
Blood samples were collected as follows:
- At termination: All surviving F0 males, all surviving F0 Reproductive phase females (no samples were obtained from animals which failed to litter) and all Recovery phase animals
Statistics:
See "Any other information on materials and methods incl. tables".
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed that were considered to be related to dietary administration of the test item.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities at any level into Toxicity animals.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males receiving 8000 ppm had statistically significantly reduced mean body weight gain between Days 1-4 of treatment and slight body weight loss between Days 36-39; however, subsequent body weight gains were similar to Controls.
Animals receiving 2000 or 4000 ppm had slightly higher group mean bodyweight gain between Days 1-46, (which was statistically significant for females) when compared with Controls.
Statistically significant group mean bodyweight loss was observed during Days 1-4 for toxicity and recovery phase females receiving 4000 or 8000 ppm, when compared with Controls. Subsequent body weight loss was also observed during Days 15-22 for females receiving 8000 ppm .
Statistically significant body weight loss was observed in reproductive phase females receiving 4000 or 8000 ppm between Days 1-4, and reduced bodyweight gain was observed in all groups of treated females during Days 4-8, when compared to Controls. Overall body weight gains for females treated at all dose levels remained slightly lower than Controls between Days 1-22 prior to pairing.
During Days 0-20 of gestation body weight gain was similar to the Control group. Body weight gain was higher during Days 1-7, and overall (Days 1-13) during the lactation phase, in all groups treated with the test item, when compared with the Controls.
During the recovery phase (Days R1-R15) the body weight gains of both the Group 4 males and females remained similar to the gains of the Controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean food consumption during Days 1-45 was similar to Controls for males receiving 2000 and 4000 ppm of the test item administered via the diet. A slight reduction in food intake was observed in male animals receiving 8000 ppm on Day 1 of treatment, however subsequent food consumption values were similar to Controls.
Toxicity phase and recovery phase females receiving 8000 ppm had slightly reduced food consumption on Day 1 to 5 of treatment when compared with Controls; however food intake was generally unaffected thereafter. Food consumption for females receiving 2000 or 4000 ppm was generally similar to that of Controls.
During Days 1 to 4 of treatment, a reduction in food consumption was observed in reproductive phase female animals receiving 8000 and to a lesser extent 4000 ppm, and on Day 2 and 4 for females receiving 2000 ppm. Following Day 4, food intake was generally similar to that of the Controls up until Day 21 prior to pairing. Females receiving 8000 ppm also showed a statistically significant reduction in food intake on Day 2 of gestation. Food intake of all groups receiving test item were statistically significantly lower on Days 16 and 17 of gestation, when compared to Controls.
Group mean food consumption during Days 1-12 of lactation for females receiving test item was generally similar to that of the Controls. However, on Day 10 of lactation all groups of treated females had statistically significant reduced food intake, when compared with Controls.
Food consumption was similar to the Controls through Days R1-R14 in the recovery phase males and females.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No effects of treatment were observed during ophthalmic examinations in Week 6 of treatment for animals receiving the test item, when comprae to Controls.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The haematological examinations in Week 6 of treatment revealed, when compared with Controls, increased white blood cell counts in both males and females treated at 4000 or 8000 ppm (statistical significance being attained at 8000 ppm). In males, this was predominantly due to increased neutrophils and lymphocytes observed at both the 4000 and 8000 ppm levels. In females, lymphocyte counts were also increased at the 4000 and 8000 ppm dose levels.
All other differences from Controls, including those that attained statistical significance, were generally small, confined to one sex, or the magnitudes were not dose-related and, consequently, were considered to represent normal biological variation.
Further hematological examinations during the recovery phase revealed a slight decrease in prothrombin time in males previously treated at 8000 ppm, when compared with the Controls. Females previously receiving 8000 ppm of test item had an increase in large unstained cells following two weeks of recovery when compared to the Control group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The biochemical examination of the blood plasma indicated an increase in creatinine and plasma cholesterol levels in animals treated at 4000 or 8000 ppm. Glucose levels were statistically significantly lower in animals receiving 2000, 4000 or 8000 ppm when compared to Control values.
For males receiving 8000 ppm, urea levels were found to be statistically significantly higher than Controls, in addition to an increase in calcium, albumin and total protein levels. Statistically significantly lower albumin or globulin ratios were observed for females receiving the test item, when compared with Controls.
All other differences from Controls, including those that attained statistical significance, were generally small, confined to one sex, or the magnitudes were not dose-related and, consequently, were considered to represent normal biological variation.
Females previously receiving 8000 ppm of test item had an increase in total protein following two weeks of recovery when compared to the Control group.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis investigations conducted in Week 6 of treatment did not reveal any findings that were considered to be related to treatment with the test item, when compared with Controls. All differences from Control values, were generally small, confined to one sex, or the extent of the difference from Controls was not dose-related and, consequently, was considered to not be associated with treatment.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory reactivity observations: In females receiving 8000 ppm one female showed a normal response to the tail pinch, one female showed an exagerated response and three females showed a weak response. This finding is not considered normal, but as a finding on its own, does not indicate a treatment related effect.

