Iodomethane

Endpoint summary

Description of key information

Oral

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

90 day rat: Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material was 5 mg/kg/day in males, and the No Observed Effect Level (NOEL) of the test material was 5 mg/kg/day in females.

90 day mouse dietary study: The no-observed-effect level (NOEL) for oral (diet) administration of the test material to mice for at least 90 days was less than 133 ppm. The no-observed-adverse-effect level (NOAEL) was 400 ppm.

1 year dog: The no-observed-adverse-effect level (NOAEL) for oral (capsule) administration of the test material to dogs for 52 consecutive weeks was 1.5 mg/kg/day.

Inhalation

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for whole-body inhalation exposure to the test material in rats for four or 13 weeks was 20 ppm. (mg/m3 from ppm = ppm x mwt(141.935)/24.45 = 116 mg/m3)

Dermal

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity of the test material when administered dermally to rats for 21 consecutive days was 30 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 NouSan No. 8147

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

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.

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

Dose / conc.:

1.5 mg/kg bw/day (nominal)

Dose / conc.:

6 mg/kg bw/day (nominal)

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.

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.

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.

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.

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

Critical effects observed:

not specified

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 February 2002 to 21 January 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 NouSan No. 8147

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 171 to 193 g and females: 138 to 160 g
- Housing: The animals were individually housed in suspended stainless steel cages [32.5 cm (D) x 19.5 cm (W) x 18 cm (H)]
- Diet: ad libitum (but not supplied on days where urine was sampled)
- Water: ad libitum
- Acclimation period: males: 7 days and females: 8 days

DETAILS OF FOOD AND WATER QUALITY: food was analysed and results showed no evidence that data would be affected.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.4 to 24.0 °C
- Humidity: 43 to 65 %
- Air changes: 15 times per hour
- Photoperiod: 12 hours of artificial light (06.00 to 18.00)

Route of administration:

oral: gavage

Details on route of administration:

The oral route was chosen in accordance with the Guidance for the Ministry of Agriculture, Forestry and Fisheries, and is considered to be one of the exposure routes.

in human beings.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test material was diluted with corn oil to make a 100 mg/mL stock solution. Prior to dosing the stock solution was diluted with corn oil to make 10, 5, 2 and 1 mg/mL.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was used as the vehicle because the test material is very slightly soluble in water and soluble in organic solvent.
- Lot/batch no: WAP7980, WAQ7752 and LDJ7800

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS CONDITIONS
Detector: Flame Ionization Detector (FID)
Column: GC-GasPro (0.32 mm i.d. x 30 m, Agilent Technologies)
Carrier Gas: Helium
Flow Rate: Adjusted to give a retention time of approximately 4.7 min for the test material (about 14 mL/min)
Column Temperature: 70 °C for 1 min, ramp at 40 °C /min to 230 °C, hold 2 min
Injection Temperature: 225 °C
Detector Temperature: 225 °C
Syringe Temperature: 100 °C
Vial Temperature: 50 °C
Injection Mode: Splitless (Sampling time: 1 min)
Injection Volume: 800 µL
Vial EQ Time: 7 min
Analysis Time: 10 min
Sample volume of vial: 1 mL

- Preparation of Standard Solutions: (From the stock standard solution, each standard solution was prepared in triplicate) Accurately 250 mg of the test material technical product was weighed and transferred to a disposable tube containing an appropriate volume of corn oil. This solution was then transferred to a 50 mL amber volumetric flask containing approximately 35 mL of corn oil and diluted to exactly 50 mL with corn oil (Stock standard solution: 5 mg/mL). Exactly 3 mL of the stock standard solution was diluted to exactly 10 mL with corn oil (STDI: 1.5 mg/mL). Exactly 1 mL of stock standard solution was diluted to exactly 10 mL with corn oil (STD2: 0.5 mg/mL).

- Preparation of System Repeatability Solution: 2 mL of the stock standard solution was diluted to exactly 10 mL with corn oil (1 mg/mL).

- Preparation of Sample Solutions: (Prepared in triplicate) An exact amount sampled was diluted with corn oil to give test material concentrations within the linearity range (0.5 to 1.5 mg/mL).

- System Repeatability: The system repeatability solution (1 mg/mL) was injected 6 times prior to analysis to confirm system repeatability. A relative standard deviation (RSD, %) of the peak area values not exceeding 15 % is acceptable.

- Concentration of Dosing Solutions: Standard solutions were injected onto the GC once followed by a single injection of each sample solution. Using CLASS-GC10, a calibration curve was constructed (least squares method) from which the test material sample solution concentrations were determined. The test material concentration in the dosing solution was calculated using the following equation: Test material concentration in the dosing solutions (mg/mL) = concentration in the sample solution (mg/mL) x dilution factor

- The dosing solutions in each concentration on the first and final dosing days were analysed by GC method, and confirmed to be within ± 15 % of the target concentration.

Duration of treatment / exposure:

90 days

Frequency of treatment:

- Once daily, seven times weekly, for 90 days (total 90 times).
- The test material was administered between 9:24 and 11:59, excepting examination days (14:35 to 15:46).

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose, plus 10 extra recovery animals per sex in the 0, 25 and 50 mg/kg/day groups.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a previous study, one female died at 50 mg/kg. Accordingly, the high dose level was set at 50 mg/kg, and 25, 10, and 5 mg/kg were set at as the lower levels, in a common ratio of approximately 2.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- All animals were observed for mortality and clinical signs at least 3 times daily during the dosing period (prior to administration, from immediately after dosing to 2 hours after dosing and 3 hours to 5 hours after dosing), once daily during the recovery period and once on the day of gross pathology.

DETAILED CLINICAL OBSERVATIONS: Yes
- Behaviour of all animals was observed once prior to the initiation of dosing, once per week for 13 weeks of dosing, and once per week for 4 weeks of recovery. Number of rearing times observed in 1 minute was counted.
- Observation items: external features (skin, fur, eyes, eyeballs, changes of visible mucosa and presence of secretion etc.), body position, body posture, (kyphosis posture etc.), function of autonomic nervous system (lacrimation, piloerection, pupil size, respiration condition, excretory condition etc.) motor coordination, abnormal gait, response to operation procedure of animals or environmental stimulation, nervous system (tremors, convulsions, muscle contraction etc.), changes of exploratory movement, stereotyped behaviour (changes of grooming, head flicking, circling etc.), abnormal behaviour (self biting, hallucinatory, abnormal vocalization etc.) and aggression

BODY WEIGHT: Yes
- All animals were weighed on the day prior to the initiation of dosing, on the first day of dosing (immediately prior to dosing), and once weekly during the dosing and recovery periods using an electronic balance.
- Body weight gain was calculated at each measurement. The weekly individual body weight measurements were used to calculate the dose of test material that each animal received for the following week (the dose of test material of Week 1 was calculated from the body weight on the first day of dosing).

FOOD CONSUMPTION:
- The food consumption of all animals was measured once prior to the initiation of dosing and once weekly during the dosing and recovery periods using an electric balance.
- Daily food consumption was calculated from the amount of food provided and that remaining on the following day.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Animals were examined once prior to the initiation of dosing, once during Week 13 of dosing, and once during Week 4 of recovery. The anterior portion and optic media of all animals were examined closely using a penlight. The ocular fundus of 5 males and 5 females of each group (lowest numbered animals) were examined using a fundus camera; however, all animals were examined prior to the initiation of dosing. The optic media and ocular fundus were observed after instillation of a mydriatic drug.

