Registration Dossier

Administrative data

Description of key information

An OECD TG 422 study is on-going on the registered substance

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Biolandes / L1117/1
- Appearance: Brown-red, solid
- Expiration date of the lot/batch: november 2019

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males: Approximately 71 days old; Females: Approximately 85 days old.
- Weight at study initiation: Males: 339-379 g; Females: 241-302 g
- Housing: Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing; 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 hematology and blood chemistry investigations and during urine collection)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (removed overnight during urine collection)
- Acclimation period: Females: six days prior to the commencement of estrous cycle evaluation; Males: six days prior to the commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 h light : 12 h dark
- Environmental Enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

IN-LIFE DATES:
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:
corn oil
Details on oral exposure:
DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet
- Correction factor: A correction factor was not required.
- Stabilizer: Corn oil (test material to corn oil ratio 5:1).
- Method of preparation: The test substance was incorporated into the diet to provide the required concentrations by initial preparation of a premix. On each occasion of the preparation of the premix, the required amount of test substance and corn oil were weighed into a suitable container. An amount of sieved diet that approximately equalled the weight of test substance was added and the mixture stirred together. A further amount of sieved diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
For the control diet, an amount of diet was added directly to the corn oil and then prepared as indicated for the premix.
- Frequency of preparation: Weekly.
- Storage of formulation: Deep-frozen (nominally -30 to -10 °C) until the day before use. Formulations were used within 28 h of removal from the freezer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 500 and 20000 ppm were analyzed to assess the stability and homogeneity of the test item in the diet matrix.
- Achieved concentration: Samples of each formulation prepared for administration in Week 1 and in the final week of treatment were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
Reproductive phase females: Three weeks before pairing, then throughout pairing and gestation until Day 12 of lactation (necropsy on Day 13 of lactation (the diet was available to the animals until the morning of necropsy)).
Toxicity phase males: Three weeks before pairing up to necropsy after minimum of six weeks.
Toxicity phase females: At least six weeks.
Recovery phase males: Three weeks before pairing up to necropsy after minimum of six weeks followed by a minimum 14-day recovery.
Recovery phase females: At least six weeks followed by a minimum 14-day recovery.
Frequency of treatment:
Continuously
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: ) following consultation with the Sponsor.

- Rationale for animal assignment: On arrival and non-selective allocation to cages.
Estrous cycles were evaluated prior to treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the reproductive phase of the study.
On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets by Study Management to ensure variations in body weight of animals did not exceed ±20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.
- Post-exposure recovery period in satellite groups: 14 days
- Other: Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
Animal Replacement: Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

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

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

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

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 male Recovery 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. Sampling was performed on the morning after overnight collection of urine.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count, Differential leucocyte count: Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count, Prothrombin time and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin, Bile acids, Urea, Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus, Total protein, Albumin and Albumin/globulin ratio (A/G Ratio).

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

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

IMMUNOLOGY: No
Sacrifice and pathology:
SACRIFICE
Time of necropsy
Toxicity phase:
F0 males and females: After Week 6 investigations were completed.
Reproductive phase females:
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 13 of lactation.
Recovery phase
F0 Males and females: After at least 14 days without treatment.
- Method of sacrifice: All adult animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination. (No animal was exposed to carbon dioxide until after completion of thyroid hormone assays).

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

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

HISTOPATHOLOGY
- Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: Initially in modified Davidson’s fluid; Eyes: In Davidson’s fluid.
- Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: All F0 animals killed or dying prematurely; Toxicity phase males and females in Groups 1 and 4 at scheduled termination.
Abnormalities: All remaining adult animals.
Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
Other examinations:
Thyroid Hormone Analysis:
At termination: F0 males, All F0 Reproductive phase females
Statistics:
See "Any other information on materials and methods incl. tables".
Water consumption and compound intake (if drinking water study):
not examined
Remarks on result:
not determinable
Remarks:
on-going study
Critical effects observed:
not specified
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening 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 ppm.

 

Reproductive females were treated daily for three weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 13 of lactation. Toxicity phase males were treated daily for three weeks before pairing up to necropsy after a minimum of six consecutive weeks. Toxicity phase females were treated daily for a minimum of six consecutive weeks. Recovery phase males were treated daily for three weeks before pairing up to necropsy after a minimum of six consecutive weeks followed by a minimum of 14 days recovery. Recovery phase females were treated daily for a minimum of six consecutive weeks followed by a minimum of 14 days recovery. A similarly constituted Control group was assigned to each phase, and received the vehicle, powdered SDS VRF1 Certified diet with corn oil, throughout the same relative treatment period. 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, thyroid hormone analysis, urinalysis, organ weight and macroscopic pathology and histopathology investigations were undertaken.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Preliminary study performed similarly to OECD Guideline 407, used as range-finder experiment for OECD 422 screening test performed in GLP laboratory.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
test item was administered for 21 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: Biolandes / L1117/1
- Appearance: Brown-red, solid
- Expiration date of the lot/batch: November 2019