Grip strength observations: The group mean forelimb grip strength value for males receiving 8000 ppm was statistically significantly low when compared with Controls. However, this value was within the Historical Control Data range. The Control values were considered atypically high, and were above the Historical Control Data range. Hindlimb grip strength values were similar to Controls and all within the Historical Control Data range.

The motor activity assessment conducted during Week 6 of treatment revealed no treatment related effects in male animals at all dose levels and females receiving 2000 or 4000 ppm.
All group mean low beam and to a lesser extent high beam activity scores for females treated at 8000 ppm were slightly high when compared with Controls, with statistical significance being attained at the 12-minute interval for high beams and the 6 and 42-minute interval for low beams. However, with the exception of the 42-minute interval (for both high and low beams) all scores were within the Historical Control Data range. The total scores were within the Historical Control Data range, therefore no effect of treatment was inferred.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The analysis of absolute and adjusted organ weights in the toxicity phase males and females that received the test item had an increase in adrenal and liver weights, when compared to the Controls, with statistical significance being attained for adjusted liver weights for animals that received 8000 ppm.
For the reproductive phase females that received 8000 ppm, the combined weight of the uterus, cervix and oviducts was low, when compared to the Controls.
In the recovery phase males, there were no treatment-related organ weight changes. The adrenal and liver weights remained slightly but not statistically significantly higher in the females previously treated at 8000 ppm, following 14 days of recovery.
All other differences from Controls were minor and were therefore attributed to normal biological variation.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination performed after 6 weeks of treatment revealed no test item-related lesions.
The macroscopic examination performed after 14 days of recovery revealed no test item-related lesions.
The incidence and distribution of all other findings were considered to be unrelated to treatment.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with the test item were seen in the adrenal glands of males and females and the kidneys of males.

Adrenal glands:
There was an increase in zona glomerulosa hypertrophy in males and females treated with the test item at 8000 ppm and in males treated with the test item at 4000 ppm. There was an increase in cortical vacuolation in males treated with the test item at 2000 ppm and above, occurring in a dose related pattern.

Kidneys:
There was an increase in cortical tubular hyaline droplets in males treated with the test item at 8000 ppm.

All other findings are considered to be incidental.

After 14 days of recovery, a complete recovery was apparent in the kidneys of males and the adrenal glands of females. However, zona glomerulosa hypertrophy was still apparent in males previously treated with Elemi Resinoid at 8000 ppm. It occurred in one female previously treated with Elemi Resinoid at 8000 ppm but this was similar to main study Control females. There was an increase in cortical vacuolation in males previously treated with Elemi Resinoid at 8000 ppm. All other histological changes were considered to be unrelated to treatment.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis:
All samples taken from adult males at termination and Day 13 of age male and female offspring in Groups 1 to 4 had concentrations that were comparable with the endogenous levels observed in the control matrix used to prepare the QC samples.
As the T4 investigations conducted indicated no treatment related effects, no further analysis of T4 or TSH was required.
Key result
Dose descriptor:
NOAEL
Effect level:
8 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
no

Formulation Analysis:

The homogeneity and stability was confirmed for the test item in SDS VRF1 Certified with a corn oil stabilizer at a ratio of test item to corn oil of 5 to 1 formulations at nominal concentrations of 500ppm and 20000 ppm during ambient temperature storage for 8 days and frozen storage for up to 15 days.

The mean concentrations of the test item in test formulations analyzed for the study were within ±10% of nominal concentrations, confirming accurate formulation.

 

Achieved Dose:

Mean achieved doses for toxicity and recovery phase males were 118, 242 or 490 mg/kg bw/day respectively at 2000, 4000 or 8000 ppm.

Mean achieved doses for toxicity and recovery phase females were 123, 239 or 501 mg/kg bw/day respectively at 2000, 4000 or 8000 ppm.

Mean achieved doses for reproductive phase females at 2000, 4000 or 8000 ppm were 126, 247 or 478 mg/kg bw/day before pairing, 139, 282 or 536 mg/kg bw/day during gestation and 309, 601 or 1186 mg/kg bw/day during Days 1-13 of lactation.