HAEMATOLOGY: Yes
- Blood was drawn from the abdominal vena cava of all animals using a syringe under anaesthesia by an intraperitoneal injection of sodium pentobarbital solution (6.48 mg/mL, 5 mL/kg) at gross pathology after the end of dosing and recovery periods. The blood samples were treated with an anticoagulant, either EDTA-2K or 3.8 % sodium citrate solution. Blood smears were prepared as a backup for measuring differential leukocytes and were discarded after the end of the study.
- For the samples treated with EDTA-2K, the following parameters were determined or calculated using a haematology system (ADVIA120, Bayer Diagnostics Manufacturing Ltd.): Erythrocyte count (RBC, dual-angle laser flow-cytometric measurement), Leukocyte count (WBC, dual-angle laser flow-cytometric measurement), Haematocrit value (Calculation), Haemoglobin concentration (Modified cyanmethaemoglobin method), Platelet count (Dual-angle laser flow-cytometric measurement), Mean corpuscular volume (MCV, dual-angle laser flow-cytometric measurement), Mean corpuscular haemoglobin (MCH, calculation), Mean corpuscular haemoglobin concentration (MCHC, calculation), Reticulocyte count (Dual-angle laser flow-cytometric measurement with RNA stain) and Differential leukocyte (Peroxidase flow-cytometric measurement and dual-angle laser flow-cytornetric measurement)- Eosinophilic leukocytes, basophilic leukocytes, monocytes, lymphocyte, neutrophilic leukocytes and large unstained cells.
- Plasma obtained by centrifugation of blood treated with an anticoagulant (3.8 % sodium citrate solution) was examined for following blood coagulation parameters with an automatic blood coagulation measuring apparatus: Prothrombin time (Light scattering method) and Activated partial thromboplastin time (Light scattering method).

CLINICAL CHEMISTRY: Yes
- Blood was collected, following blood sampling for haematology at gross pathology after the end of dosing and recovery periods, from the abdominal aorta of all animals using syringe. After stabilisation at room temperature for 40 to 60 minutes, serum was obtained by centrifugation.
- The following parameters were determined using an automatic analyser: Aspartate aminotransferase (ASAT, JSCC transferable method), Alanine aminotransferase (ALAT, JSCC transferable method), Alkaline phosphatase (ALP, JSCC transferable method), y-GTP (JSCC transferable method), Lactate dehydrogenase (LDH, Wroblewski-La due method), Creatinine Phosphokinase (JSCC transferable method), Total bilirubin (Vanadate oxidation method), Total protein (Biuret method), Albumin (BCG method), Total cholesterol (COD-HDAOS method), Triglyceride (GPO-HDAOS method, Glycerol blanking method), Phospholipid (Choline oxidase DAOS method), Glucose (Hexokinase .GIcK.G-6-PDH method), Blood urea nitrogen (BUN, crease-GIDH method), Creatinine (Creatininase F-DAOS method), Inorganic phosphorus (PNP.XDH method), Calcium (Ca, MXB method), Sodium (Na, electrode method), Potassium (K, electrode method) and Chloride (Cl, electrode method).
- The following parameters were determined using an automatic electrophoresis apparatus: Protein fractions (Electrophoresis method): Albumin ratio, α1-globulin ratio, α2-globulin ratio, β-globulin ratio, γ-globulin ratio, A/G ratio.

URINALYSIS: Yes
- Fresh urine were collected from all animals by the compulsory method, and 4-hour preserved urine was collected from 5 males and 5 females of each group (lowest numbered animals) using a metabolic cage once during Week 13 of dosing and once during Week 4 of recovery. The colour was judged visually, and the following parameters were examined using test paper and an automatic urine analyser: pH, protein, glucose, ketone body, bilirubin, urine occult blood and urobilinogen.
- Urine volume was measured using a measuring cylinder and urine specific gravity using a urinary refractometer. Additionally, urine sediments were collected from the 4-hour preserved urine by centrifugation, and stained with Sternheimer-Malbin. The following parameters were examined microscopically: erythrocytes, leukocytes, crystals, epithelial cells, bacteria, urinary casts, sperm and others.

NEUROBEHAVIOURAL EXAMINATION: Yes
- The sensorimotor reaction to irritation [auditory irritation, visual irritation and proprioceptive irritation], grip strength, and amount of spontaneous activity of all animals were examined once prior to the initiation of dosing, once during Week 13 of dosing and once during Week 4 of recovery.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Animals that died were weighed and necropsied as soon as possible after death. Organs and tissues were examined macroscopically. Surviving animals were weighed (after being fasted for 16 to 18 hours) on the day following the final day of the dosing or recovery period, and euthanised by exsanguination following blood sampling for the examination, under anaesthesia by an intraperitoneal injection of sodium pentobarbital solution (6.48 mg/mL, 5 mL/kg). The organs and tissues were observed macroscopically, and prepared for subsequent microscopic examination. Representative photographs were taken of abnormalities that were considered to have been induced by the test material.

- Organ weights: The following organs of all animals were weighed with an electronic balance: liver, kidneys (bilateral), adrenals (bilateral), testes (bilateral), ovaries (bilateral), thymus, lungs (bilateral, including bronchi), spleen, heart, and brain (including cerebellum and brain stem). Relative organ weights were calculated from the absolute weight and body weight on the day of gross pathology.

HISTOPATHOLOGY: Yes
- The organs and tissues of all animals listed below were fixed in 10 % neutral buffered formalin. The eyeballs and optic nerves were fixed in a solution mixture of formaldehyde and glutaraldehyde, and the testes were fixed in Bouin's fluid. The organs and tissues were vacuum-packed and stored: Heart, aorta (thorax), spleen, thymus, femoral bone (left) and sternal bone (bone and bone marrow, decalcified specimens), submandibular lymph node* *, mesenteric lymph node, trachea, lungs (including bronchi)*, tongue, oesophagus, stomach (fore and glandular), small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), pancreas, liver, kidneys*, urinary bladder, testes*, epididymides*, seminal vesicles, coagulating glands, prostate, ovaries*, uterus, vagina, pituitary, thyroids*, parathyroids (It was not possible to prepare slide specimens for the left sides of Animal Nos. 9, 14, 17, 73, 91 and 94; right sides of Animal Nos. 5, 6, 11, 68, 94 and 96)*, adrenals*, cerebrum, cerebellum, brain stem, spinal cord (cervix, thorax and lumbar region), sciatic nerves**, lacrimal glands*, skeletal muscle (quadriceps femoris)**, skin (gluteal area)**, mammary gland (relevant area, only female)**, eyeballs (including optic nerve and retina)*, Harderian glands*, and gross lesions. (*= Left and right organs were examined.* *= Left and right organs were collected. If there were no gross lesions, only the left was examined.)
- All organs and tissue listed above of animals in the control and 50 mg/kg groups and test material-related abnormal lesions (liver, animal Nos.: 33, 57, 73, 138, 151, and 155) in each group were trimmed, embedded in paraffin, sectioned, stained with haematoxylin-eosin, and examined microscopically.
- The liver, stomach (fore and grandular), submandibular glands (including uniforate sublingual gland) and thymus (only female) in the 25, 10, and 5 mg/kg groups were trimmed, embedded in paraffin, sectioned, stained with hematoxylin-eosin, and examined microscopically.
- Representative photographs of changes which were suspected to be test material-related were taken.

Statistics:

