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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 November 2012 to 18 February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Appearance: off white powder
- Storage conditions: ambient, protected from light

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8-9 weeks (males); 7-8 weeks (females) - treatment initiation
- Weight at study initiation: 296 - 356 g (males); 187 - 240 g (females) at treatment initiation.
- Housing: Animals were housed in polycarbonate cages with stainless steel grid tops and solid bottoms, with approximate dimensions of 61 x 43.5 x 24 cm. The animals were initially housed 2 or 3 per cage. A few days prior to pairing for mating, males were transferred to individual cages with a stainless steel grid insert. After mating, the males were re-housed with their original cage mates. Mated females were transferred to individual solid bottom cages. White paper tissue was supplied as nesting material from Day 20 of gestation.
- Diet: ad libitum
- Water: tap water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 22 °C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 10 air changes per hour (minimum)
- Photoperiod (hrs dark / hrs light): a 12 hour light/dark cycle was maintained
- Environmental enrichment: Chewing objects and hiding devices were provided to the animals as appropriate for psychological/environmental enrichment.

IN-LIFE DATES: From 26 November 2012 to 11 January 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% (w/v) Carboxymethylcellulose (high viscosity) in Milli-Q water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS
- The dosing formulations were prepared at weekly intervals, stored in a refrigerator set to maintain 4°C and dispensed daily. All formulations were used within the 8 day stability period that had been previously established. The dosing formulations were stirred for at least 30 minutes prior to and throughout dosing.
Any residual volumes were discarded following the completion of dosing each day.
On Day 38 of study, the refrigerator holding the test material was outside of the set range (2-8 °C) for approximately 12 hours, reaching a maximum temperature of 11 °C. As stability of the test material formulations has been established for up to 8 days at ambient temperature, this deviation was considered not to have impacted the integrity of the study.

- Dose volume: 10 mL per kg body weight.

- The volume administered to each animal was determined on each day by the weight of that animal as measured at the time of administration, except during late gestation; from Day 16 of gestation until when parturition was complete, the dose volume was determined by the weight of the animal on Day 16 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From each formulation, duplicate (0.1 mL) samples of top, middle and bottom samples were taken at each sampling time point and sent to the analytical laboratory for analysis (duplicate middle samples only obtained from Control formulations). Additional triplicate samples from the top, middle and bottom (middle sample only from control) were also taken as back-up samples. The results of the sample concentration analyses were considered acceptable if all results were within ± 10 % of theoretical concentration. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentration of ≤ 10 % for each group.

- Preparation of test solutions: approximately 0.1 mL of formulation was weighed into a digestion vessel and digested in approximately 20 mL of 69 % nitric acid. Once digested, the samples were transferred to a 100 mL volumetric flask with washings made to volume in 2 % nitric acid. The samples were further diluted, as necessary, in 2 % nitric acid to give sample solution concentrations within the range of the calibration standard used. Samples and standards were analysed by ICP-OES.

- Preparation of calibration standards: 20 mg of test material was added to a digestion vessel and digested in approximately 20 mL of 69 % nitric acid. Once digested the solution was transferred to a 100 mL volumetric flask with washings and made to volume in 2 % nitric acid to obtain a stock solution with a concentration of 0.200 mg/mL cerium trifluoride. The stock solution was diluted volumetrically in 2 % nitric acid to obtain target concentrations of approximately 0.00100, 0.00200, 0.00400, 0.00600, 0.00800 and 0.0100 mg/mL cerium trifluoride.

- ICP-OES conditions:
> Instrument: Perkin Elmer Optima 8000 ICP-OES
> Elements and wavelength: Ce (418.660 nm)
> No. of replicates: 3
> Delay time: 60 seconds
> Read time: set to auto
> Minimum read time: 1 second
> Maximum read time: 5 seconds
> Points per peak: 7
> Plasma conditions:
Plasma: 8 L/min
Auxiliary: 0.2 L/min
Nebuliser: 0.7 L/min
RF power: 1300 Watts
Plasma viewing mode: radial
> Peristatic pump sample flow rate: 1.50 mL/min
> Wash:
Frequency: between samples
Flow rate: 1.50 mL/min
Normal time: 30 seconds
Wash solvent: Milli-Q water
> Diluent: 2 % nitric acid
> Microwave digestion:
Ramp: 15 min to 180 °C
Hold: 15 min at 180 °C
Cool down: 30 min

- Calculations: a calibration curve was constructed by plotting the mean peak areas of the standards (including the calibration blank) against the relevant cerium trifluoride concentration in mg/mL. The concentration of cerium trifluoride in the test samples was determined by interpolation of this line.