Achieved doses generally maintained the interval between dietary concentrations. As expected, the achieved doses declined slightly as the animals mature - this is more obvious in males.

Conclusions:
Based on the results of this study it is concluded that the NOAEL of test item for systemic toxicity was 8000 ppm (mean achieved doses of 490 mg/kg bw/day for males, 501 mg/kg bw/day for toxicity phase 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 2000, 4000 and 8000 ppm.

 

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.

A similarly constituted Control group was assigned to each phase, and received the vehicle, untreated diet, throughout the same relative treatment period.

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

The mean concentrations of test item in test formulations analyzed for the study were within ±10% of nominal concentrations, confirming accurate formulation.

There were no treatment-related mortalities at any level. No effects that could be clearly related to treatment were observed on sensory reaction, grip strength, motor activity, ophthalmic examination and urinalysis parameters. In addition there were no treatment-related clinical signs observed.

No adverse effects of treatment were evident on the circulating levels of thyroxine. No significant changes were identified at the microscopic examination of thyroid and pituitary glands and the testes and ovaries.

There was an initial reduction of body weight gain/ body weight loss in toxicity phase females receiving 4000 ppm and in both sexes receiving at 8000 ppm, associated with an initial reduction in food consumption during this period, when compared to the Controls.

Haematological findings at termination included increased white blood cell counts in animals treated at 4000 or 8000 ppm, which was predominantly due to increased neutrophils and lymphocytes in males and increased lymphocytes observed at these levels and following a 14 day recovery period lymphocytes levels were still slightly elevated.

Biochemical effects of the blood plasma indicated an increase in creatinine and plasma cholesterol levels in animals treated at 4000 or 8000 ppm. Glucose levels were statistically significantly lower in animals receiving 2000, 4000 or 8000 ppm. Urea levels were found to be statistically significantly higher than Controls for males receiving 8000 ppm, in addition to an increase in calcium, albumin and total protein levels. Statistically significantly lower albumin or globulin ratios were observed for females receiving test item, when compared with Controls. Following a 14 day recovery period females previously receiving 8000 ppm maintained an increase in total protein, when compared to the Control group, but all other parameters were similar to Controls.

The analysis of absolute and adjusted organ weights in the toxicity phase males and females that received the test item had an increase in adrenal and liver weights, when compared to the Controls, with statistical significance being attained for adjusted liver weights for animals that received 8000 ppm. In the recovery phase the adrenal and liver weights remained slightly but not statistically significantly higher in the females previously treated at 8000 ppm, following 14 days of recovery.

Changes considered to be related to administration of the test item were present in the adrenal glands of males and females and kidneys of males. There was an increase in zona glomerulosa hypertrophy seen in the adrenals of males and females treated at 8000 ppm and in males treated at 4000 ppm, and was persistent in males after 14 days of recovery. Vacuolation of the adrenal gland cortex was increased in males given the test item at 2000 ppm and above in a dose-related incidence. This persisted after 14 days of recovery.

There was an increase in renal cortical tubular hyaline droplets in males treated with the test item at 8000 ppm.

Dietary administration of test item in males and toxicity phase females for 6 weeks and reproductive phase females for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation at levels up to 8000 ppm was generally well tolerated and there were no treatment-related mortalities at any level.

There were initial effects on bodyweight and food consumption at the start of treatment, but this had no impact on overall bodyweight and showed full recovery. Test item showed no evidence of being an endocrine disruptor and did not affect thyroid hormone levels.

Administration of test item was associated with minimal to slight changes in the adrenal glands of males and females and kidneys of males receiving 8000 ppm. Slight changes were noted in the adrenal glands of males treated at 2000 ppm and above that persisted in the adrenal glands of males after a 14-day recovery period. However, these changes are considered likely to be non-adverse.