Functional examination (apart from sensorimotor reaction to irritation), food consumption, body weight, urine volume, urine specific gravity, haematological, blood chemistry and organ weight (absolute and relative weights) data were analysed for homogeneity of variance by Bartlett's test. When homogeneity of variance existed, a one way analysis of variance was applied. When the results were significant, Dunnett's test was applied to compare the means of the treatment and control group. When the variances by Bartlett's test were heterogeneous, the Kruskal-Wallis's H-test was performed after rank conversion. When the result was statistically significant, the mean ranks among the groups were compared using Dunnett's type test. Urinalysis (excepting quantitative data) and gradable, ophthalmological, gross pathological and histopathological examination data were analysed by the Exact rank sum test, and non-gradable data was analysed by Fisher's exact test between the control and test article groups. A computer was used for these statistical analyses (p<0.05). Data related to clinical signs, detailed observations of condition, functional examination (only sensorimotor reaction to irritation) were not analysed.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Salivation was observed beginning on Day 5 in the animals that died on study. Soft stool, a trace of reddish rhinorrhoea, reddish eye gum, and dirty lower belly by urine from Day 75, and a decrease in spontaneous activity from Day 79 were observed in 1 female. The animal was confirmed to be dead on the morning of Day 81.
- Lacrimation, dirty lower belly by urine, prone position, and a decrease in spontaneous activity were observed in 1 female from Day 82, and the animal died on Day 83 after dosing. Lacrimation and a decrease in spontaneous activity were observed in 2 females on Day 85, and these animals were confirmed to be dead on the morning of Day 86.
- Salivation was sporadically observed in all males and surviving 15 females at 50 mg/kg from Day 3 to the final dosing day. Reddish rhinorrhoea, reddish eye gum, and dirty lower belly by urine were observed in 1 female between Days 82 and 84. Reddish rhinorrhoea, dirty lower belly by urine and a decrease in spontaneous activity were observed in 1 female on Day 86. Salivation was sporadically observed in 18 males and 19 females at 25 mg/kg from Day 6 to the final dosing day. Salivation was sporadically observed in 5 males and 4 females at 10 mg/kg from Day 7 to the final dosing day. -Reddish eye gum was observed in 1 male on Days 67 and 68.
- No abnormalities were observed in any animal at 5 mg/kg.
- Defect of the teeth was observed in 1 female in the control group between Days 79 and 85.
- No abnormalities were observed in any group during the recovery period.
- No significant differences in the detailed observations of condition were observed in any group compared with the control group during the dosing or recovery periods.

Mortality:

mortality observed, treatment-related

Description (incidence):

Four females died at 50 mg/kg on Days 81, 83, and 86 during the dosing period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Suppression of body weight gain was observed in males at 50 mg/kg at Weeks 1, 2, 5, 6, 8 and 10 during the dosing period, and a decrease in body weight was observed from Week 1 to the final dosing day. No differences were observed in females at 50 mg/kg compared with the control group during the dosing period.
- No test material-related changes were observed at 5, 10, or 25 mg/kg.
- A decrease in body weight was observed in females at 25 mg/kg at Week 13, and an increase in body weight gain was observed at Week 1. Suppression of body weight gain was observed in females at 10 mg/kg at Week 5, and an increase in body weight gain was observed at Week 6. Suppression of body weight gain was observed in females at 5 mg/kg at Week 3, and an increase in body weight gain was observed at Week 9. However, no differences were observed in body weight, or the changes were not dose-dependent. Accordingly, these changes were judged to be toxicologically insignificant.
- An increase in body weight gain was observed in males at 50 mg/kg at Week 2 of recovery. However, no differences were observed at 25 mg/kg compared with the control group during the recovery period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- A decrease in food consumption was observed in males at 50 mg/kg at Weeks 2 and 4, and an increase in food consumption was observed at Weeks 11 and 13 during the dosing period.
- A decrease in food consumption was observed in males at 25 mg/kg at Weeks 2 and 4, and a decrease in food consumption at Week 13 and an increase in food consumption at Week 8 were observed in females during the dosing period.
- An increase in food consumption was observed in males at 10 mg/kg at Week 8, and a decrease in food consumption was observed in females at Week 13 during the dosing period.
- A decrease in food consumption was observed in males at 5 mg/kg at Weeks 2 and 4, and in females at Week 13 during the dosing period.
- No differences were observed in any group compared with the control group during the recovery period.

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 abnormalities were observed in any group at Week 13 of the dosing period and Week 4 of the recovery period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- No test material-related changes were observed in any group at the end of the dosing or recovery periods.
- An increase basophilic leukocyte ratio was observed in females at 25 mg/kg at the end of the dosing period compared with the control group. This change was judged to be toxicologically insignificant as they were not dose-dependent.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- High alkaline phosphatase, total bilirubin, total protein, albumin, phospholipid, calcium, and sodium were observed in males at 50 mg/kg during the end of dosing period, and high α2-globulin ratio, [β-globulin ratio and a low A/G ratio were observed in females.
- High total bilirubin, total protein, calcium, and sodium were observed in males at 25 mg/kg.
- High chloride was observed in males and females at 25 mg/kg, and high sodium was observed in females. Low alkaline phosphatase was observed in females at 10 mg/kg. However, these changes were judged to be toxicologically insignificant as they were not dose-dependent.
- No test material-related changes were observed in the examination at the end of the recovery period.
- A high α2-globulin ratio was observed in females at 50 mg/kg. However, this change was judged to be toxicologically insignificant as it was within the range of background (fatal).

Urinalysis findings:

no effects observed

Description (incidence and severity):