Concentration of cerium fluoride in formulations (mg/mL) = (C x D x P) / W

Where
C = concentration of the sample solution obtained by interpolation from the calibration line (mg/mL)
D = dilution factor
P = density of the formulation (g/mL)
W = weight of aliquot (g)

- The limit of detection (LOD) estimated from an analyte response at the level of 3.3 times the ratio of the standard deviation of the blank to the slope of the calibration curve, was estimated to be 0.00000633 mg/mL cerium trifluoride.

- The limit of quantification (LOQ) estimated from an analyte response at the level of 10 times the ratio of the standard deviation of the blank to the slope of the calibration curve, was estimated to be 0.0000192 mg/mL cerium trifluoride.
Duration of treatment / exposure:
The males were dosed daily for 4 weeks, starting 2 weeks prior to mating. The females were dosed daily 2 weeks prior to mating, throughout mating and through to at least Day 4 of lactation, with the following exceptions:
Animals 60 (100 mg/kg/day) and 78 (1000 mg/kg/day) were not dosed on Day 0 of lactation due to still being in the processing of littering. Dosing recommenced on Day 1 of lactation for these animals.
Animal 50 (0 mg/kg/day) was not dosed on Day 0 of lactation due to still being in the processing of littering; this animal was found dead later the same day.
Frequency of treatment:
Animals were dosed once daily.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
other: nominal conc.
No. of animals per sex per dose:
10 males and 10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected after a review of existing relevant toxicological data. Including a separate 14-day dose range finding study, where dose levels up to 1000 mg/kg/day produced no adverse reaction to treatment.
- Animal assignment: On arrival from the suppliers, the animals were housed in cages which were suspended on a series of racks. Male and female cages were racked separately. Cages were allocated to treatment group by the use of randomly sequenced numbers in such a way that each complete rack contained representatives from all treatment groups.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked for viability early morning and as late as possible each day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week starting in pretrial, all animals received a detailed clinical examination.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded one week prior to the start of treatment. From the start of treatment, the individual body weights were recorded daily.
* Animals 50 (0 mg/kg/day), 60 (100 mg/kg/day) and 78 (1000 mg/kg/day) were not weighed on Day 0 of lactation due to still being in the processing of littering.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was measured for both sexes weekly, starting 1 week prior to dosing until pairing for mating.
After mating, the female food consumption was measured over Days 0-7, 7-14 and 14-20 of gestation and Days 0-4 of lactation. Male food consumption did not recommence after pairing for mating.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretrial, week -1 (all animals); week 4 (all males); shortly prior to sacrifice (all females).
- The eyes were examined using an indirect ophthalmoscope after the application of a mydriatic agent (1 % Tropicamide, Mydriacyl®). The following areas were evaluated: anterior, lenticular and fundic areas.

HAEMATOLOGY: Yes. Samples for haematology were collected via the tail vein. Blood was then transferred into plastic tubes containing anticoagulant.
- Time schedule for collection of blood: week 4 (males); ca day 4 of lactation (females).
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: first 5 animals per group (males); first 5 animals per group which reared their litter to day 3 of lactation (females).
- Approximately 0.5 mL of blood was taken into tubes containing EDTA and analysed for the following: Red blood cell count, Haemoglobin concentration, Haematocrit, Mean corpuscular volume, Red blood cell distribution width, Mean corpuscular haemoglobin concentration, Mean corpuscular haemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unclassified cells.

COAGULATION
Samples for coagulation were collected via the tail vein. Blood was then transferred into plastic tubes containing anticoagulant.
- Time schedule for collection of blood: week 4 (males); ca day 4 of lactation (females)
- Animals fasted: No
- How many animals: first 5 animals per group (males); first 5 animals per group which reared their litter to day 3 of lactation (females)
- Blood samples (0.9 mL) were taken into tubes containing 0.1 mL trisodium citrate (3.8% (w/v)). The final sample volume was as close as possible to 1.0 mL to give a final concentration of 0.38 % (blood to citrate ratio of 9:1). The citrated blood samples were centrifuged and the plasma separated into plastic tubes and analysed for the following: Activated partial thromboplastin time, Fibrinogen, Prothrombin time.