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Desiccated, refrigerated (nominally 2-8 °C), protected from light, under nitrogen.
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Males: 70 to 77 days; Females: 84 to 91 days
- Weight at study initiation: Males: 339-383 g; Females: 237-267 g
- Housing: Animals were housed in groups of 4/sex in polycarbonate cages
- Diet: SDS VRF1 Certified powdered diet, ad libitum (removed overnight before blood chemistry collection)
- Water: Potable water from public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 7 days

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

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

IN-LIFE DATES: March 2018
Route of administration:
oral: feed
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: A correction factor was not required.
- Method of preparation:
On each occasion of the preparation of the premix the required amount of test substance and corn oil were weighed into a suitable container. An amount of sieved diet that approximately equaled the weight of test substance was added and the mixture stirred together. A further amount of sieved diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
For the control diet, an amount of diet was added directly to the corn oil and then prepared as indicated for the premix.
- Frequency of preparation: Weekly
- Storage of formulation: Deep-frozen (nominally -20 °C) for 15 days.

STABILITY AND HOMOGENEITY
Before the commencement of treatment, the suitability of the proposed mixing procedure and the homogeneity and stability of the test material in the carrier diet, was determined as part of the main OECD 422 study (Envigo Study Number: ). Initial stability of the formulations was been confirmed for one day at ambient temperature for formulations between 500 ppm and 20000 ppm.
Stability of the formulations was confirmed as follows:
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No analysis of the formulated diets was performed on this study, however, samples of the first preparations were stored at -20 °C.
Duration of treatment / exposure:
21 days
Frequency of treatment:
Continuously
Dose / conc.:
0 ppm
Dose / conc.:
3 500 ppm
Dose / conc.:
7 500 ppm
Dose / conc.:
15 000 ppm
No. of animals per sex per dose:
5
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dietary concentrations used in this study (0, 3500, 7500 and 15000 ppm) were selected in conjunction with the Sponsor. In a recent acute oral toxicity study conducted according to OECD guideline 423 in rats, the LD50 of the test item was found to be higher than 2000 mg/kg bw. Based on these results, 15000 ppm (equivalent to 1000 mg/kg bw/day) was selected as the highest dietary concentration in order to assess the palatability and the systemic toxicity of this test item.
- Rationale for animal assignment: Randomly allocated on arrival.
Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
- Animal Replacement
On Day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed ±20% of the mean for the appropriate sex. 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 - Two males and two females.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Day 1, 4, 8, 11, 15, 18 and 21 to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded daily throughout the study from Day -3 and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded daily throughout the study from Day -3.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 3 from all animals
- Animals fasted: Yes, blood samples were collected after overnight withdrawal of food
- Animals were held under light general anaesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin, Bile acids, Urea, Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus, Total protein, Albumin, Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Dry smears: Daily smears were taken for 21 days, using cotton swabs moistened with saline. Smears were subsequently examined to establish the duration and regularity of the estrous cycle.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; Animals were killed following 21 days of treatment by carbon dioxide asphyxiation and subjected to detailed necropsy. 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. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. The retained tissues were checked before disposal of the carcass.