- No test material-related changes were observed in any group at Week 13 of the dosing period and Week 4 of the recovery period.
- Low protein was observed in males at 25 and 50 mg/kg at Week 13 of the dosing period, when compared with the control group. This was judged to be toxicologically insignificant, because all individual values were within the range of the background data.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- No significant differences were observed in any group compared with the control group during the dosing or recovery periods.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- No test material-related changes were observed in any group at the end of the dosing or recovery periods.
- The following organ weight changes were observed at the end of the dosing period. Low absolute weight of the kidney (right) was observed in females at 10 mg/kg. High relative weight of the testis (right, bilateral) and liver were observed in males at 50 mg/kg. High relative weight of the liver was observed in females at 25 and 50 mg/kg. High relative weight of the spleen was observed in females at 50 mg/kg at the end of the recovery period. These were judged to be toxicologically insignificant, because they were observed only in the relative weight and were not dose-dependent.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- The following changes were observed in 4 females that died at 50 mg/kg: adhesion in the abdominal organs in 2 females, ascites in the abdominal cavity and reddening of the thymus, dark reddening of the endometrium and enlargement of the adrenal in 1 female, black focus of the mucosa in the glandular stomach and dark reddening in the thymus in 2 females, reddening of the mucosa in the glandular stomach, thickening of the mucosa in the forestomach, reddening of the mucosa in the duodenum, black of the jejunum and reddening of the liver in I female, external findings in 2 females.
- The following changes were observed in the surviving animals at 50 mg/kg: adhesion in the abdominal organs in 1 male and 2 females, thickening and/or attaching of the greyish white material of the mucosa in the forestomach, enlargement of the liver in 2 females, yellowish brown focus of the liver in 3 females, reddening and red foci in the stomach in 1 female.
- Adhesion in the abdominal organs was observed in 2 females at 25 mg/kg. No test material-related changes were observed at 5 and 10 mg/kg.
- Red focus in the lung was observed in 1 male at 50 mg/kg. Discolouration in the liver was observed in 2 males at 5 mg/kg, and in 1 male at 10 mg/kg. Accentuated lobular pattern in the liver was observed in 1 male at 10 mg/kg. White foci in the liver was observed in 1 male at 5 mg/kg, and in 4 males at 10 mg/kg, and in 2 males and 2 females at 25 mg/kg, and in 3 males at 50 mg/kg. Dilatation renal pelvis in the kidney was observed 1 male at 25 mg/kg. Small size of the testis was observed in 2 males at 5 mg/kg. Hydrometra was observed in 2 females at 5 mg/kg, and in 1 female at 10 mg/kg. These were judged to be toxicologically insignificant, because they were not dose-dependent, and they were observed in the control group.
- Adhesion in the abdominal organs and thickening of the mucosa in the forestomach were observed in 1 female in the examination at the end of the recovery period.
- No test material-related changes were observed at 25 mg/kg.
- White focus in the liver was observed in 2 males and 1 female at 25 mg/kg, and in 3 males and 1 female at 50 mg/kg. Accentuated lobular pattern in the liver was observed in 2 males at 25 mg/kg. Discolouration in the liver was observed in 2 males at 25 mg/kg. Red focus in the thymus was observed in 1 male at 25 mg/kg. Incomplete retraction in the liver was observed in 1 male at 50 mg/kg. Hydrometra in the uterus was observed in 3 females at 25 mg/kg and 2 females at 50 mg/kg. These changes were judged to be toxicologically insignificant as they were not dose-dependent, or were also observed in the control group and the background data.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- The following changes were observed in the 4 females that died at 50 mg/kg: decrease in the number of the myeloid cells and decrease in the number of the megakaryocytes in the femoral bone marrow (left) and sternal bone marrow in 3 females, decrease in the number of the lymphocytes in the mesenteric lymph node and submandibular lymph node (left) in 3 females, necrosis of the lymphocytes in the mesenteric lymph node in 1 female, necrosis of the lymphocytes in the submandibular lymph node (left) in 2 females, necrosis of the lymphocytes in the thymus in 4 females, haemorrhage in the thymus in 2 females, decrease in the number of the lymphocytes in the cortex in the thymus in 2 females, mineralization in the cortex in the thymus in 1 female, decrease in the number of the lymphocytes in the white pulp in the spleen in 4 females, necrosis of the lymphocytes in the white pulp in the spleen in 1 female, increase in the number of the macrophages in the red pulp and thickening of the capsule associated with necrosis and inflammatory cell infiltration in the spleen in 1 female, dilatation of the sinusoid and proliferation of the Kupffer cells in the liver in 2 females, brown pigment in the liver in 1 female, hyperkeratosis and hyperplasia of the squamous epithelium in the forestomach in 4 females, oedema of the submucosa in the forestomach and glandular stomach in 2 females, necrosis/ulceration of the squamous epithelium in the forestomach in 2 females, inflammatory cell infiltration in the submucosa in the glandular stomach in 2 females, inflammatory cell infiltration in the submucosa of the forestomach in 1 female, adhesion in 2 females, associated with granulation tissue in 1 female, haemorrhage in the hepatic tissue and necrosis of hepatocytes in 1 female, squamous metaplasia of the ductal epithelium in the submandibular glands in 4 females, and haemorrhage in the cortex in the adrenal (right) in 1 female.
- The following changes were observed at 50 mg/kg for surviving animal at the end of the dosing period; brown pigment in the liver in 1 female, focal haemorrhage in the liver in 1 male, focal necrosis of the hepatocyte in 2 males, focal haemorrhage and fibrous in 1 male and 1 female in the liver (gross abnormal site), focal necrosis of the hepatocyte in 2 males and 1 female in the liver (gross abnormal site), bile duct proliferation in 2 females in the liver (gross abnormal site), giant cell granuloma in 1 female in the liver (gross abnormal site), hyperkeratosis and hyperplasia of the squamous epithelium in the forestomach in all males and females, oedema in the submucosa in the forestomach in 4 females, oedema in the submucosa of the glandular stomach in 2 females, fibrosis of the submucosa in the forestomach in 3 females, necrosis/ulceration of the squamous epithelium in the forestomach in 2 females, haemorrhage in the forestomach, inflammatory cell infiltration in the submucosa in the forestomach and glandular stomach in 1 female, lymphocyte infiltration in the submucosa in the glandular stomach in 1 female, decrease of the granules in the granular ducts in the submandibular gland in 5 males and 4 females, squamous metaplasia of the ductal epithelium in the submandibular gland in all males and females, neutrophil infiltration in the ductal epithelium in the submandibular gland in 3 males and 1 female, neutrophil accumulation in the ductal lumen in the submandibular gland in 1 male and 1 female, squamous metaplasia of the ductal epithelium in the sublingual gland in 4 females, decrease of the lymphocytes in the cortex in the thymus in 1 female, adhesion in the abdominal organs in 1 male.
- The following changes were observed at 25 mg/kg at the end of the dosing period; adhesion in the abdominal organs in 2 females, focal necrosis of the hepatocytes in the liver in 1 male, hyperkeratosis or hyperplasia of the squamous epithelium in the forestomach in 8 males and 6 females, inflammatory cell infiltration of the submucosa in the forestomach/glandular stomach in 2 females, oedema of the submucosa in the forestomach in 1 female, necrosis/ulceration of the squamous epithelium in the forestomach in 1 female, decrease of the granules in the granular ducts in the submandibular gland in 3 males and 2 females, squamous metaplasia of the ductal epithelium in the submandibular gland in all males and females, neutrophil infiltration in the ductal epithelium in the submandibular gland in 2 males and 2 females, neutrophil accumulation in the ductal lumen in the submandibular gland in 1 female.
- The following changes were observed at 10 mg/kg at the end of the dosing period; hyperkeratosis or hyperplasia of the squamous epithelium in the forestomach in 3 males, focal haemorrhage and necrosis of the hepatocytes in the liver in 1 male, squamous metaplasia of the ductal epithelium in the submandibular gland in 1 male and 2 females, squamous metaplasia of the ductal epithelium in the sublingual gland in 1 female.
- The following changes were observed at 5 mg/kg at the end of the dosing period; focal haemorrhage and necrosis of the hepatocytes in the liver in 1 male.
- The following changes were observed at the end of the recovery period; adhesion in the abdominal organs in 1 female at 50 mg/kg, hyperkeratosis and hyperplasia of the squamous epithelium in the forestomach in 1 female at 50 mg/kg, necrosis of the hepatocytes in the liver in 1 male at 50 mg/kg, focal haemorrhage and necrosis of the hepatocytes in the liver in 1 male at 25 mg/kg.
- Other histopathological findings, were judged to be spontaneous because they were observed in the control group or the background data.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

From these results, test material-related effects on the liver and digestive system were observed in males and females at 10 mg/kg and above in this study. Effects on the liver were observed only one male at 5 mg/kg. These changes almost recovered during the recovery period. Accordingly, it was considered that under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material was 5 mg/kg/day in males, and the No Observed Effect Level (NOEL) of the test material was 5 mg/kg/day in females.

Key result

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

other: Based on histopathological changes in the forestomach and submandibular glands at 10 mg/kg/day.

Key result

Dose descriptor:

NOEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

 

Table 1: Table to show the selected pathology findings among rats killed after 13 weeks of dosing. The table shows incidence of finding and total of animals examined for that particular endpoint in parenthesis

Dose (mg/kg)	0	5	10	25	50	0	5	10	25	50
	Males					Females
Stomach- hyperkeratosis	0 (10)	0 (10)	3 (10)	8 (10)	10 (10)	0 (10)	0 (10)	0 (10)	6 (10)	7 (7)
Stomach- hyperplasia	0 (10)	0 (10)	3 (10)	7 (10)	10 (10)	0 (10)	0 (10)	0 (10)	6 (10)	7 (7)
Submandibular gland- squamous metaplasia	0 (10)	0 (10)	1 (10)	10 (10)	10 (10)	0 (10)	0 (10)	2 (10)	10 (10)	7 (7)

Conclusions:

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material was 5 mg/kg/day in males and the No Observed Effect Level (NOEL) of the test material was 5 mg/kg/day in females.

Executive summary:

The repeated dose oral toxicity of the test material was investigated in accordance with the standardised guidelines EPA OPPTS 870.3100 and JMAFF 12 NouSan No. 8147, under GLP conditions.

The test material was administered orally to 10 male and 10 female Crj : CD (SD) IGS rats per group once daily for 90 days at dose levels of 0 (control), 5, 10, 25 and 50 mg/kg to evaluate the toxicity potential of the test material in corn oil. Ten males and 10 females were allocated to the control and 25 and 50 mg/kg groups to assess reversibility during a 28-day recovery period. The control group received corn oil in the same manner as the test material.

Four females died at 50 mg/kg on Days 81, 83, and 86. In the animals that died, salivation was sporadically observed from Day 5, and it was accompanied by prone position, a trace of reddish rhinorrhoea, reddish eye gum, lacrimation, soft stool, dirty around anus and a decrease in spontaneous activity in some animals. A decrease in food consumption, suppression of body weight gain, and a decrease in body weight were also observed. In gross pathology, adhesion of the abdominal organs in the abdominal cavity, reddening or black foci of the mucosa in the glandular stomach, thickening of the mucosa in the forestomach, ascites in the abdominal cavity, and reddening or dark reddening of the thymus were observed in the animals that died. Histopathology revealed that these changes were hyperkeratosis, hyperplasia, necrosis, ulceration, inflammatory cell infiltration in the squamous epithelium in the forestomach, necrosis of the lymphocyte, haemorrhage, a decrease in the number of the lymphocytes in the thymus, decreases in the number of myeloid cells and the megakaryocytes in the femoral and sternal bone marrow, a decrease or necrosis of the lymphocytes in the white pulp in the spleen, and mesenteric and submandibular lymph nodes, an increase in the number of the macrophages in the red pulp in the spleen, and squamous metaplasia of the ductal epithelium in the submandibular glands.