CLINICAL CHEMISTRY: Yes. Samples for clinical chemistry were collected via the tail vein. Blood was then transferred into plastic tubes containing anticoagulant.
- Time schedule for collection of blood: week 4 (males); ca day 4 of lactation (females)
- Animals fasted: No
- How many animals: first 5 animals per group (males); first 5 animals per group which reared their litter to day 3 of lactation (females)
- Blood samples (1.0 mL) were taken into tubes containing lithium heparin which were then centrifuged and the plasma was analysed for the following: Urea, Glucose, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatise, Creatine phosphokinase, Lactate dehydrogenase, Sodium, Potassium, Chloride, Total protein, Albumin, Globulin, Albumin/globulin ratio, Cholesterol, Creatinine, Total bilirubin, Calcium, Phosphate.

DETAILED FUNCTIONAL OBSERVATIONS
- Time schedule: weekly from pre-treatment (week -1)
- Cageside observations included: Prostration, Lethargy, Writhing, Circling, Breathing abnormalities, Gait abnormalities, Tremor, Fasciculation, Convulsions, Biting (of cage components or self mutilating), Vocalisations, Piloerection and Ease of removal from the cage.
Body temperature was taken directly from an implanted electronic chip and recorded. In the event the microchip produced a value that was considered spurious a rectal temperature was used to check the value; in these instances the rectal temperature has been reported.
Condition of the eyes, checked for:
Pupillary function
Miosis
Mydriasis
Exophthalmos
Encrustation
Lacrimation.
Condition of the coat, presence of salivation and overall ease of handling were also assessed.

- Observations in a standardized area (2 min observation):
Latency (time to first locomotory movement)
Level of mobility
Rearing
Grooming
Urination/defecation
Arousal (level of alertness)
Posture, tremor/convulsions, vocalisation, piloerection – recorded as for cageside observations
Palpebral closure
Gait abnormalities
Stereotypy (excessive repetition of behaviours) and/or unusual behaviours.

- Functional Tests (Full Examination)
The following additional functional assessments were performed on 5 males per group during week 4 and on 5 females per group during lactation (the first 5 females per group to rear their litter to day 3 of lactation). These assessments were performed at an approximately standardised time of day and prior to blood sampling.
> Reaction to sudden sound (click above the head).
> Reaction to touch on the rump with a blunt probe.

> Grip strength:
A strain gauge was used, to which is attached a wire pull-mesh. Once the animal had gripped the mesh, the body was pulled until its grasp was broken; the strain gauge recorded the force required. The procedure was repeated 3 times for the forelimbs and 3 times for the hindlimbs, and the mean fore and hind grip strengths calculated.

> Pain perception:
This was assessed by measurement of the tail flick response. The apparatus used shone a calibrated infra-red heat source onto the tail and automatically measured the reaction time of the animal (accurate to 0.1 s). It was ensured that no visible injury to the tail was caused during this test.

> Landing Foot Splay:
Corn oil was applied to the hind paws of each animal. The animal was then held in a horizontal, prone position with the nose ca 30 cm above a bench surface covered with absorbent paper. When the animal was calm, it was dropped. The distance between the prints of the central footpads was measured and the average measurement recorded. The procedure was repeated 3 times.

> Motor activity:
Each animal was placed in an individual monitoring cage, scanned by a motion sensor utilising infra-red pyroelectric detectors. Movement was detected in 3 dimensions anywhere in the cage, and was differentiated into large and small movements. Each animal was monitored for one session of 1 h for males and 0.5 h for females. Activity counts were recorded over successive periods of 5 min each.

> Other physical/functional abnormalities:
Any other abnormality not already recorded in the above screening battery.
Sacrifice and pathology:
The males were killed when mating was completed and the animals had been dosed for at least 4 weeks.
The females and litters were killed between Day 5 and 7 of lactation.
Animals 10 days of age or more were killed by exposure to carbon dioxide followed by exsanguination. Animals less than 10 days of age were killed by intra-peritoneal injection of sodium pentobarbitone.

UNSCHEDULED DEATHS
Premature decedents were necropsied with a view to diagnosis of the cause of the animal’s condition or cause of death. An external examination was followed by inspection of the cranial, thoracic and abdominal contents, and the tissues (as specified for histology, below) were collected.

