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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:
27 August 2012 to 03 December 2012
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

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
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
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: Pink powder
- Storage conditions: Ambient/dark

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 (initiation of dosing).
- Weight at study initiation: 329 - 379 g (males) and 202 - 258 g (females) at initiation of dosing.
- 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: rat and mouse breeder diet ad libitum
- Water: water from the public supply, ad libitum
- Acclimation period: 13 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23 °C
- Humidity (%): 38 - 74 %
- 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 03 September 2012 to 19 October 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1 % (w/v) Carboxymethylcellulose in Milli-Q water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS
The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4 °C, and dispensed daily. They were prepared by adding an appropriate amount of vehicle to the required amount of test material and were mixed by a high shear mixer and magnetic stirring until a visibly homogenous formulation was obtained. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes prior to dosing and continuously during dosing. All formulations were used within the 8-day stability period that had been previously established.
Dose volume: 10 mL per kg body weight.
Concentration in vehicle: 10, 30 and 100 mg/mL for the 100, 300 and 1000 mg/kg dose levels, respectively.
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 sets of top, middle and bottom samples (ca 0.5 mL) were taken at each sampling time point and analysed by ICP-OES (duplicate middle samples only obtained from control formulations). An additional sample from the top, middle and bottom (middle sample only from control) were also taken as back up samples. The results of the sample concentration were considered acceptable if they 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: Samples were digested in approximately 10 mL of 69 % nitric acid in a microwave. Once digested, the samples were transferred to 50 mL volumetric flasks with washings and made to volume in 2 % nitric acid. The samples were further diluted in 2 % nitric acid to give sample solution concentrations within the range of the calibration standards 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 a microwave in approximately 10 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 dierbium trioxide. 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 dierbium trioxide.

- ICP-OES conditions:
> Instrument: Perkin Elmer Optima 8000 ICP-OES
> Element and wavelength: Er (369.265 nm)
> No. of replicates: 3
> Delay time: 60 seconds
> Read time: set to auto
> Minimum read time: 2 seconds
> Maximum read time: 10 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
> Peristaltic 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: 12 min to 150 °C
Hold: 10 min at 150 °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 dierbium trioxide concentration in mg/mL. The concentration of dierbium trioxide in the test samples was determined by interpolation of this line.

Concentration of dierbium trioxide 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): 0.00000269 mg/mL dierbium trioxide, estimated as 3.3 times the ratio of the standard deviation of the calibration blank to the slope of the calibration curve.
- The limit of quantification (LOQ): 0.00000814 mg/mL dierbium trioxide, estimated as 10 times the ratio of the standard deviation of the calibration blank to the slope of the calibration curve.
Duration of treatment / exposure:
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). Several females were not dosed on their respective days of parturition due to the animals starting to give birth prior to the commencement of dosing that day.
Frequency of treatment:
Animals were dosed once daily.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg/day
Basis:
actual ingested
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 conducted at the testing facility, 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. The cages 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. During pre-trial Animal 66 (Group 3 female) was replaced with spare Animal 83 due to findings in the pre-trial ophthalmic examinations.

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 pre-trial, all animals received a detailed clinical examination including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.
All animals were examined for reaction to treatment daily during the course of the study. The onset, intensity and duration of any signs were recorded.

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.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was measured for both sexes weekly, starting 1 week prior to dosing until pairing for mating.
After pairing, 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 measurement did not recommence after pairing for mating.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-trial, 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 after careful cleaning of the sampling site to avoid any possible contamination.
- 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 cell volume, red blood cell distribution width, mean cell haemoglobin concentration, mean cell 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 and other cells (as appropriate).

COAGULATION: Yes
Samples for coagulation were collected via the tail vein after careful cleaning of the sampling site to avoid any possible contamination.
- 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 and prothrombin time.

CLINICAL CHEMISTRY: Yes
Samples for clinical chemistry were collected via the tail vein after careful cleaning of the sampling site to avoid any possible contamination.
- 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 and phosphate.

DETAILED FUNCTIONAL OBSERVATIONS
- Time schedule: weekly from pre-treatment (week -1).
- Cageside observations included checking for the following: 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.
Condition of the eyes was also assessed, checked for: pupillary function (reaction to visual stimulus), miosis, mydriasis, exophthalmos, encrustation and lacrimation.
Condition of the coat, presence of salivation and overall ease of handling were also assessed.

- Observations in a standardized area (2 min observation) included checking for the following: latency (time to first locomotory movement), level of mobility, rearing, grooming, urination/defecation, arousal (level of alertness), posture, tremor/convulsions, vocalisation, piloerection, palpebral closure, gait abnormalities and 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: reaction to sudden sound (click above the head), reaction to touch on the rump with a blunt probe, grip strength, pain perception, landing foot splay, motor activity and any other physical/functional abnormalities not already recorded in the screening battery.
Sacrifice and pathology:
TERMINAL PROCEDURES
The males were killed when mating was completed and the animals had been dosed for at least 4 weeks (Study Day 30).
The females and litters were killed between Days 5 and 7 of lactation (Study Days 43, 45 and 47).
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.

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 reproductive tracts of all females were examined.

ORGAN WEIGHTS: Yes
The following organs were weighed at necropsy for all adult animals surviving to terminal kill: brain, epididymis, adrenal gland, pituitary gland, prostate gland, thyroid gland, heart, kidney, liver, lung, ovary, spleen, testis, thymus and 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:
Aortic artery, blood smear* (animals sacrificed prematurely only), bone marrow smear# , bone marrow (femur, sternum), bone (femur, rib and sternum), brain, cervix, epididymides, eyes~, adrenal glands, harderian glands~ , lacrimal glands, mammary gland, parathyroid glands, pituitary gland, prostate gland, salivary glands, seminal vesicle gland, thyroid glands, 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, ureters, urinary bladder, uterus and 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 nasal cavity, blood smears and bone marrow smears), were processed to paraffin wax block from selected animals. 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). The testes of all male animals were processed to wax impregnation. This was done in order to maintain tissue integrity as the testes must be removed from fixative and processed to wax impregnation within 72 hrs. A PAS-Haematoxylin stained slide was prepared from the testes and epididymides of the males.
Statistics:
Unless otherwise stated, 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 and food consumption, haematology, coagulation, clinical chemistry and selected 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, i.e. 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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results" for information.
Mortality:
mortality observed, treatment-related
Description (incidence):
See "Details on results" for information.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results" for information.
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:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results" for information.
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 examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Animal 1 (Group 1 male) was killed prematurely on study Day 19 due to adverse clinical observations including enlarged and discoloured hind feet and dragging hind limbs continuously. The death of this animal was not related to treatment.
Treatment at levels up to 1000 mg/kg/day in males produced no clinical observations that were considered related to treatment with test material.
In females dosed at 1000 mg/kg/day there was a slight increase in incidence in several clinical observations such as body hunched, piloerection and walking on tip toes. These clinical observations were observed at this dose level only and were evident from around the time of parturition onwards. These observations are typical of females giving birth however, the increased incidence of these signs indicate that these clinical observations are related to treatment with the test material. In females at levels up to 300 mg/kg/day there were no clinical observations that were considered to be related to treatment with test material.
Clinical results can be seen in Table 2.

BODY WEIGHT AND WEIGHT GAIN
In males dosed up to 1000 mg/kg/day the group mean body weight gains were similar to the control for the duration of the study.
At levels up to 1000 mg/kg/day the group mean body weight gain for females prior to mating and throughout gestation were similar to the control. At 1000 mg/kg/day there was a 36 % reduction in female group mean body weight gain over Days 1-4 of lactation, when compared to the control. At levels up to 300 mg/kg/day the group mean body weight gain during lactation was similar to the control. Bodyweight measurements can be seen in Tables 3 and 4.

FOOD CONSUMPTION
Group mean food consumption in all treated males was similar to the control. During pre-trial the group mean food consumption in the females from treated groups was slightly lower than the control, however, was similar to the control from Day 1 of dosing until pairing for mating. The group mean food consumption for females throughout gestation and lactation was also similar to controls.

OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmoscopy findings which were considered to be related to treatment with the test material. All findings were considered to be typical of rats of the age and strain used.

HAEMATOLOGY AND COAGULATION
In treated males all haematology and coagulation parameters were similar to controls. Any slight intergroup differences were considered incidental and not related to treatment with the test material.
In females at 1000 mg/kg/day slight differences in several haematology parameters were noted when compared to controls. There was an apparent slight reduction in group mean haemoglobin concentration, red blood cell count and haematocrit and an associated increase in reticulocyte count, when compared to the control. There were also slight reductions noted in all white blood cell parameters with the exception of basophils, when compared to controls. These differences were minor and did not achieve statistical significance. Due to the inconsistency in this pattern of effect within the animals dosed at 1000 mg/kg/day and the small degree of change in each parameter when compared to controls, these changes cannot be definitively attributed to treatment. In treated females all coagulation parameters were similar to controls and any slight intergroup differences were considered incidental and not related to treatment with the test material.

CLINICAL CHEMISTRY
In males dosed at 300 mg/kg/day and above there was a dose-related decrease in LDH, when compared to controls. At 300 mg/kg/day the group mean LDH was reduced by 31 % and at 1000 mg/kg/day the group mean LDH was reduced by 36 %, compared to the control. In males, the mean LDH level in the historical control data for animals of this age and strain is 179.3 U/L which is comparable to the control group mean level of 170 U/L. Therefore, due to the clear dose related decrease at 300 mg/kg/day and above this reduction in LDH is considered to be treatment-related.
In females dosed at 1000 mg/kg/day there was a 21 % decrease in group mean LDH, when compared to controls. This did not achieve statistical significance however 3/5 females had levels lower than the control range and the group mean level of 157 U/L shows a 25 % reduction when compared to the historical control data level of 208.3 U/L. Therefore it cannot be discounted that this slight decrease in LDH may be related to treatment. All other clinical chemistry parameters in males and females were similar to controls. Any slight intergroup differences were considered incidental and too small to be attributed to treatment with the test material.
Results can be seen in Table 5.

DETAILED FUNCTIONAL OBSERVATIONS
The type and distribution of neurotoxicity clinical observations in both males and females did not indicate any effect of treatment. In all dose groups, all detailed functional observations in males and females were comparable to controls for the duration of the study. Any slight intergroup differences were considered too small to be attributed to treatment. Slight inter-group differences in motor activity were intermittent and considered incidental, therefore could not be associated with treatment with the test material.

ORGAN WEIGHTS
No test material-related organ weight changes were noted. There were isolated organ weight values that were different from their respective controls. 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/or related to difference of sexual maturity and unrelated to administration of the test material.

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

HISTOPATHOLOGY
No test material-related microscopic findings were noted. The microscopic findings observed were considered incidental and of the nature commonly observed in this strain and age of rat. The incidence and severity of microscopic findings were similar in control and treated animals and, therefore, were considered unrelated to administration of the test material.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No evidence of systemic toxicity observed at the highest dose tested.
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Increased incidence of clinical signs observed at 1000 mg/kg bw/day around the time of parturition and reduced bodyweight gain during days 1-4 of lactation.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Dose formulation Analysis

The results of the analyses of the group 2-4 dosing formulations prepared for use on day 1 of dosing were found to be below the acceptance criteria (% difference from nominal: group 2, -48.3 %, group 3, -53 % and group 4, -11.9 %). An Out Of Specification (OSS) investigation was carried out to try and source any issue/error that may have led to these spurious results. The investigation was inconclusive and so the system was cleaned out and performance checks were conducted on the instrumentation prior to the original samples being re-run along with the back-up samples. The analysis of back-up and original samples showed improved results but many were still out of specification. Following further investigation it was concluded that there was evidence that the analytical samples were not prepared appropriately prior to analysis causing inadequate sample digestion in all of the day 1 samples.

A further analysis time point was added to formulations prepared for use on week 2 to confirm the investigation conclusions and confirm that all formulations are accurate and homogenous. 

Results

The results of the analysis of dosing formulations prepared for use on week 2 and week 4 of dosing were all found to be within the ± 10 % acceptance criteria specified in the analytical method. This indicates an acceptable accuracy of formulation for the duration of the study and the spurious results obtained on day 1 samples were due to an analytical error and not an error in the formulation procedure. The low coefficient of variation (< 5 %) indicated that the formulations were homogenous.

Test solutions were also found to be stable over at least 8 days, when stored at ambient temperature or 2-8 °C in the dark.

Results can be seen in Table 6.

Table 2: Group Incidence of Clinical Observations

Males

Observation/finding

Group / Dose level (mg/kg/day)

1 / 0

2 / 100

3 / 300

4 / 1000

Abnormal coloured/discoloured skin on hind feet

1

0

0

0

Both feet enlarged/markedly enlarged

1

0

0

0

Sparse hair

0

1

0

0

Scab(s)

0

0

0

2

Killed prematurely

1

0

0

0

Females

Observation/finding

Group / Dose level (mg/kg/day)

1 / 0

2 / 100

3 / 300

4 / 1000

Sparse hair/ bald areas

1

2

1

1

Staining on skin

0

0

0

2

Scab(s)

1

0

1

0

Flaky skin on both fore feet

0

1

0

0

Lesion on tail (dry)

0

1

0

0

Body hunched

0

0

0

4

Eyes partially closed

0

0

0

1

Walking on tip toes

0

0

0

2

Piloerection

0

0

0

4

Dark discharge, vagina

0

0

0

1

Pale discoloured skin, extremities

0

0

0

1

 

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

Group/sex

Day

Change

-7

0

7

14

21

28

0-28

1M

291

358

402

430

455

484

126

2M

292

356

396

428

442

472

115

3M

287

349

394

424

444

472

123

4M

286

354

397

427

448

479

126

 

Table 4: Body weights (g): Group Mean Values (Females)

Group/sex

Day (prior to mating)

Change

Day (during gestation)

Change

Day (during lactation)

-7

0

7

14

0 - 14

0

7

14

16

20

0 - 20

1

4

1F

210

240

260

275

35

284

320

362

386

437

153

304

334

2F

204

232

249

267

35

275

316

363

385

431

156

294

322

3F

203

227

248

265

38

271

308

356

376

425

154

296

323

4F

203

234

252

269

35

275

313

360

381

441

166

307

326

 

Table 5: Clinical Chemistry (Group Mean Values)

Group

/sex

ALP

(U/L)

ALT

(U/L)

AST

(U/L)

LDH

(U/L)

CPK

(U/L)

Urea

(mmol/L)

Glu

(mmol/L)

T.Bil

(µmol/L)

Chol

(mmol/L)

TP

(g/L)

Alb

(g/L)

Glob

(g/L)

AG

-R

Na

(mmol/L)

K

(mmol/L)

Cl

(mmol/L)

Phos

(mmol/L)

Ca

(mmol/L)

Crea

(µmol/L)