In the surviving animals at 50 mg/kg, salivation was sporadically observed in all males and 15 females from Day 3 to the final dosing day. A trace of reddish rhinorrhoea, reddish eye gum, dirty lower belly by urine, and a decrease in spontaneous activity were observed in 2 females between Days 82 and 86. Suppression of body weight gain and a decrease in body weight were observed in males from Week 1, and a decrease in food consumption was observed at Weeks 2 and 4. No abnormalities were observed in males or females in body weight or food consumption during the recovery period. In blood chemistry, high alkaline phosphatase, total bilirubin, total protein, albumin, phospholipid, calcium, and sodium in males, high a2-globulin ratio, 13-globulin ratio, and low A/G ratio in females were observed in the examination at the end of the dosing period. In gross pathology at the end of the dosing period, adhesion in the abdominal organs in males and females, thickening and attaching of the greyish white material of the mucosa in the forestomach, enlargement of the liver, yellowish brown focus of the liver, reddening and red focus in the stomach in females were observed. Adhesion in the abdominal organs and thickening of the mucosa in the forestomach were observed in only 1 female at the end of recovery period. In histopathology at the end of the dosing period, hyperkeratosis, hyperplasia, necrosis/ulceration of the squamous epithelium in the stomach, haemorrhage in the forestomach, oedema in the submucosa of the glandular stomach, fibrosis of the submucosa in the forestomach, inflammatory cell infiltration in the submucosa in the forestomach and glandular stomach, lymphocyte infiltration in the submucosa in the glandular stomach, haemorrhage in the forestomach, and adhesion in the abdominal organs were observed. Focal necrosis of the hepatocyte, focal haemorrhage and fibrosis, bile duct proliferation, and giant cell granuloma in the liver were observed. In the submandibular glands, a decrease in the granules in the granular ducts, squamous metaplasia and neutrophil infiltration of the ductal epithelium, and neutrophil accumulation were observed, and squamous metaplasia of the ductal epithelium in the sublingual gland was also observed. A decrease in the lymphocytes in the cortex in the thymus was observed. Adhesion in the abdominal organs and thickening in the mucosa in the forestomach were observed in 1 female at the end of the recovery period.

Salivation was sporadically observed in 18 males and 19 females at 25 mg/kg from Day 6 to the end of the final dosing day. In blood chemistry at the end of the dosing period, high total bilirubin, total protein calcium, and sodium were observed in males. Adhesion in the gastrointestinal tracts was observed in females in gross pathology at the end of the dosing period. Hyperkeratosis, hyperplasia, and necrosis/ulceration of the squamous epithelium in the forestomach, inflammatory cell infiltration and oedema of the submucosa in the forestomach, and adhesion in the abdominal organs were observed in histopathology at the end of the dosing period. Squamous metaplasia and neutrophil infiltration in the ductal epithelium in the submandibular gland, focal necrosis of the hepatocyte and a decrease in the granules in the granular ducts were observed in 1 male. Focal haemorrhage and necrosis of the hepatocyte were observed in 1 male at the end of the recovery period.

Salivation was sporadically observed in 5 males and 4 females at 10 mg/kg from Day 7 to the final dosing day. Reddish eye gum was observed in 1 male on Days 67 and 68. Hyperkeratosis and hyperplasia of the squamous epithelium in the stomach, focal haemorrhage and necrosis in the hepatocyte in males and squamous metaplasia of the ductal epithelium in the submandibular or sublingual gland in males and females were observed in histopathology at the end of the dosing period.

Focal haemorrhage and necrosis of the hepatocyte were observed in 1 male at 5 mg/kg in histopathology at the end of the dosing period. No other abnormalities were observed in any other examination parameter.

From these results, test material-related effects on the liver and digestive system were observed in males and females at 10 mg/kg and above in this study. Effects on the liver were observed in only one male at 5 mg/kg. These changes were almost recovered during the recovery period. Accordingly, it was considered that under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material was 5 mg/kg/day in males, and the No Observed Effect Level (NOEL) of the test material was 5 mg/kg/day in females.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 August 2002 to 26 March 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 NouSan No. 8147

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

CD-1

Remarks:

Crl: CD-1(ICR)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: 23.5 to 30.3 g for males and from 19.4 to 26.1 g for females
- Fasting period before study: no
- Housing: Upon arrival, all animals were housed three per cage by sex for approximately three days. Thereafter, all animals were housed individually in clean, wire-mesh cages suspended above cage-board.
- Diet: 20 g every two days
- Water: ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY: No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.2 to 21.7 °C
- Humidity: 32.4 to 42.8 %
- Photoperiod: Light timers were set to provide a 12-hour light (6 a.m. to 6 p.m.)/12-hour dark photoperiod.

Route of administration:

oral: feed

Details on route of administration:

The selected route of administration was oral (diet) since the intended use of the test material indicated that oral ingestion is a potential route of human exposure.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: weekly
- The control and test diets were prepared on a weight/weight basis as follows. Appropriate amounts of control or test material were weighed into tared weighing vessels. Appropriate amounts of diet for each group were then weighed into separate tared weighing vessels. The control or test material and diet were then added to a V-blender and mixed for 15 minutes. The control diet was prepared first and the blender was thoroughly washed and dried prior to preparation of the test diets. The quantity of control material used to prepare the diet for the control group was equal to the quantity of test material required to prepare diet for the 1200 ppm group.
- Storage temperature of food: The test diets were prepared weekly, divided into aliquots following mixing and stored frozen (approximately -20°C) until used. Each aliquot was used for two days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SUMMARY
- A sample of the bulk Microencapsulated test material was collected during the week prior to the initiation of test diet administration and analysed for percent load of the test material in the microcapsules. The percent load was found to be 3.13 % and was used to calculate the amount of Microencapsulated test material for each test diet. Additional samples of the test material were taken during study weeks 2, 4 and 11 and analysed to verify percent load (2.68, 2.81 and 3.83 %, respectively). The average of the percent load for study weeks 2 and 4 (2.74 %) was used to formulate the test diets for study weeks 5-11.
- Stability of the test material in the basal diet was assessed in a separate study. Homogeneity analysis of the 133 and 400 ppm test diets was not conducted since they were within the range of dose levels analysed on previous study. Prior to the initiation of test diet administration, samples (approximately 100 g each) for homogeneity determination were collected from the top, middle and bottom strata of the 1200 ppm dietary formulation. In addition, samples (approximately 100 g each) of each diet preparation (including the control) were collected weekly throughout the study and frozen. Duplicate samples were collected during study weeks 0, 1, 3, 7 and 12. Analyses for test material concentration were conducted on one set of samples collected during these study weeks.
- The test material formulations were found to be homogeneous and contained the amounts of test material specified in the protocol.

GAS CHROMATOGRAPHY
- Instrument: Hewlett Packard 5890A (Series II) gas chromatograph equipped with an ECD detector, a HP Headspace analyser and ChemStation data system, or equivalent
- Column: Alltech EC Wax, 30 m x 0.53 mm ID (1.0 μm film thickness)
- Temperature (Program): 65 °C for 1.0 minute, ramp at 1 °C/minute to 70 °C, hold 0 minutes
- Carrier gas: Helium set at 2.1 psi (EPC constant flow on)
- Injector temperature: 225 °C
- Injection volume: 1 mL, splitless
- Detector: ECD at 225 °C

HEADSPACE PARAMETERS
- Zone temp: Oven 53 °C, Loop 120 °C and Transfer Line 121 °C
- Event Times: GC cycle time 6.5 minutes, vial EQ time 6.0 minutes, pressurisation time 0.20 minutes, loop fill time 0.20 minutes, loop EQ time 0.05 minutes and inject time 0.20 minutes.

- PREPARATION OF CALIBRATION AND QUALITY CONTROL STOCK SOLUTIONS: The stock solutions were prepared by transferring 86.0 μL of test material (density = 2.28) into a 250 mL volumetric flask containing approximately 200 mL of toluene. The contents were diluted to a final volume of 250 mL with toluene and thoroughly mixed. This stock was prepared at the concentration of 0.8 mg/mL.

- PREPARATION OF CALIBRATION AND QUALITY CONTROL SAMPLES: Calibration and quality control (QC) samples were prepared for analysis by transferring the appropriate volumes of the appropriate stock solutions into 500 mL plastic screw cap jars containing 40 g of untreated rodent diet.
-PREPARATION OF PERCENT LOAD SAMPLES: Samples were prepared for analysis by transferring approximately 100 mg of test material microcapsules into a 500 mL plastic screw cap jar containing 40 g untreated rodent diet.

- DIET FORMULATION ANALYSIS: Samples were prepared for analysis by transferring 40 g of diet formulation into a 500 mL plastic screw cap jar.

- PROCESSING OF SAMPLES: Calibration, QC and experimental samples were processed for analysis by adding 200 mL of deionised (DI) water and 100 mL of toluene into the 500 mL plastic screw cap jar containing 40 g of diet and any applicable fortifications. The samples were tightly capped and shaken for one hour. An aliquot of the toluene phase (approximately 2 mL) was transferred to a 5 mL glass test tube, capped and centrifuged for 10 minutes at 2500 rpm. An aliquot (200 μL for the low range and 20 μL for the high range) of the toluene extract was transferred to a headspace vial containing 1.8 mL (low range) or 1.98 mL (high range) of toluene, and the samples were capped and handshaken to mix before analysis on the GC.

- 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 calculated from the regression equation using a PC spreadsheet program. 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 3.2 to 3.6 minutes. The analysis time required for each run was approximately 10 minutes.
- Determinations of the test material (w/w) % load in microcapsules were performed periodically. The concentration of test material in the microcapsules resulted in values of 2.32 to 3.13 % and 3.71 to 3.83 % test material load (w/w).
- A representative formulation was prepared on August 23, 2002 for the evaluation of homogeneity in the diet admixes. Samples from the top, middle and bottom of the formulation batch were collected and analysed. The overall mean concentration of the homogeneity samples had an RSD of less than 10 % and was within 15 % of the target concentration and, therefore, met all formulation homogeneity and concentration criteria.
- The analysed dosing formulations had concentrations within the required 15 % of the target dose concentrations except for this Group 4 formulation prepared on 9/16/02. This formulation was re-prepared on 9/18/02 and, when analysed, resulted in a concentration within acceptable limits.

Duration of treatment / exposure:

90 days

Frequency of treatment:

New feed (approximately 20 grams) was supplied every other day and animals were allowed to eat it ad. libitum.

Dose / conc.:

133 ppm

Dose / conc.:

400 ppm

Dose / conc.:

1 200 ppm

No. of animals per sex per dose:

20 animals per sex per dose

Control animals:

yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
- All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. Clinical examinations were performed once daily. All significant findings were recorded. Detailed physical examinations were conducted on all animals weekly, beginning one week prior to test material administration, and prior to the scheduled necropsy. The daily clinical observation did not have to be conducted on days when detailed physical examinations were performed.

BODY WEIGHT: Yes
- Individual body weights were recorded weekly, beginning at least one week prior to test material administration. Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights were recorded prior to the scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Individual food consumption was recorded every other day, beginning approximately one week prior to test material administration.
- Food intake was calculated as g/animal/day for the corresponding body weight intervals.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

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

CLINICAL PATHOLOGY:
- Blood for clinical pathology evaluations (haematology and serum chemistry) were collected from all animals/sex/group at the scheduled necropsy (study week 13). Blood was collected from the vena cava under isoflurane anaesthesia at the time of necropsy. Five animals/sex/group were used for evaluation of haematology parameters and the remaining animals/sex/group were used for evaluation of serum chemistry parameters.
- Urine samples for urinalysis were collected over an approximate 24-hour time period in the last week of the dosing period from five animals/sex/group using metabolism cages. Urine was collected chilled (on ice).

HAEMATOLOGY PARAMETERS: Total Leukocyte Count (White Cells), Erythrocyte Count (Red Cells), Hemoglobin, Hematocrit, Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (Platelet), Differential Leukocyte Count (-Percent and Absolute, -Neutrophil, -Lymphocyte, -Monocyte, -Eosinophil and -Basophil), Platelet Estimate and Red Cell Morphology (RBC Morphology).

CLINICAL CHEMISTRY PARAMTERS: 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).

URINALYSIS PARAMETERS: pH, Colour (CLOR), Appearance (APP), Protein (PRO), Glucose (GLU), Bilirubin (BIL), Urobilinogen (URO), Ketones (KET), Occult Blood (BLD), Leukocytes (LEU) and Nitrites (NIT)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY:
- A complete necropsy was conducted on all animals. Animals were euthanised by isoflurane anaesthesia 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 viscera.
- 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, Brain (Cerebrum (2 levels) and Cerebellum with pons/medulla), Coagulating gland, 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), Harderian glands (2), Heart, Kidneys (2), Larynx, Lacrimal glands [exorbital (2)], Liver, Lungs (including bronchi, fixed by inflation with fixative), Lymph nodes (Mandibular and Mesenteric), Mammary gland (females only), Nasal cavity, Ovaries with oviducts (2), Pancreas, Parathyroids, Peripheral nerve (sciatic), Pharynx, Pituitary, Prostate, Salivary glands [mandibular (2)], Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Testes (2, Fixed in Bouin’s solution), Thymus, Thyroid, Tongue, Trachea, Urinary bladder, Uterus with cervix, Vagina, Zymbal’s gland and Gross lesions (when possible).
- The following organs were weighed from all animals at the scheduled necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries with oviducts, Spleen, Testes, Thymus, Thyroid with parathyroids and Uterus. Paired organs were weighed together. Thyroid/parathyroids were weighed following fixation. 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 listed in gross pathology from all animals in the control and 1200 ppm groups at the scheduled necropsy. In addition, the oesophagus, thyroid and all gross lesions were examined from the 133 and 400 ppm groups.

Statistics:

- 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.
- 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 statistically significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test article-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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test material-related clinical observations consisted of decreased defecation and faeces smaller than normal in the 1200 ppm group males and the 400 and 1200 ppm group females. These changes in excreta generally occurred during the first five weeks of the study. There were no other test material-related clinical observations. All other findings were noted with similar incidence in the control group, were limited to single animals and/or were findings commonly observed in mice of this age and strain.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male and one female in the 1200 ppm group were found dead on study days 60 and 9, respectively. No significant clinical findings were noted in either animal prior to death; however, both deaths were considered to be test material-related. All other animals survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related lower mean body weights were noted in the 400 and 1200 ppm group males and females. Mean cumulative body weight gains in the 1200 ppm group males and females were lower (statistically significant at p < 0.01) throughout the dosing period when compared to the control group. Mean cumulative body weight gains in the 400 ppm group males and females were also lower throughout the study (occasionally statistically significant at p < 0.05 for females). As a result of these lower cumulative body weight gains, mean body weights in the 400 and 1200 ppm groups were significantly (p<0.05 or p<0.01) lower throughout the study. By the end of the dosing period, mean body weights in the 1200 ppm group males and females were 17 and 11 % lower than the control group, respectively. Mean body weights in the 400 ppm group males and females were also 8 and 6 % lower than the control group by the end of the dosing period, respectively.
- One male in the 1200 ppm group had the lowest body weight of all males prior to being found dead on study day 56.
- There were no other remarkable changes in body weight.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Test material-related lower food consumption was noted in the 400 and 1200 ppm group males and females. Throughout the study, mean food consumption in the 400 and 1200 ppm group males and females was significantly (p < 0.05 or p < 0.01) lower than the control group values. During study week 0, mean food consumption in the 1200 ppm group males and females was 21 to 37 % lower than the control group. Mean food consumption in the 400 ppm group was 6 to 29 % lower than the control group during study week 0. By the end of the dosing period, mean food consumption in the 1200 ppm group was still 9 % lower than the control group.
- As a result of the lower food consumption, lower test material consumption was noted for the 1200 ppm group males and females. Actual doses of test material for this group were 165.8 and 153.3 mg/kg/day for males and females, respectively, during the first week of the study. As food consumption improved, compound consumption improved to 205.6 and 223.6 mg/kg/day for males and females, respectively, during study week 12. Average test material consumption, calculated as mg/kg/day for each study group, was as follows:
- 133 ppm group: males- 23.6 mg/kg/day and females: 26.8 mg/kg/day
- 400 ppm group: males- 65.3 mg/kg/day and females: 79.2 mg/kg/day
- 1200 ppm group: males- 212.0 mg/kg/day and females: 221.6 mg/kg/day

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. All findings were considered spontaneous and/or incidental in nature and unrelated to test material administration.