GROSS PATHOLOGY: Yes
All adult animals surviving to scheduled euthanasia were subjected to a complete necropsy examination which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
* The left adrenal gland from Male 33 (1000 mg/kg/day) was lost at necropsy in error. Given that there were no test material related differences in adrenal weights, or macro/microscopic pathology findings (indeed there were no findings in the right adrenal gland from this animal itself), the loss of this one adrenal gland from this single animal was considered not to have impacted the integrity of the study.

ORGAN WEIGHTS: Yes
The following organs were weighed at necropsy for all adult animals surviving to terminal kill: Brain, Epididymides, Adrenal glands, Pituitary gland, Prostate gland, Thyroid glands, Heart, Kidneys, Liver, Lung, Ovaries, Spleen, Testes, Thymus, Uterus.

HISTOPATHOLOGY: Yes
Representative samples of the following tissues were collected from all adult animals and preserved in 10 % neutral buffered formalin, unless otherwise indicated:
Aorta, Blood smear* (Animals sacrificed prematurely only), Bone marrow smear# , Bone marrow (femur, sternum), Femur, Rib, Sternum, Brain, Cervix, Epididymides, Eyes~, Adrenals, Harderian glands~ , Lacrimal glands, Mammary gland, Parathyroid glands, Pituitary gland, Prostate gland, Salivary glands, Seminal vesicle, Thyroids, Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidneys, Large intestine (caecum, colon, rectum), Larynx, Liver, Lung, Mandibular lymph node, Mesenteric lymph node, Skeletal muscle, Nasal cavity, Optic nerve~, Sciatic nerve, Oesophagus, Ovaries, Oviducts, Pancreas, Pharynx, Skin, Small intestine (duodenum, ileum, jejunum), Spinal cord, Spleen, Stomach, Testes+, Thymus, Tongue, Trachea, Ureter x2, Urinary bladder, Uterus, Vagina.
* Air dried
# Fixed in methanol and then air dried
~ Preserved in Davidson’s fixative
+ Preserved in Modified Davidson’s fixative

- Bone Marrow Smears
Duplicate bone marrow smears were taken at necropsy from all adult animals. Both bone marrow smears were stained using May-Grunwald-Giemsa as the Romanowsky stain. Bone marrow smears were not evaluated.

- Histopathology
The tissues, as listed above (except animal identification, nasal cavity, blood smears and bone marrow smears), were processed to paraffin wax block from selected animals. The testes of all male animals were processed to wax impregnation. Sections were cut 4-6 µm thick, stained with haematoxylin and eosin from 5 males and 5 females in the Control and High dose groups (the same animals that were used for laboratory investigations). An additional PAS-Haematoxylin stained slide was prepared from the testes and epididymides of the selected males.
Statistics:
All statistical tests were two-sided and performed at the 5 % significance level using in house software. Pairwise comparisons were only performed against the control group.
Selected body weight, food consumption, haematology, coagulation, clinical chemistry and selected functional observation battery (FOB) and motor activity data was analysed for homogeneity of variance using the ‘F Max' test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student's t test ie pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student's t test).
Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate. In addition, organ weights as a percentage of terminal body weight were also analysed using ANOVA as an exploratory analysis.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
no treatment-related effects
Mortality:
no mortality observed
Description (incidence):
no treatment-related effects
Body weight and weight changes:
no effects observed
Description (incidence and severity):
no treatment-related effects
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
no treatment-related effects
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
no treatment-related effects
Haematological findings:
no effects observed
Description (incidence and severity):
no treatment-related effects
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
no treatment-related effects
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
no treatment-related effects
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
no treatment-related effects
Gross pathological findings:
no effects observed
Description (incidence and severity):
no treatment-related effects
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
no treatment-related effects
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
Animal 50 (0 mg/kg/day) was found dead on Day 0 of lactation. The animal had given birth to 16 live and 1 dead pups prior to being found dead, but had displayed no previous clinical signs. There were no necropsy findings in this animal. The reason(s) for death are not known, but as this was a Control animal it did not reflect an effect of treatment.
There were no clinical signs during the course of the study that were considered to be related to administration of test material.

BODY WEIGHT AND WEIGHT GAIN
Body weight gains were similar between Control animals and animals receiving test material.

FOOD CONSUMPTION
Food consumption was similar between Control animals and animals receiving test material.

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmic findings that were considered to be related to administration of test material.

HAEMATOLOGY
There were no differences in haematological or coagulation parameters in Control animals and animals receiving test material that were considered to be related to the test material.