Based on the results of this study it is concluded that the No-Observed-Adverse-Effect- Level (NOAEL) for systemic toxicity is 8000 ppm (mean achieved doses of 490 mg/kg bw/day for males, 501 mg/kg bw/day for toxicity phase females).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Adopted 29 July 2016
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Resinoid of Canarium luzonicum (Burseraceae) obtained from the exudate by cyclohexane extraction
EC Number:
946-584-9
Molecular formula:
Not applicable (UVCB)
IUPAC Name:
Resinoid of Canarium luzonicum (Burseraceae) obtained from the exudate by cyclohexane extraction
Test material form:
other: pale yellow paste
Details on test material:
- Name: Elemi resinoid
- Storage condition of test material: Room temperature, in the dark
- Expiration date of the lot/batch: 15 December 2017
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor - 1002888716
- Appearance: Paste, pale yellow
- Expiration date of the lot/batch: 15 December 2017

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Dry area, closed containers, room temperature 15 to 25°C

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) 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.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males: 69 to 76 days old; Females: 83 to 90 days old.
- Weight at study initiation: Males: 339-391 g; Females: 237-305 g
- Housing: Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing; Cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Number of animals per cage: Pre-pairing: up to five animals of one sex; Pairing: one male and one female; Males after mating: up to five animals; Gestation: one female; Lactation: one female + litter
- Diet: SDS VRF1 Certified powdered diet, ad libitum (removed overnight before blood sampling for thyroid hormone, hematology and blood chemistry investigations)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (removed overnight during urine collection)
- Acclimation period: Females: 20 days before the commencement of treatment; Males: six days before the commencement of treatment.

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: 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 (returned on Day 13 of lactation, after dispatch of the litter)] and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study [except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced at the same time as the cages.

IN-LIFE DATES: 19 July 2017 to 10 October 2017

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: feed
Details on exposure:
DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet
- Stabilizer: Corn oil (test material to corn oil ratio 5:1).
- Correction factor: A correction factor was not required.
- Method of preparation: The required amount of test item was weighed out into a suitable container. With magnetic stirring, the required amount of solvent was added until the test item was fully dispersed. The dispersion was added to the appropriate amount of plain diet, followed by the addition of a further appropriate amount of plain diet.
The bulk was stirred well and then the solvent evaporated off in a fume hood, until constant weight was achieved.
The required amount of corn oil was then added to the mixture and the bulk mixed firstly using a mechanical grinder, and then with a Turbula mixer after making to the required weight with plain diet.
Appropriate amounts of plain diet was then added to aliquots of this pre-mix, with mixing, until the requisite test item concentration was achieved.
- Frequency of preparation: Weekly.
- Storage of formulation: Frozen (-10 to -30 °C). Stability and homogeneity of the formulations was confirmed for 15 days when stored frozen (-10 to -30°C) and for eight days at ambient temperature (15°C to 25°C).
Details on mating procedure:
- Animals: Toxicity phase and Recovery phase males paired 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.
- Duration of pairing: Up to 2 weeks
- Daily checks for evidence of maiting: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
- Day 0 of gestation: When positive evidence of mating was detected.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the diet matrix at concentrations of 500 ppm and 20000 ppm.
- Achieved concentration: Samples of each formulation prepared for administration in Week 1 and the final week of treatment were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
- Reproductive phase (females): Three weeks before pairing, then throughout pairing and gestation until Day 12 of lactation (necropsy on Day 13 of lactation (the treated diet was available to the animals until the morning of necropsy)).
- Toxicity phase (males): Three weeks before pairing up to necropsy after a minimum of six weeks.
- Toxicity phase (females): A minimum of 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 were not dosed.
Frequency of treatment:
Continuously
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Group 1 (control)
Dose / conc.:
2 000 ppm
Remarks:
Group 2 (low dose)
Dose / conc.:
4 000 ppm
Remarks:
Group 3 (mid dose)
Dose / conc.:
8 000 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: Dietary levels were selected following the completion of the preliminary toxicity study (Envigo Study number: SN57HH) following consultation with the Sponsor.
Effects on
body weight and food consumption were seen at 7500 ppm in males at the start of treatment, from which they did not completely recover. The same trend was observed at 10000 ppm in males and females, with slight recovery at the end of treatment. These effects were considered likely to be related to the palatability of test item. Also, high kidneys and liver weights (mean increase of 9 and 28% from controls, respectively) were observed in males at 10000 ppm and even in liver weights at 5000 ppm (mean increase of 15% from controls).
The high dietary concentration of 8000 ppm was expected to elicit initial mean body weight loss or stasis and initial low food consumption and potential adverse effects in target organs, especially in males. The lowest dietary concentration of 2000 ppm was expected to be a no effect level for effects on body weight and food consumption. The intermediate dietary concentration of 4000 ppm allowed evaluation of any concentration related trends and provides a geometric progression of dietary concentrations.

- Rationale for animal assignment: On arrival and non-selective allocation to cages. Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 days cycles were not allocated to the study. On Day 1 of study all animals were weighed and body weights were reviewed 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.

- Rationale for selecting satellite groups: Females within the toxicity and recovery groups were not paired.

- Post-exposure recovery period in satellite groups: 14 days

- Identification of animals: Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.