Haematological findings:

no effects observed

Description (incidence and severity):

- Haematology parameters were unaffected by test material administration. There were no statistically significant changes when the control and test material-treated groups were compared.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum chemistry parameters were unaffected by test material administration. There were no statistically significant changes when the control and test material-treated groups were compared.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis parameters were unaffected by test material administration. There were no statistically significant changes when the control and test material-treated groups were compared.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related increases in mean absolute and/or relative thyroid/parathyroid weights were noted in the 133, 400 and/or 1200 ppm groups. Mean absolute and relative (to final body weight and to brain weight) thyroid/parathyroid weights were significantly (p < 0.05 or p < 0.01) increased in the 133, 400 and 1200 ppm group males when compared to the control group. Thyroid/parathyroid weights are summarised in Table 1.
- There were no other test material-related changes in organ weights. Mean relative (to final body weight) brain weight was significantly (p<0.01) increased in the 1200 ppm group males. However, this change was attributed to the lower mean body weights in this group and was not considered to be a direct effect of the test material. Mean absolute and relative (to brain weight) adrenal gland weights in the 400 and 1200 ppm group females were decreased (statistically significant at p < 0.05) compared to the control group. Mean absolute and relative (to brain weight) kidney weights were decreased (statistically significant at p < 0.01 or p < 0.05) in the 1200 ppm group males. In addition, mean absolute ovary/oviduct weight in the 1200 ppm group females was significantly decreased (statistically at p < 0.05) when compared to the control group. Mean absolute epididymides weight in the 400 ppm group males was significantly (p < 0.05) decreased when compared to the control group. These organ weight variations were considered to be spontaneous and did not have histopathological correlates. Therefore, the changes were not considered test material-related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test material-related macroscopic findings in the animals that died early or in the animals that survived to the scheduled necropsy. All macroscopic findings were considered spontaneous and/or incidental in nature and unrelated to test material administration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- One male and one female in the 1200 ppm group were found dead on study days 60 and 9, respectively. Oesophageal hyperkeratosis was noted microscopically in the female. There were no test material-related microscopic findings in the male. However, both deaths were considered to be the result of test material administration.
- Test material-related microscopic changes were noted in the thyroid glands and the oesophagus in the 133, 400 and 1200 ppm groups. Microscopic findings in the thyroid glands of the 133, 400 and 1200 ppm group males and females consisted of increased accumulations of follicular colloid with corresponding attenuation (compression and/or atrophy) of follicular lining cells. Colloid accumulations were generally minimal to mild in all affected animals, and there was little difference in the incidence and severity among the 133, 400 and 1200 ppm groups. These findings in the thyroid were most likely an adaptive response to test material. The incidence of microscopic findings in the thyroid gland is summarised in Table 2.
- Test material-related oesophageal changes were characterised by an increase in the thickness of keratin deposits (hyperkeratosis) along the mucosal surface, without distinct morphologic alteration of the subjacent squamous epithelium. The incidence and severity of hyperkeratosis increased in a dose-related manner. Mild hyperkeratosis was noted in one male and one female in the 1200 ppm group. Hyperkeratosis in all animals in the 133 and 400 ppm groups and most animals in the 1200 ppm group was of minimal severity. In addition, hyperkeratosis in one male and two females in the 400 ppm group was associated with mild to moderate submucosal chronic inflammation. Increased oesophageal keratin in the 1200 ppm group may have corresponded to the slight reduction in food consumption. Hyperkeratosis likely resulted from local irritation of the test material upon the oesophageal mucosa. The incidence of microscopic findings in the oesophagus is also summarised in Table 2.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CONCLUSIONS

Haematology, serum chemistry and urinalysis parameters were unaffected by test material administration. No test material-related ophthalmic findings were noted, and there were no test material-related macroscopic changes.

Test material-related effects noted in the 1200 ppm group consisted of:
- Death of one male and one female.
- Clinical signs of decreased defecation and faeces smaller than normal during study weeks 1-5.
- Decreased mean body weight and mean cumulative body weight gains; mean body weight was 11 to 17 % lower than the control group by study week 13.
- Decreased mean food consumption throughout the study (most severe during study weeks 1 to 5).
- Increased mean absolute and relative thyroid/parathyroid weights.
- Microscopically observed increased accumulations of follicular colloid with corresponding attenuation of follicular lining cells in the thyroid gland and hyperkeratosis in the oesophagus.

Test material-related effects noted in the 400 ppm group consisted of:
- Clinical signs of decreased defecation and faeces smaller than normal during study weeks 1-5.
- Decreased mean body weight and mean cumulative body weight gains; mean body weight was 6 to 8 % lower than the control group by study week 13.
- Decreased mean food consumption throughout the study (most severe during study weeks 1 to 5).
- Increased mean absolute and relative thyroid/parathyroid weights.
- Microscopically observed increased accumulations of follicular colloid with corresponding attenuation of follicular lining cells in the thyroid gland and hyperkeratosis in the oesophagus.

Test material-related effects noted in the 133 ppm group consisted of:
- Increased mean absolute and relative thyroid/parathyroid weights.
- Microscopically observed increased accumulations of follicular colloid with corresponding attenuation of follicular lining cells in the thyroid gland and hyperkeratosis in the oesophagus.

Based on the increase in thyroid weights with correlating accumulation of follicular colloid and hyperkeratosis in the oesophagus in the 133 ppm group, the no-observed-effect level (NOEL) for oral (diet) administration of the test material to mice for at least 90 days was less than 133 ppm. The no-observed-adverse-effect level (NOAEL) was 400 ppm. The NOAEL is based on the reductions in body weight of less than 10 %, with corresponding decreased food consumption and clinical signs. The thyroid changes were most likely an adaptive response and therefore, not considered adverse. Hyperkeratosis in the oesophagus is likely a result of local irritation and therefore, not considered adverse.

Key result

Dose descriptor:

NOAEL

Effect level:

400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOEL

Effect level:

< 133 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Table 1: Thyroid/Parathyroid Weights (% Increase From Control Group)

Parameter	133 ppm		400 ppm		1200 ppm
	M	F	M	F	M	F
Absolute weight	34**	137	41**	55	34*	55
Relative to final body weight	38**	139	50**	61	56**	67
Relative to brain weight	39**	133	44**	52	40**	60

** = Significantly different from the control group at p < 0.01 using Dunnett’s test.

* = Significantly different from the control group at p < 0.05 using Dunnett’s test.

Table 2: Selected Microscopic Findings At The Scheduled Necropsy

Parameter		0 ppm		133 ppm		400 ppm		1200 ppm
		M	F	M	F	M	F	M	F
Thyroid: Increased Colloid	Minimal	1/9	1/10	3/10	5/10	7/10	8/10	6/9	6/9
	Mild	0/9	0/10	5/10	4/10	2/10	1/10	2/9	3/9
Thyroid: Cyst, ultimobranchial	Minimal	0/9	0/10	0/10	0/10	0/10	0/10	1/9	1/9
	Mild	1/9	0/10	0/10	0/10	0/10	0/10	2/9	0/9
Oesophagus: Hyperkeratosis	Minimal	0/10	0/10	1/10	2/10	7/10	7/10	8/9	7/9
	Mild	0/10	0/10	0/10	0/10	0/10	0/10	1/9	0/9
Oesophagus: Inflammation chronic	Minimal	0/10	0/10	0/10	0/10	0/10	2/10	0/9	0/9
	Mild	0/10	0/10	0/10	0/10	1/10	0/10	0/9	0/9

Conclusions:

Under the conditions of this study the the no-observed-effect level (NOEL) for oral (diet) administration of the test material to mice for at least 90 days was less than 133 ppm. The no-observed-adverse-effect level (NOAEL) was 400 ppm.