CLINICAL CHEMISTRY
Mean LDH and CPK activities were lower than Controls in males at 1000 mg/kg/day (31 % and 47 % of Control mean, respectively), however given that intra-group variation was notably high and that the activities at 1000 mg/kg/day were within the historical background control range, these findings were considered incidental.
Mean glucose levels were higher than Controls in males at 300 and 1000 mg/kg/day (16 % and 22 % higher, respectively). The mean value at 300 mg/kg/day was within the historical background control range, whilst the mean value at 1000 mg/kg/day was very slightly above the background range; however it should be noted that Animal 34 at 1000 mg/kg/day had a glucose level notable higher than the other animals at this dose level. The changes in glucose levels were therefore were considered incidental.
All other apparent differences in clinical chemistry parameters were considered to be incidental due to large intra-group variation and/or the absence of a clear dose response relationship.

NEUROBEHAVIOUR
There were no neurotoxicity clinical observations during the course of the study that were considered to be related to administration of test material.
Detailed functional observations in animals receiving test material were similar to Control animals throughout the course of the study.
Motor activity values in animals receiving test material were similar to Control animals. Any slight intergroup differences were considered to be too minor and/or transient to be attributed to the test material.

ORGAN WEIGHTS
No test material-related organ weight changes were noted. There were isolate organ weight values that were different from Control. There were, however, no patterns, trends or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and unrelated to administration of test material.
It was noted that thyroid weights were higher than Control in males at 1000 mg/kg/day, however this was considered to be a consequence of a low mean value in Controls, as the mean value at 1000 mg/kg/day was within the historical background control range for absolute thyroid weights.

GROSS PATHOLOGY
No test material-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat on this type of study, and/or were of similar incidence in Control and treated animals and, therefore, were considered unrelated to administration of test material.

HISTOPATHOLOGY
A higher incidence of minimal chronic progressive nephropathy was recorded in males receiving 1000 mg/kg/day (2/5) relative to Controls (0/5). This is a progressive change that occurs spontaneously in rats at a higher incidence in males. The lesions in these animals were small, focal and with evidence of chronicity, and therefore were considered unrelated to administration of test material.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in Control and treated animals and, therefore, were considered unrelated to administration of test material.

Effect levels

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 2: Body weights (g): Group Mean Values (Males)

Group/sex

Day

Change

-7

0

7

14

21

28

0-28

1M

259

323

363

402

426

447

124

2M

259

319

356

394

413

433

114

3M

261

321

361

395

414

434

113

4M

266

332

379

417

444

467

135

Table 3: Body weights (g): Group Mean Values (Females)

Group/sex

Day (prior to mating)

Change

Day (during gestation)

Change

Day of lactation*

-7

0

7

14

0 - 14

0

7

14

16

20

0 - 20

1

4

1F

191

217

233

247

31

249

285

327

346

389

140

280

301

2F

189

214

228

241

27

250

287

327

345

391

141

277

303

3F

188

216

232

248

32

253

293

332

351

396

143

278

302

4F

184

206

222

239

32

242

277

319

340

378

136

266

293

* animals with litters surviving to Day 4 or after only

Applicant's summary and conclusion

Conclusions:
Treatment with the test material at levels up to 1000 mg/kg/day was not associated with any changes in clinical observations, body weight gain, food consumption, ophthalmoscopy findings, neurotoxicity findings, clinical pathology parameters (haematology, coagulation or clinical chemistry), organ weights, gross necropsy findings or histopathogical findings.
In conclusion, under the conditions of this study the No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day, for both males and females.
Executive summary:

The oral repeat dose toxicity of the test material was determined in a GLP study which was conducted in accordance with the standardised guideline OECD 422. The study also placed an emphasis on neurological effects as a specific endpoint to identify any neurotoxic potential of the test material. During the study groups of 10 male and 10 female rats were administered test material at dose levels of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day. The males were treated for 2 weeks prior to mating, then through mating, until the day prior to necropsy (ca 4 weeks of treatment). Females were treated for 2 weeks prior to mating, then through mating, gestation and until at least Day 4 of lactation (ca 6 weeks of treatment).

The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, ophthalmology, detailed functional tests and observations, clinical pathology parameters (haematology, coagulation and clinical chemistry), organ weights, and gross and microscopic pathological examinations.

There were no signs of toxicity noted in any of the parameters, or endpoints, that were evaluated. Therefore, under the conditions of this study the No Observed Effect Level (NOEL) was considered to be 1000 mg/kg/day, for both males and females.