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

HISTOPATHOLOGY: No; but tissues with macroscopic abnormalities were preserved for microscopic examinations in 10% neutral buffered formalin for microscopic examination (except testes: In modified Davidson’s fluid).
Other examinations:
MICRONUCLEUS TEST
- Positive control
Five bone marrow smears previously prepared from rats administered cyclophosphamide (Envigo Study Number ) will be stained and coded along with the bone marrow smears prepared in this study.
- Preparation of bone marrow smears
The following will be performed by Necropsy personnel: On completion of the treatment period, the first five surviving Toxicity phase males and females from each group will be euthanised by carbon dioxide inhalation, followed by exsanguination. One femur will be dissected from each animal and the proximal head removed.
The following will be performed by Cell and Molecular Sciences personnel: The contents of the femur from each animal will be flushed out and pooled in a total volume of 3 mL pre-filtered foetal calf serum. The cell suspension will be sedimented by centrifugation, the supernatant will be discarded and the cells will be re-suspended in a small volume of fresh foetal calf serum. A small drop of the suspension will be transferred to a glass microscope slide and a smear prepared. At least four smears will be prepared from each animal. At least one smear from each animal will be stained and examined, the remaining smears being held temporarily in reserve in case of technical problems with the first smear.
- Fixation and staining of slides
Fixed for a minimum of 10 minutes in methanol and allowed to air-dry. Rinsed in purified water. Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes. Washed in purified water for 5 minutes. Rinsed in cold tap water for 2 minutes. Stored at room temperature until required. Immediately prior to scoring, slides are wet mounted with glass coverslips using purified water.
- Microscopic examination
At least 5 slides (prepared in a separate study (Envigo Study Number )) from animals treated with Cyclophosphamide, a well characterised clastogen, will be stained and coded along with the bone marrow smears prepared from this study.
Coded slides will be examined by fluorescence microscopy and 4000 polychromatic erythrocytes per animal will be examined for the presence of micronuclei. Usually only one smear is examined per animal, any remaining smears being held temporarily in reserve in case of technical problems with the first smear.
The proportion of polychromatic erythrocytes will be assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes will be recorded.
- Acceptance criteria: The following criteria will be applied for assessment of assay acceptability:
1. The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database.
2. Concurrent positive control or scoring controls should induce responses that produce a significant increase compared with the concurrent negative control.
3. A maximum tolerated dose or maximum feasible dose has been achieved.
4. Adequate number of cells and doses have been analysed.
5. Each treated and control group should include at least 5 analysable animals.
- Analysis of data: The results obtained for each treatment group will be compared with the results obtained for the vehicle control group using non-parametric statistical methods. Analysis will be performed for the study using data from all animals that survive to scheduled termination to maximise the power of statistical analysis. If less than five animals per group survive to scheduled termination the study may be repeated.
For the proportion of polychromatic erythrocytes at 24 hours, an asymptotic one-tailed Jonckheere’s test for trend (Jonckheere 1954) with “step-down” will be used on Groups 1 to 4 for a decrease from control. Exact one-tailed Wilcoxon pairwise tests (Wilcoxon 1945), for a decrease from control, will also be carried out on Group 1 (control) versus Groups 2, 3, 4 and 5 (positive control slides).
For incidences of micronucleated polychromatic erythrocytes at 24 hours, an exact one-tailed Linear-by-Linear association test (Cytel 1995) with “step-down” will be used on Groups 1 to 4 for an increase from control. Also, exact one-tailed pairwise Permutation tests (Cytel 1995), for an increase from control, will be carried out on Group 1 (control) versus Groups 2, 3, 4 and 5 (positive control slides). Statistical significance will be declared at the 5% level for all tests.
Quasar (version 1.4) and/or SAS (version 9.1.3) and/or StatXact 3 in-house statistical analysis packages may be used for statistical analysis.
- Evaluation and Interpretation of Results:
Providing that all acceptability criteria are fulfilled, the test article is considered clearly negative if, in all experimental conditions examined:
a) None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control,
b) There is no dose-related increase when evaluated by an appropriate trend test,
c) All results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits),
and d) Bone marrow exposure to the test substance(s) occurred.

Evidence of exposure of the bone marrow to a test substance may include a depression of the polychromatic to normochromatic erythrocyte ratio or measurement of the plasma or blood levels of the test substance. Negative results indicate that, under the test conditions, the test chemical does not produce micronuclei in the polychromatic erythrocytes of the test species.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly positive if:
a) At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control,
b) This increase is dose-related when evaluated with an appropriate trend test,
and c) Any of these results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
When conducting a dose-response analysis, at least three treated dose groups should be analysed. Statistical tests should use the animal as the experimental unit. Positive results in the micronucleus test indicate that a test chemical induces micronuclei, which are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species.
There is no requirement for verification of a clear positive or clear negative response.
In cases where the response is not clearly negative or positive and in order to assist in establishing the biological relevance of a result (e.g. a weak or borderline increase), the data should be evaluated by expert judgement and/or further investigations of the existing experiments completed. In some cases, analysing more cells could be useful.
In rare cases, even after further investigations, the data will preclude making a conclusion that the test chemical produces either positive or negative results, and the study will therefore be concluded as equivocal.
Statistics:
Summary statistics (e.g. means and standard deviations) presented in this report were calculated from computer-stored individual raw data. Group mean values and standard deviations were frequently calculated using a greater number of decimal places than presented in the appendices. It is, therefore, not always possible to derive exact group values from the data presented in the appendices.
For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Key result
Remarks on result:
not determinable
Remarks:
on-going study
Key result
Critical effects observed:
not specified
Executive summary:

In a repeated dose toxicity range-finding study, groups of Crl:CD(SD) rats (5/sex/dose) received test item orally, via the diet at concentrations of 3500, 7500 or 15000 ppm. A similarly constituted control group received the vehicle, basal diet with added corn oil. During the study, clinical condition, body weight, food consumption, oestrous cycles, blood chemistry, organ weight, macropathology investigations as well as a micronucleus test were undertaken.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification