Iodomethane

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 January 2002 to 19 August 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Version / remarks:

1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3150 (90-Day Oral Toxicity in Non-rodents)

Version / remarks:

1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 NouSan No. 8147

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Iodomethane

EC Number:

200-819-5

EC Name:

Iodomethane

Cas Number:

74-88-4

Molecular formula:

CH3I

IUPAC Name:

iodomethane

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Iodomethane
- Physical state: liquid
- Analytical purity: 99.7%
- Impurities (identity and concentrations): 0.2 % water and <0.1 % methanol
- Composition of test material, percentage of components: not stated
- Isomers composition: not stated

Test animals

Species:

dog

Strain:

Beagle

Details on species / strain selection:

The animal model, the beagle dog, is recognised as appropriate for toxicity studies and is a widely used breed for which significant historical control data are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 months
- Weight at study initiation: males: 9.4 to 10.9 kg and females: 6.2 to 7.7 kg.
- Fasting period before study: no
- Housing: The animals were housed individually in clean, stainless steel cages that were cleaned
daily during the acclimation period and throughout the study. The animals were allowed regular opportunity for exercise and social interaction.
- Diet: Approximately 400 g was offered once daily, replenished approximately three hours after dose administration. During the study, several animals were supplemented with additional food to stimulate appetite.
- Water: ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.9 to 20.8 °C
- Humidity: 33.5 to 52.6 %
- Photoperiod: Light timers were set to provide a 12-hour light (6 a.m. to 6 p.m.)/12-hour dark photoperiod.

Administration / exposure

Route of administration:

oral: capsule

Details on route of administration:

The selected route of administration was oral (capsule) since this is the standard for assessment of toxicity in beagle dogs.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

CAPSULE PREPARATION
- For the control group, the appropriate number of size 13 capsules were filled (in the animal room) with the appropriate amount of corn oil using a syringe (without a needle).

- The test material was formulated in corn oil (v/v) based on the specific gravity of 2.28 g/mL. The appropriate amount of vehicle was dispensed into a 30 mL amber jar, using the maximum volume feasible for this size vial to minimize headspace. The appropriate amount of the test material was added by injection, using a syringe (without a needle), below the surface of the vehicle. The vials were immediately capped and gently inverted to ensure mixing. The time of completion was recorded. The appropriate number of size 13 capsules were dispensed to be filled in the animal room.
- In the animal room, 1-cc or 3-cc syringes were filled with the appropriate amount of the test material formulation. All syringes were filled without using needles, and all were filled before the first capsule was filled for each group. As soon as a capsule was filled, it was immediately administered to the dog. This approach minimized the possibility of evaporation of the test material from the corn oil solution.

-The test material formulations were prepared daily throughout the study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

COLLECTION OF SAMPLES
- Samples for dose concentration confirmation were collected once during study weeks 0, 1, 3, 7 and 12. Additional samples were collected during study week 1 due to analytical results that were outside the acceptable range in all test material groups. On the days when analysis was scheduled, an extra vial of each concentration was prepared and used for analysis, instead of sampling from the vial intended for dosing, so that head space that might affect the concentration would not be introduced into the dosing formulations used in the animal room.
- The test material formulations were found to contain the amounts of test material specified in the protocol, with the exception of the formulations prepared on February 13, 2002 (study week 1). The 1.5, 6.0 and 15 mg/kg/day formulations were 40.2, 64.7 and 150 %, respectively, of target on that day.

GAS CHROMATOGRAPHY

Instrument: Hewlett Packard 5890A (Series II) gas chromatograph equipped with an FID detector, a
HP Headspace analyzer and chemstation
Column: J & W Scientific GS-GasPro, 30 m x 0.316 mm ID (0.25 μm film thickness)
Temperature (Program): 7 0°C for 1.0 minutes, ramp at 40 °C/minute to 230 °C, hold 2 minutes
Carrier gas: Helium set at 12psi at 70 °C (EPC constant flow on)
Injector temperature: 225 °C
Injection volume: 1 mL splitless
Detector: FID at 225 °C
Retention time: Approximately 4.5 minutes

HEADSPACE PARAMETERS
Zone temperature Oven 50°C
Loop 100°C
Transfer Line 100°C
Event Times GC cycle time 10 minutes
Vial EQ time 7 minutes
Pressurization time 0.20 minutes
Loop fill time 0.20 minutes
Loop EQ time 0.05 minutes
Inject time 0.20 minutes

PREPARATION OF CALIBRATION STOCK SOLUTIONS: The calibration stock solutions were prepared by transferring (under the surface of the oil) the appropriate amount of the test material into approximately 7 mL of corn oil in a 10 mL volumetric flask. The contents were brought to volume with corn oil and thoroughly mixed by gentle inversion. These stocks were prepared in the nominal concentration range of 10 to 200 mg/mL.

PREPARATION OF QUALITY CONTROL SAMPLES: The quality control (QC) stock solutions were prepared as above. These stocks were prepared in the nominal concentrations of 15 and 150 mg/mL.

SAMPLE PROCESSING: Calibration, QC and formulation samples were prepared for analysis by transferring 30 μL of the sample into a headspace vial containing 970 μL of corn oil and thoroughly
mixing the contents by gentle inversion.

CONCENTRATION QUANTITATION
A calibration curve was constructed for each set of analyses. The test material peak area (y) and the theoretical concentrations of the calibration standards (x) were fit with a least squares regression analysis to the ln-quadratic function: ln(y) = a × [ln(x)]^2 + b × ln(x) + c
Concentrations were back-calculated from the results of the regression analysis using a PC spreadsheet program (Microsoft® Excel). The concentration data were transferred to another Excel spreadsheet, where appropriate summary statistics, i.e., means, standard deviations (SD), relative standard deviations (RSD), and percent relative error (%RE) were calculated and presented in tabular form.

RESULTS AND DISCUSSION: Under the described chromatographic conditions, the retention time of the test material was approximately 4.5 minutes. The total analysis time required for each run was approximately 7 minutes. The analysed concentrations were within 15 % of the target dose concentrations, except those prepared on 2/13/2002 (all groups), which did not meet the requirements for concentration acceptability mentioned above. The initial analysis of the formulation made on 2/6/02 was technically a valid run (2/3 of the QC samples within specification). However, the data suggests an instrument malfunction resulting in decreased response from injection #21-#29. These formulation samples were reprocessed and analysed on 2/7/02, resulting in all concentrations within acceptable limits. Results can be seen in Table 1.

Duration of treatment / exposure:

7 days per week for a minimum of 90 days

Frequency of treatment:

Approximately the same time every day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

6 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

15 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected based on results from a prior dose range-finding study, which demonstrated intolerance at 30 mg/kg/day, but tolerance at 15 mg/kg/day.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- The animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. All animals were also observed prior to dose administration and approximately two hours (± 30 minutes) following dose administration (designated as 2-hours post-dosing for report presentation purposes). All significant findings were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Detailed physical examinations were conducted on all animals weekly, beginning one week prior to test material administration and prior to the scheduled necropsy.

BODY WEIGHT: Yes
- Individual body weights were recorded weekly, beginning approximately one week prior to test material administration (study week -1). Mean body weights were calculated and mean body weight changes were calculated for each corresponding interval. Final body weights (non-fasted) were recorded prior to the scheduled necropsy.

FOOD CONSUMPTION:
- Individual food consumption was recorded daily, beginning approximately one week prior to test material administration (study week -1) and the weekly averages reported for the corresponding body weight intervals. Food intake was calculated as g/animal/day.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Ocular examinations were conducted on all animals prior to the initiation of dose administration (study week -1) and at the end of the treatment period (study week 13). All ocular examinations were conducted using an indirect ophthalmoscope and a slit lamp bio microscope, preceded by pupillary dilation with an appropriate mydriatic agent.

CLINICAL PATHOLOGY
- Blood and urine samples for clinical pathology evaluations (haematology, serum chemistry and urinalysis) were collected from all dogs prior to the initiation of dose administration (study week -1), during study week 6 and during the last week of dosing (study week 12).
- Samples for haematology were collected into tubes containing potassium EDTA as the anticoagulant. Samples for coagulation parameters were collected into tubes containing sodium citrate as the anticoagulant. Samples for serum chemistry were collected without anticoagulants. The animals were fasted overnight prior to blood collection while in metabolism cages for urine collection. Blood was taken from the jugular vein.

HAEMATOLOGY:
- Parameters evaluated: Total Leukocyte Count (White Cell), Erythrocyte Count (Red Cells), Haemoglobin, Haematocrit, Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular Haemoglobin Concentration (MCHC), Platelet Count (Platelet), Prothrombin Time (Pro Time), Activated Partial Thromboplastin Time (APTT), Differential Leukocyte Count -Percent and Absolute, -Neutrophil, -Lymphocyte, -Monocyte, -Eosinophil, -Basophil, Platelet Estimate and Red Cell Morphology (RBC Morphology).

CLINICAL CHEMISTRY:
- Parameters evaluated: Albumin, Total Protein, Globulin, Albumin/Globulin Ratio (A/G Ratio), Total Bilirubin (Total Bili), Urea Nitrogen, Creatinine, Alkaline Phosphatase (Alkaline Phos’tse), Alanine Aminotransferase (Alanine Transfer), Aspartate Aminotransferase (Aspartat Transfer), Gamma Glutamyltransferase (Glutamyl Transfer), Glucose, Total Cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium and Triglycerides (Triglyceride).
- Serum hormones: Thyroid Stimulating Hormone (TSH), Triiodothyronine (T3) and Thyroxide (T4).

URINALYSIS:
- Parameters evaluated: Specific Gravity (SG), pH, Urobilinogen (URO), Total Volume (TVOL), Colour (CLOR), Appearance (APP), Protein (PRO), Glucose (GLU), Ketones (KET), Bilirubin (BIL), Occult Blood (BLD), Leukocytes (LEU), Nitrites (NIT) and Microscopy of Sediment.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- A complete necropsy was conducted on all animals. Animals were euthanised by an intravenous injection of sodium pentobarbital followed by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal and pelvic cavities including contents. The following tissues and organs were collected and placed in 10 % neutral buffered formalin (except as noted):
Adrenal glands (2), Aorta, Bone with marrow- Femur and Sternum, Bone marrow smear (not placed in formalin), Brain (Cerbrum level 1, cerebrum level 2, cerebellum with medulla/pons), Epididymides (2, fixed in Bouin’s solution), Eyes with optic nerve (2, fixed in Davidson’s solution), Gallbladder, Gastrointestinal tract (Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon and Rectum), Heart, Kidneys (2), Larynx, Liver (sections of two lobes), Lungs (including bronchi, fixed by inflation with fixative), Lymph nodes- Mandibular and Mesenteric, Nose, Ovaries (2), Pancreas, Peripheral nerve (sciatic), Pharynx, Pituitary, Prostate, Salivary glands [mandibular (2)], Skeletal muscle (rectus femoris), Skin (with mammary gland), Spinal cord (cervical, midthoracic, lumbar), Spleen, Testes (2, fixed in Bouin’s solution), Thymus, Thyroid/parathyroids (2), Trachea, Urinary bladder, Uterus with cervix, Vagina and Gross lesions (when possible).
- The following organs were weighed from all animals at the scheduled necropsy: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver with gallbladder, Ovaries, Spleen, Testes, Thymus,Thyroid with parathyroids and Uterus. Paired organs were weighed together. Organ to final body weight and organ to brain weight ratios were calculated.

HISTOPATHOLOGY: Yes
- After fixation, protocol-specified tissues were trimmed according to standard operating procedures and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned at four to eight microns, mounted on glass microscope slides and stained with haematoxylin and eosin. Microscopic examination was performed on all tissues collected, from all animals, with the following exception. The mammary gland was not examined from the females in Groups 2 and 3. Missing tissues were identified as missing, not found at trimming, not found after recut, not in plane of section or other reasons as appropriate.

Statistics:

- All statistical tests were performed using appropriate computing devices or programs.
- Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1 and 5 %, comparing each test material-treated group to the control group by sex. Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Statistical analyses were not conducted if the number of animals was two or less. Due to the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ by ± 1 in the last figure.
- Body weight, body weight change, food consumption, clinical pathology and organ weight data were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistical significance (p<0.05), Dunnett's test was used to compare the test material-treated groups to the control group. Clinical pathology values for white blood cell types that occur at a low incidence (i.e., monocytes, eosinophils and basophils) were not subjected to statistical analysis.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related clinical findings were observed in the 6.0 and 15 mg/kg/day group males and females. Emesis was observed in the 6.0 and 15 mg/kg/day groups. The incidence of abnormal excreta was increased in the 15 mg/kg/day group. In addition, injected sclera occurred with greater frequency than in control group animals in the 1.5, 6.0 and 15 mg/kg/day group animals. This finding represents dilatation of the blood vessels in the eye, was most likely pharmacological and was not considered toxicologically significant.
- Test material-related clinical observations in the 15 mg/kg/day group consisted of emesis and possibly injected sclera. The incidence of abnormal excreta (soft faeces and mucoid faeces) was increased in this group compared to that in the control group. At the time of dosing, increased salivation and head shaking were observed. In the 6.0 mg/kg/day group, test material-related clinical findings consisted of emesis, injected sclera and salivation.
Emesis occurred frequently throughout the study; however, the severity of emesis gradually improved beginning approximately two weeks after initiation of dosing, indicating some acclimation to an apparent gastrointestinal irritation caused by the test material. Individual animals (three animals in the 6.0 mg/kg/day group and two animals in the 15 mg/kg/day group) were also affected by periods of emesis, accompanied by weight loss and decreased food consumption.
- Injected sclera was observed in the 1.5 mg/kg/day group animals. This finding was considered test material-related, but not toxicologically significant. No other test material-related clinical findings were observed.

Mortality:

mortality observed, treatment-related

Description (incidence):

- Test material-related moribundity was observed in one male in the 15 mg/kg/day group. One male in the 15 mg/kg/day group was euthanised in extremis on study day 48. Clinical observations prior to euthanasia included emaciation, hypo activity, mucoid faeces, emesis, dehydration and limited food consumption. The moribund condition of this animal was attributed to the test material. All other animals survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test material-related effects on mean body weights.
- Body weight losses were noted for individual animals during periods of frequent emesis, decreased food consumption and hypoactivity.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test material-related effects on mean food consumption values. Decreased food consumption was noted for individual animals during periods of frequent emesis, body weight loss and/or hypoactivity.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

- There were no test material-related ophthalmic findings.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test material-related effects on haematology parameters. However, several statistically significant (p < 0.05 or p < 0.01) differences from the control group values were observed at the study week 6 evaluation. Higher mean platelet counts were observed in the 15 mg/kg/day group males and higher white blood cell counts were noted in the 6.0 mg/kg/day group females. Since similar effects were not noted in the other sex and no dose-related trends were evident, they were not considered to be test material-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related decreases in mean albumin and/or total protein levels were observed in the 15 mg/kg/day group males and females for study weeks 6 and 12. Lower mean albumin and total protein levels in the 6.0 mg/kg/day group females at study week 12 may have been related to treatment.
- Mean albumin levels in the 15 mg/kg/day group males and females were lower than the control group values at the study week 6 and 12 evaluations. The changes in this group were accompanied by lower mean total protein levels for the males and females at the study week 12 evaluation and for the females at the study week 6 evaluation. The differences from the control group values were statistically significant (p < 0.05 or p < 0.01).
- Other statistically significant (p < 0.05 or p < 0.01) differences were observed, but were not considered to be treatment-related due to the lack of a dose response or temporal-related trends or similar effects in the opposite sex. These changes included lower mean alanine aminotransferase and calcium levels (study weeks 6 and 12, respectively) in the 15 mg/kg/day group males and slightly higher sodium levels (study week 12) for the 6.0 mg/kg/day group males. Mean albumin and total protein levels in the 6 mg/kg/day group females were also significantly (p < 0.05 or p < 0.01) lower than the control group values at the study week 12 evaluation. However, they were not test material-related since the values were comparable to the pre-test levels, while the control group values increased slightly.
- There were no test material-related effects on the serum hormones (TSH, T3 and T4) evaluated in this study.

Urinalysis findings:

no effects observed

Description (incidence and severity):

- There were no test material-related effects on urinalysis parameters.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test material-related effects on organ weights. Statistically significant differences (p < 0.05 or p < 0.01) from the control group were observed, but were not considered test material-related due to lack of dose-related trends, similar differences in the opposite sex and/or microscopic correlates. These differences consisted of reduced mean absolute and relative (to brain weight) heart weight, decreased mean relative (to brain weight) kidney and epididymides weights in the 6.0 mg/kg/day group males and increased mean relative (to brain weight) liver/gallbladder weight in the 15 mg/kg/day group females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related macroscopic findings were observed in the 15 mg/kg/day group male that was euthanized in extremis on study day 48. Eroded areas in the stomach, dark red areas in the entire length of the intestinal tract and red discoloration of the cortico-medullary junction of the kidneys were noted.
- This animal also had a small prostate gland, small testes and enlarged, dark red mediastinal lymph nodes; these findings were not considered test material-related.
- No test material-related macroscopic findings were observed at the scheduled necropsy.
- Findings in the test material-treated groups occurred similarly in the control group, were not dose-related, occurred infrequently and/or are common findings in laboratory dogs.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related microscopic findings were observed in the stomach and/or oesophagus in the 15 mg/kg/day group male that was euthanised in extremis and at the scheduled necropsy in one 6.0 mg/kg/day group male and one 15 mg/kg/day group male and female. Possible test material-related changes in the olfactory epithelium were noted in the 6.0 and 15 mg/kg/day group females.
- In the 15 mg/kg/day group male that was euthanised in extremis, test material-related findings included chronic inflammation in the stomach, correlating to eroded areas noted macroscopically, and haemorrhage in the cecum and rectum, correlating to dark red areas. The rectum also had chronic inflammation. Other affected areas of the intestinal tract (duodenum, jejunum, ileum and colon) did not have distinctive histological findings correlating to dark red areas, but congestion near or slightly above background levels may have been the cause. The remaining microscopic findings for this animal were considered to be random findings.
- At the scheduled necropsy, ulceration and/or chronic active inflammation of the stomach were observed in one male in each of the 6.0 and 15 mg/kg/day groups. This finding consisted of a marked response at the oesophageal opening of the stomach in one animal and a much smaller lesion including the fundic area of the stomach in the other. Mild ulceration of the oesophagus was observed in one 15 mg/kg/day group female, suggesting an effect either directly related to treatment or secondary due to dosing.
- Previous rat inhalation studies demonstrated a test material-induced effect on the olfactory epithelium, resulting in degenerative changes. Therefore, the nasal cavities and olfactory epithelium were examined in the current, oral administration (capsule) study in dogs. At nasal level 4, at the roof of the dorsal meatus, the olfactory epithelium was occasionally observed undergoing degeneration, sometimes leading to squamous metaplasia for both control and treated dogs. In male dogs, there appeared to be no differences between the control and treated dogs. However, a slight increase in olfactory epithelial degeneration at this level was observed for the 6.0 and 15 mg/kg/day group females, suggesting a possible test material-related effect. In light of numerous instances of emesis seen during the study, a possible secondary effect of slight aerosol exposure to the test material is suggested as a possible cause of the olfactory epithelium degeneration, rather than a systemic effect. A slight increase in cysts of the respiratory epithelium at nasal level 2 was observed for two females in the 15 mg/kg/day group that may suggest a test material-related effect.
- Due to the nature of the test material, the thyroid glands were examined carefully. Although some variety in the size and number of thyroid follicles was observed, there did not appear to be any evidence of thyroid follicular hyperplasia/degeneration that was previously observed in the rat inhalation range-finding study. Colloid within thyroid follicles of the test material-treated dogs also appeared adequate.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CONCLUSIONS
- Body weights, food consumption and haematology, serum hormone and urinalysis parameters were unaffected. No ophthalmic lesions indicative of a toxic effect were observed.
- No test material-related changes were noted in the 1.5 mg/kg/day group.
- Test material-related effects noted in the 15 mg/kg/day group consisted of: euthanasia of one male due to moribund condition, increases in clinical findings (emesis, salivation, head shaking, soft or mucoid faeces and possibly injected sclera). Injected sclera was attributed to the test material, but not considered to be adverse, lower mean albumin and total protein levels at study weeks 6 and 12 and microscopic changes in the stomach, oesophagus and/or cecum and rectum (ulceration, chronic active inflammation and/or haemorrhage in two male and one female) and olfactory epithelium degeneration at nasal level 4 and cysts of the respiratory epithelium at nasal level 2 (females only).
- Test material-related effects noted in the 6.0 mg/kg/day group consisted of: increases in emesis, salivation prior to and following dosing and injected sclera. Injected sclera was attributed to the test material, but not considered to be adverse and gastric ulceration (one male) and olfactory degeneration at nasal level 4 (females).
- Test material-related effects noted in the 1.5 mg/kg/day group consisted of: increases in injected sclera. Injected sclera was attributed to the test material, but not considered to be adverse
- Based on the results of this study, the no-observed-effect level (NOEL) for oral (capsule) administration of the test material to dogs for a minimum of 90 days was less than 1.5 mg/kg/day. The no-observed-adverse-effect level (NOAEL) was 1.5 mg/kg/day.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

1.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

other: Due test material related decreases in mean albumin and total protein levels in anaimals dosed at 15 mg/kg/day and increased incidence of emesis in both the 6 and 15 mg/kg/day dose gps, the NOAEL was considered 1.5 mg/kg/day for both genders.

2

Key result

Dose descriptor:

NOEL

Effect level:

< 1.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study the NOAEL was considered to be 1.5 mg/kg/day for male and female dogs.

Executive summary:

The repeated dose oral toxicity of the test material was investigated in accordance with the standardised guidelines OECD 409, OPPTS 870.3150 and JMAFF 12 NouSan No. 8147, under GLP conditions.

The toxicity potential of the test material in corn oil when administered orally in capsules to dogs for a minimum of 90 days was evaluated to assist in the dose selection for a chronic toxicity study. The test material in the vehicle, corn oil, was administered orally via capsules once daily, seven days per week, for a minimum of 90 days at dosage levels of 1.5, 6.0 and 15 mg/kg/day. A concurrent control group received capsules containing corn oil on a comparable regimen. Each group consisted of four males and four females. The animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily and detailed physical examinations were performed weekly. Individual body weights were recorded weekly. Food consumption was recorded daily and reported weekly. Clinical pathology evaluations (haematology, serum chemistry, serum hormones and urinalysis) were performed prior to the initiation of dose administration (study week -1) and during study weeks 6 and 12 (last week of the dosing period). Ophthalmic examinations were performed during study weeks -1 and 13. Complete necropsies were performed on all dogs, and selected organs were weighed at the scheduled necropsies. All tissues were examined microscopically from all animals.

Body weights, food consumption and haematology, serum hormone and urinalysis parameters were unaffected. No ophthalmic lesions indicative of a toxic effect were observed. No test material-related changes were noted in the 1.5 mg/kg/day group. Test material-related effects noted in the 15 mg/kg/day group consisted of: euthanasia of one male due to moribund condition, increases in clinical findings (emesis, salivation, head shaking, soft or mucoid faeces and possibly injected sclera). Injected sclera was attributed to the test material, but not considered to be adverse, lower mean albumin and total protein levels at study weeks 6 and 12 and microscopic changes in the stomach, oesophagus and/or cecum and rectum (ulceration, chronic active inflammation and/or haemorrhage in two male and one female) and olfactory epithelium degeneration at nasal level 4 and cysts of the respiratory epithelium at nasal level 2 (females only). Test material-related effects noted in the 6.0 mg/kg/day group consisted of: increases in emesis, salivation prior to and following dosing and injected sclera. Injected sclera was attributed to the test material, but not considered to be adverse and gastric ulceration (one male) and olfactory degeneration at nasal level 4 (females). Test material-related effects noted in the 1.5 mg/kg/day group consisted of: increases in injected sclera. Injected sclera was attributed to the test material, but not considered to be adverse.

Under the conditions of this study the NOAEL was considered to be 1.5 mg/kg/day for male and female dogs.