1M

194

66

83

170

179

6.8

7.84

1.7

1.7

66

42

23

1.8

143

5.1

104

2.39

2.74

30

2M

184

62

81

157

154

6.5

8.14

1.7

1.7

66

42

25

1.8

142

5.0

103

2.33

2.72

27

3M

202

65

84

118*

134*

7.1

8.09

1.7

1.5

67

43

24

1.8

142

5.2

103

2.43

2.74

28

4M

194

66

79

108#

163

7.2

8.19

1.7

1.9

68

43

25

1.7

143

5.1

103

2.48

2.76

30

1F

93

96

82

198

134

9.6

6.09

1.8

2.0

62

41

21

1.9

143

5.6

103

1.80

2.78

40

2F

135

94

85

169

129

9.2

5.85

1.7

2.0

61

40

22

1.9

142

5.4

103

1.63

2.83

39

3F

95

95

86

172

120

9.9

6.65

1.7

2.0

66

41

24

1.7

141

5.4

104

1.29

2.73

41

4F

102

107

88

157

114

9.5

6.19

1.7

2.1

62

40

23

1.7

143

5.4

104

1.47

2.76

35

ALP = Alkaline Phosphatase; ALT= Alanine Aminotransferase; AST = Aspartate Aminotransferase; LDH = Lactate Dehydrogenase; CPK = Creatine Phosphokinase; Glu = Glucose; T.Bil = Total Bilirubin; Chol = Cholesterol; TP = Total Protein; Alb = Albumin; Glob = Globulin; AG-R = Albumin Globulin Ratio; Na= Sodium; K= Potassium; Cl= Chloride; Phos = phosphate; Ca = Calcium; Crea = Creatinine.

* p < 0.01

# p < 0.001

 

Table 6: Formulation Analysis Results

Day 1

Dose

group

Nominal conc.

(mg/mL)

Mean found

(mg/mL)

% Difference

from nominal

1

0

0

n/a

2

10

5.17

-48.3

3

30

14.1

-53.0

4

100

88.1

-11.9

Day 1 (re-run of original samples and back-up samples)

Dose

group

Nominal conc.

(mg/mL)

Mean found

(mg/mL)

% Difference

from nominal

2

10

9.47

-5.3

3

30

18.8

-37.3

4

100

96.6

-3.4

2 (back-up)

10

10.3

3.0

3 (back-up)

30

10.3

-65.7

4 (back-up)

100

88.0

-12.0

Week 2

Dose

group

Nominal conc.

(mg/mL)

Mean found

(mg/mL)

% Difference

from nominal

1

0

0

n/a

2

10

9.78

-2.2

3

30

29.5

-1.7

4

100

103

3.0

Week 4

Dose

group

Nominal conc.

(mg/mL)

Mean found

(mg/mL)

% Difference

from nominal

1

0

0

n/a

2

10

10.1

1.0

3

30

29.6

-1.3

4

100

105

5.0

 

Applicant's summary and conclusion

Conclusions:
In males, treatment with the test material at 300 mg/kg bw/day and above was associated with a dose-related decrease in Lactate Dehydrogenase (LDH). This was the only clinical chemistry parameter affected and there were no other signs of toxicity noted in the animals. Therefore, the significance of the decrease in LDH could not be established.
At 1000 mg/kg bw/day in females, there was an increased incidence of clinical observations around the time of parturition. A 36.7 % decrease in group mean bodyweight gain over Days 1-4 of lactation was also noted along with a slight decrease in LDH, when compared to controls.
Treatment at 100 mg/kg bw/day in both sexes resulted on no significant changes in any of the parameters assessed that were considered to be indicative of a reaction to treatment.
In conclusion, under the conditions of the study, the male systemic No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg bw/day. As demonstrated in the study, the female systemic No Observed Effect Level (NOEL) was considered to be 300 mg/kg bw/day.
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 Sprague-Dawley Crl:CD (SD) strain rats were administered the test material at dose levels of 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day. Another group of 10 male and 10 female rats was dosed with the vehicle (1 % w/v carboxymethylcellulose in Milli-Q water) following the same dosing regimen as the treated animals and was used as control.

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).

In males, treatment with the test material at 300 mg/kg bw/day and above was associated with a dose-related decrease in Lactate Dehydrogenase (LDH). This was the only clinical chemistry parameter affected and there were no other signs of toxicity noted in the animals. Therefore, the significance of the decrease in LDH could not be established.

At 1000 mg/kg bw/day in females, there was an increased incidence of clinical observations around the time of parturition. A 36.7 % decrease in group mean bodyweight gain over Days 1-4 of lactation was also noted along with a slight decrease in LDH, when compared to controls.

Treatment at 100 mg/kg bw/day in both sexes resulted on no significant changes in any of the parameters assessed that were considered to be indicative of a reaction to treatment.

In conclusion, under the conditions of the study, the male systemic No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg bw/day. As demonstrated in the study, the female systemic No Observed Effect Level (NOEL) was considered to be 300 mg/kg bw/day.