- 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: Body weight range extreme Four males and two females
Irregular estrous cycles Five females
Acyclic One female
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day, by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

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 the 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; Day 1, 4, 7 and 13 of lactation.

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 28), but recommenced for males on Day 29. 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 relevant phase.

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 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: Yes
- Time schedule for collection of blood: Week 6: Five lowest numbered surviving Toxicity phase males and females in each group; Recovery: 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 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.
- Blood samples were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant.
- 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, 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 (T-Prot and U-Prot), Creatinine (T-Creat and U-Creat), Glucose (T-Gluc and U-Gluc), Sodium, Potassium, Chloride
A microscopic examination of the urine sediment was performed: Epithelial cells, Leucocytes (WBC), Erythrocytes (RBC), Casts and Other abnormal components (A)
The slide was also examined for abnormalities in spermatozoa and crystals.

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

IMMUNOLOGY: No

OTHER: Mating Procedure
- Animals: Toxicity phase and Recovery phase males paired with Reproductive phase females (Toxicity and Recovery phase females were not paired for mating).
- Pairing commenced: After a minimum of three weeks of treatment.
- Male/female ratio: 1:1 from within the same treatment groups.
- Duration of pairing: Up to two weeks.
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
- Day 0 of gestation: When positive evidence of mating was detected.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

OTHER: 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):
Dry and wet smears were taken as follows:
- Dry smears: Reproductive phase females: from beginning of treatment until animals were paired for mating, using cotton swabs.
- Wet smears: Using pipette lavage during the following phases: For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study / After pairing until mating / For four days before scheduled termination (all Reproductive phase, Toxicity phase and Recovery phase females).
Litter observations:
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to 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 viable litter: Day 25 after mating.
°F0 females killed at termination - 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.
Reproductive Phase Females: Each uterine horn Number of implantation sites was counted and confirmed if none were visible at visual inspection for non-pregnant
females.

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: Toxicity phase males and females in Groups 1 and 4 at scheduled termination.
°Abnormalities: All remaining adult animals.
°Processing - Kidneys: toxicity phase males in Group 2 and 3 and all Recovery phase males.
°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 with an assessment of stomach for milk content was performed. Abnormal tissues retained.
- F1 offspring on Day 4 of age:
Blood sampling required
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia.
- F1 offspring on Day 13 of age
Blood sampling required
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Thyroid glands were preserved from one male and one female in each litter.
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
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
- Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
- Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering) x 100
- Lactation index (%) = (Number of live offspring on Day 13 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 (before and after blood sampling) and 13 of age.
- Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed that were considered to be related to dietary administration of the test item.

One Control female (1F. 62) was killed due to reasons of animal welfare on Day 20 after mating. Clinical signs for this female comprised a swollen, firm mass on the lower ventral abdomen. It was considered that the mass would later impair suckling behaviour for the Control female, thus this death was not treatment related.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities at any level into Toxicity animals.

One Control female (1F. 62) was killed due to reasons of animal welfare on Day 20 after mating.

Two further Control females (Animals No’s. 61 and 66) were killed on Day 25 of gestation due to them failing to litter. At necropsy it was found that Animal 61 was not pregnant, and Animal 66 was pregnant with one dead pup in utero. Neither animal had any adverse macroscopic findings.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males receiving 8000 ppm had statistically significantly reduced mean body weight gain between Days 1-4 of treatment and slight body weight loss between Days 36-39; however, subsequent body weight gains were similar to Controls.
Animals receiving 2000 or 4000 ppm had slightly higher group mean bodyweight gain between Days 1-46, (which was statistically significant for females) when compared with Controls.
Statistically significant group mean bodyweight loss was observed during Days 1-4 for toxicity and recovery phase females receiving 4000 or 8000 ppm, when compared with Controls. Subsequent body weight loss was also observed during Days 15-22 for females receiving 8000 ppm .
Statistically significant body weight loss was observed in reproductive phase females receiving 4000 or 8000 ppm between Days 1-4, and reduced bodyweight gain was observed in all groups of treated females during Days 4-8, when compared to Controls. Overall body weight gains for females treated at all dose levels remained slightly lower than Controls between Days 1-22 prior to pairing.
During Days 0-20 of gestation body weight gain was similar to the Control group. Body weight gain was higher during Days 1-7, and overall (Days 1-13) during the lactation phase, in all groups treated with the test item, when compared with the Controls.
During the recovery phase (Days R1-R15) the body weight gains of both the Group 4 males and females remained similar to the gains of the Controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean food consumption during Days 1-45 was similar to Controls for males receiving 2000 and 4000 ppm of the test item administered via the diet. A slight reduction in food intake was observed in male animals receiving 8000 ppm on Day 1 of treatment, however subsequent food consumption values were similar to Controls.
Toxicity phase and recovery phase females receiving 8000 ppm had slightly reduced food consumption on Day 1 to 5 of treatment when compared with Controls; however food intake was generally unaffected thereafter. Food consumption for females receiving 2000 or 4000 ppm was generally similar to that of Controls.
During Days 1 to 4 of treatment, a reduction in food consumption was observed in reproductive phase female animals receiving 8000 and to a lesser extent 4000 ppm, and on Day 2 and 4 for females receiving 2000 ppm. Following Day 4, food intake was generally similar to that of the Controls up until Day 21 prior to pairing. Females receiving 8000 ppm also showed a statistically significant reduction in food intake on Day 2 of gestation. Food intake of all groups receiving test item were statistically significantly lower on Days 16 and 17 of gestation, when compared to Controls.
Group mean food consumption during Days 1-12 of lactation for females receiving test item was generally similar to that of the Controls. However, on Day 10 of lactation all groups of treated females had statistically significant reduced food intake, when compared with Controls.
Food consumption was similar to the Controls through Days R1-R14 in the recovery phase males and females.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No effects of treatment were observed during ophthalmic examinations in Week 6 of treatment for animals receiving the test item, when comprae to Controls.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The haematological examinations in Week 6 of treatment revealed, when compared with Controls, increased white blood cell counts in both males and females treated at 4000 or 8000 ppm (statistical significance being attained at 8000 ppm). In males, this was predominantly due to increased neutrophils and lymphocytes observed at both the 4000 and 8000 ppm levels. In females, lymphocyte counts were also increased at the 4000 and 8000 ppm dose levels.
All other differences from Controls, including those that attained statistical significance, were generally small, confined to one sex, or the magnitudes were not dose-related and, consequently, were considered to represent normal biological variation.
Further hematological examinations during the recovery phase revealed a slight decrease in prothrombin time in males previously treated at 8000 ppm, when compared with the Controls. Females previously receiving 8000 ppm of test item had an increase in large unstained cells following two weeks of recovery when compared to the Control group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The biochemical examination of the blood plasma indicated an increase in creatinine and plasma cholesterol levels in animals treated at 4000 or 8000 ppm. Glucose levels were statistically significantly lower in animals receiving 2000, 4000 or 8000 ppm when compared to Control values.
For males receiving 8000 ppm, urea levels were found to be statistically significantly higher than Controls, in addition to an increase in calcium, albumin and total protein levels. Statistically significantly lower albumin or globulin ratios were observed for females receiving the test item, when compared with Controls.
All other differences from Controls, including those that attained statistical significance, were generally small, confined to one sex, or the magnitudes were not dose-related and, consequently, were considered to represent normal biological variation.
Females previously receiving 8000 ppm of test item had an increase in total protein following two weeks of recovery when compared to the Control group.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis investigations conducted in Week 6 of treatment did not reveal any findings that were considered to be related to treatment with the test item, when compared with Controls. All differences from Control values, were generally small, confined to one sex, or the extent of the difference from Controls was not dose-related and, consequently, was considered to not be associated with treatment.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory reactivity observations: In females receiving 8000 ppm one female showed a normal response to the tail pinch, one female showed an exagerated response and three females showed a weak response. This finding is not considered normal, but as a finding on its own, does not indicate a treatment related effect.

Grip strength observations: The group mean forelimb grip strength value for males receiving 8000 ppm was statistically significantly low when compared with Controls. However, this value was within the Historical Control Data range. The Control values were considered atypically high, and were above the Historical Control Data range. Hindlimb grip strength values were similar to Controls and all within the Historical Control Data range.

The motor activity assessment conducted during Week 6 of treatment revealed no treatment related effects in male animals at all dose levels and females receiving 2000 or 4000 ppm.
All group mean low beam and to a lesser extent high beam activity scores for females treated at 8000 ppm were slightly high when compared with Controls, with statistical significance being attained at the 12-minute interval for high beams and the 6 and 42-minute interval for low beams. However, with the exception of the 42-minute interval (for both high and low beams) all scores were within the Historical Control Data range. The total scores were within the Historical Control Data range, therefore no effect of treatment was inferred.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with the test item were seen in the adrenal glands of males and females and the kidneys of males.

Adrenal glands:
There was an increase in zona glomerulosa hypertrophy in males and females treated with the test item at 8000 ppm and in males treated with the test item at 4000 ppm. There was an increase in cortical vacuolation in males treated with the test item at 2000 ppm and above, occurring in a dose related pattern.

Kidneys:
There was an increase in cortical tubular hyaline droplets in males treated with the test item at 8000 ppm.

All other findings are considered to be incidental.

After 14 days of recovery, a complete recovery was apparent in the kidneys of males and the adrenal glands of females. However, zona glomerulosa hypertrophy was still apparent in males previously treated with Elemi Resinoid at 8000 ppm. It occurred in one female previously treated with Elemi Resinoid at 8000 ppm but this was similar to main study Control females. There was an increase in cortical vacuolation in males previously treated with Elemi Resinoid at 8000 ppm. All other histological changes were considered to be unrelated to treatment.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis:
All samples taken from adult males at termination and Day 13 of age male and female offspring in Groups 1 to 4 had concentrations that were comparable with the endogenous levels observed in the control matrix used to prepare the QC samples.
As the T4 investigations conducted indicated no treatment related effects, no further analysis of T4 or TSH was required.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Prior to treatment all females had a regular oestrus cycle of 4 to 5 days. During the treatment period all females had a regular oestrus cycle of 4 to 5 days, with the exception of one female receiving 2000 ppm which had an extended estrous cycle.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
All females had a pre-coital interval of 1-4 days, with the exception of two Control females, which had pre-coital intervals of 9-12 or 13-14 days. At termination, toxicity and recovery phase females showed estrous and all reproductive females were in diestrus.
Gestation length and gestation index were considered unaffected by treatment at any dose level.

Estrous cycles, pre-coital interval and mating performance and fertility was considered unaffected by treatment with Elemi Resinoid, when compared with Controls.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
8 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
The distribution of signs at physical examination showed no relationship to parental treatment.
Mortality / viability:
no mortality observed
Description (incidence and severity):
Litter size was not affected by treatment with the test item. The number of females raising their litters to Day 13 of age was 7, 10, 10 and 10 for Control, 2000, 4000 and 8000 ppm, respectively. Total litter size on Day 1 was statistically significantly lower for females receiving 4000 or 8000 ppm when compared with Controls, however there was no difference in live litter size on Day 1 when compared with Controls, and the values at 4000 or 8000 ppm were within the Historical Control Data range, so no effect of treatment was inferred.
Group mean post implantation survival index, mean live birth index, viability index and lactation index were unaffected by treatment with the test item.
There were no effects on sex ratio that were associated to treatment with the test item.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean offspring bodyweight and mean bodyweight gain for male and female offspring of parents treated with the test item was considered unaffected by treatment when compared with Controls.
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 conclusive effect on ano-genital distance in the males and females offspring of parents treated with the test item.
No nipples were observed in male offspring of parents treated with the test item.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of offspring that either died prematurely or at scheduled termination on Day 13 of age did not reveal any findings that could be attributed to treatment.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Generation:
F1
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Formulation Analysis:

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limits of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.

The homogeneity and stability was confirmed for the test item in SDS VRF1 Certified with a corn oil stabilizer at a ratio of test item to corn oil of 5 to 1 formulations at nominal concentrations of 500ppm and 20000 ppm during ambient temperature storage for 8 days and frozen storage for up to 15 days.

The mean concentrations of the test item in test formulations analyzed for the study were within ±10% of nominal concentrations, confirming accurate formulation.

 

Achieved Dose:

Mean achieved doses for toxicity and recovery phase males were 118, 242 or 490 mg/kg bw/day respectively at 2000, 4000 or 8000 ppm.

Mean achieved doses for toxicity and recovery phase females were 123, 239 or 501 mg/kg bw/day respectively at2000, 4000 or 8000 ppm.

Mean achieved doses for reproductive phase females at 2000, 4000 or 8000 ppm were 126, 247 or 478 mg/kg bw/day before pairing, 139, 282 or 536 mg/kg bw/day during gestation and 309, 601 or 1186 mg/kg bw/day during Days 1-13 of lactation.

Achieved doses generally maintained the interval between dietary concentrations. As expected, the achieved doses declined slightly as the animals mature - this is more obvious in males.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the NOAEL of test item for systemic toxicity was 8000 ppm (mean achieved doses of 490 mg/kg bw/day for males, 501 mg/kg bw/day for toxicity phase females, 478 mg/kg bw/day for females during pre-paring phase, 536 mg/kg bw/day for females during gestation and 1186 mg/kg bw/day for females during lactation). The NOAEL of test item for reproductive/developmental effects was concluded to be 8000 ppm.
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 2000, 4000 and 8000 ppm.

 

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). Selected F1 offspring were killed on Day 4 of age for blood sampling collection for thyroid hormone analysis. The remaining F1 offspring were killed on Day 13 of age. The offspring received no direct administration to the test item; any exposure was in utero or via the milk.

A similarly constituted Control group was assigned to each phase, and received the vehicle, untreated diet, throughout the same relative treatment period.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, 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, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

The mean concentrations of test item in test formulations analyzed for the study were within ±10% of nominal concentrations, confirming accurate formulation.

There were no treatment-related mortalities at any level. No effects that could be clearly related to treatment were observed on sensory reaction, grip strength, motor activity, ophthalmic examination and urinalysis parameters. In addition there were no treatment-related clinical signs observed, no effects on estrous cycles, pre-coital interval, mating performance and fertility, gestation length, litter size, offspring survival or bodyweight.

No adverse effects of treatment were evident on the circulating levels of thyroxine. No significant changes were identified at the microscopic examination of thyroid and pituitary glands and the testes and ovaries. All offspring were macroscopically normal; in particular no effects were seen on the external genitalia. Ano-genital distances were not affected by treatment, and there were no nipples in male offspring.

There was an initial reduction of body weight gain/ body weight loss in toxicity and reproductive phase females receiving 4000 ppm and in both sexes receiving at 8000 ppm, associated with an initial reduction in food consumption during this period, when compared to the Controls.

Haematological findings at termination included increased white blood cell counts in animals treated at 4000 or 8000 ppm, which was predominantly due to increased neutrophils and lymphocytes in males and increased lymphocytes observed at these levels and following a 14 day recovery period lymphocytes levels were still slightly elevated.

Biochemical effects of the blood plasma indicated an increase in creatinine and plasma cholesterol levels in animals treated at 4000 or 8000 ppm. Glucose levels were statistically significantly lower in animals receiving 2000, 4000 or 8000 ppm. Urea levels were found to be statistically significantly higher than Controls for males receiving 8000 ppm, in addition to an increase in calcium, albumin and total protein levels. Statistically significantly lower albumin or globulin ratios were observed for females receiving the test item, when compared with Controls. Following a 14 day recovery period females previously receiving 8000 ppm maintained an increase in total protein, when compared to the Control group, but all other parameters were similar to Controls.

The analysis of absolute and adjusted organ weights in the toxicity phase males and females that received the test item had an increase in adrenal and liver weights, when compared to the Controls, with statistical significance being attained for adjusted liver weights for animals that received 8000 ppm. For the reproductive phase females that received 8000 ppm, the combined weight of the uterus, cervix and oviducts was low, when compared to the Controls. In the recovery phase the adrenal and liver weights remained slightly but not statistically significantly higher in the females previously treated at 8000 ppm, following 14 days of recovery.

Changes considered to be related to administration of the test item were present in the adrenal glands of males and females and kidneys of males. There was an increase in zona glomerulosa hypertrophy seen in the adrenals of males and females treated at 8000 ppm and in males treated at 4000 ppm, and was persistent in males after 14 days of recovery. Vacuolation of the adrenal gland cortex was increased in males given the test item at 2000 ppm and above in a dose-related incidence. This persisted after 14 days of recovery.

There was an increase in renal cortical tubular hyaline droplets in males treated with test item at 8000 ppm.

Dietary administration of test item in males and toxicity phase females for 6 weeks and reproductive phase females for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation at levels up to 8000 ppm was generally well tolerated and there were no treatment-related mortalities at any level.

There were initial effects on bodyweight and food consumption at the start of treatment, but this had no impact on overall bodyweight and showed full recovery. There were no effects on parental reproductive parameters or fertility. All offspring were macroscopically normal. The test item showed no evidence of being an endocrine disruptor and did not affect thyroid hormone levels.

Administration of test item was associated with minimal to slight changes in the adrenal glands of males and females and kidneys of males receiving 8000 ppm. Slight changes were noted in the adrenal glands of males treated at 2000 ppm and above that persisted after a 14-day recovery period. However, these changes are considered likely to be non-adverse.

Based on the results of this study it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity is 8000 ppm (mean achieved doses of 490 mg/kg bw/day for males, 501 mg/kg bw/day for toxicity phase females, 478 mg/kg bw/day for females during pre-paring phase, 536 mg/kg bw/day for females during gestation and 1186 mg/kg bw/day for females during lactation). The NOEL for reproductive and developmental toxicity is 8000 ppm.