Executive summary:

The repeated dose oral toxicity of the test material was investigated in accordance with the standardised guidelines OECD 408, EPA OPPTS 870.3100 and JMAFF 12 NouSan No. 8147, under GLP conditions.

The objectives of the study were to evaluate the toxicity potential of the test material when administered orally via the diet to mice for 90 days and to assist in the selection of doses for a carcinogenicity study.

The test material was administered continuously in the diet to three groups mice for a minimum of 90 days. Dosage levels were 133, 400 and 1200 ppm. A concurrent control group received the control material, Placebo Microcapsules, on a comparable regimen. All four groups consisted of 10 animals/sex/group. All 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 and food consumption was recorded every other day. Clinical pathology evaluations (haematology, serum chemistry and urinalysis) were performed at the time of the scheduled necropsy (study week 13). Ophthalmic examinations were performed during study weeks -1 and 12. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues were examined microscopically from all animals.

Haematology, serum chemistry and urinalysis parameters were unaffected by test material administration. No test material-related ophthalmic findings were noted, and no test material-related macroscopic changes were observed at the scheduled necropsy.

Test material-related effects noted in the 1200 ppm group consisted of: Death of one male and one female. Clinical signs of decreased defecation and faeces smaller than normal during study weeks 1-5. Decreased mean body weight and mean cumulative body weight gains; mean body weight was 11 to 17 % lower than the control group by study week 13. Decreased mean food consumption throughout the study (most severe during study weeks 1 to 5). Increased mean absolute and relative thyroid/parathyroid weights. Microscopically observed increased accumulations of follicular colloid with corresponding attenuation of follicular lining cells in the thyroid gland and hyperkeratosis in the oesophagus.

Test material-related effects noted in the 400 ppm group consisted of: Clinical signs of decreased defecation and faeces smaller than normal during study weeks 1-5. Decreased mean body weight and mean cumulative body weight gains; mean body weight was 6 to 8 % lower than the control group by study week 13. Decreased mean food consumption throughout the study (most severe during study weeks 1 to 5). Increased mean absolute and relative thyroid/parathyroid weights. Microscopically observed increased accumulations of follicular colloid with corresponding attenuation of follicular lining cells in the thyroid gland and hyperkeratosis in the oesophagus.

Test material-related effects noted in the 133 ppm group consisted of: Increased mean absolute and relative thyroid/parathyroid weights. Microscopically observed increased accumulations of follicular colloid with corresponding attenuation of follicular lining cells in the thyroid gland and hyperkeratosis in the oesophagus.

Based on the increase in thyroid weights with correlating accumulation of follicular colloid and hyperkeratosis in the oesophagus in the 133 ppm group, the no-observed-effect level (NOEL) for oral (diet) administration of the test material to mice for at least 90 days was less than 133 ppm. The no-observed-adverse-effect level (NOAEL) was 400 ppm. The NOAEL is based on the reductions in body weight of less than 10 %, with corresponding decreased food consumption and clinical signs. The thyroid changes were most likely an adaptive response and therefore, not considered adverse. Hyperkeratosis in the oesophagus is likely a result of local irritation and therefore, not considered adverse.

Reference 4

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 July 2002 to 3 November 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4100 (Chronic Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 NouSan No. 8147

Deviations:

no

GLP compliance:

yes

Limit test:

no

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: 9.0 to 10.7 kg for the males and 8.1 to 9.4 kg for the females.
- Fasting period before study: no
- Housing: The animals were housed individually in 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 of diet was offered once daily, replenished approximately 3 hours after dose administration. Diet was supplemented with different diets in in order to stimulate appetite.
- Water: ad libitum
- Acclimation period: 13 days
- All animals were immunised prior to arrival at the testing laboratory. The immunisations included rabies, Bordetella, distemper, hepatitis, leptospirosis (DHL), parvovirus, parainfluenza and oral papilloma virus.

DETAILS OF FOOD AND WATER QUALITY: No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- Temperature: 18.8 to 21.9 °C
- Humidity: 32.0 to 62.8 %
- Photoperiod: Light timers were set to provide a 12-hour light (6 a.m. to 6 p.m.)/12-hour dark photoperiod.

Route of administration:

oral: capsule

Details on route of administration:

The selected route of administration was oral (oil in capsule) since this is anticipated to be a potential route of human exposure.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- 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:

SUMMARY
- The formulation in corn oil is a solution, thus it was not necessary to assess homogeneity.
- In a previous study, it was shown that the test material in corn oil was stable in a concentration range of 15-500 mg/mL.
- Samples for dose concentration confirmation were collected once during study weeks 0, 1, 3, 7 and 12, and at 6, 9 and 11 months. 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 contained the amounts of test material specified in the protocol.

GAS CHROMATOGRAPHY
- Instrument: Hewlett Packard 5890A (Series II) gas chromatograph equipped with an FID detector, a HP Headspace analyser
- Column: J & W Scientific GS-GasPro, 30 m × 0.316 mm ID (0.25-μm film thickness)
- Temperature (Program): 70 °C for 1.0 minute, ramp at 40 °C/minute to 230 °C, hold 2 minutes
- Carrier gas: Helium set at 12 psi (EPC constant flow on)
- Injector temperature: 225 °C
- Injection volume: 1 mL splitless
- Detector: FID at 225 °C
- Retention time: Approximately 4.2 minutes

HEADSPACE PARAMETERS
- Zone temperature: Oven 50 °C, Loop 100 °C and Transfer Line 100 °C
- Event Times GC cycle time 10 minutes, Vial EQ time 7 minutes, Pressurisation time 0.20 minutes, Loop fill time 0.20 minutes, Loop EQ time 0.05 minutes and 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 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 concentration range of 10 to 200 mg/mL.

PREPARATION OF QUALITY CONTROL STOCK SOLUTIONS: The quality control (QC) stock solutions were prepared as above. These stocks were prepared in the concentrations of 15 and 120 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. 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 test material was approximately 4.2 minutes. The total analysis time required for each run was approximately 7 minutes.
- The analysed formulations met the SOP requirements for concentration acceptability for suspension formulations, i.e., the analysed concentrations were within 15 % of the target dose concentrations, except for that of the Group 2 formulation prepared on July 30, 2002 (117 % of target). This formulation was re-prepared and reanalysed on August 1, 2002, and the results met acceptance criteria.

Duration of treatment / exposure:

7 days per week for a minimum of 52 weeks

Frequency of treatment:

Daily

Dose / conc.:

1.5 mg/kg bw/day (nominal)

Dose / conc.:

6 mg/kg bw/day (nominal)

Dose / conc.:

12 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4 dogs/sex/dose

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS:
- 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 2 hours (± 30 minutes) following dose administration (designated as 2 hours post-dosing for report presentation purposes).
- Detailed physical examinations were conducted on all animals weekly, beginning 1 week prior to test material administration and prior to the scheduled necropsy.

BODY WEIGHT: Yes
- Individual body weights were recorded weekly, beginning approximately 1 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 (fasted) were recorded prior to each scheduled necropsy.

FOOD CONSUMPTION
- Individual food consumption was recorded daily, beginning approximately 1 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.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Ocular examinations were conducted on all animals prior to the initiation of dose administration (study week -1) and near the end of the treatment period (study week 51). All ocular examinations were conducted using an indirect ophthalmoscope and slit lamp bio-microscope (or other suitable equivalent equipment) 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 25 and during the last week of dosing (study week 52). The animals were fasted overnight prior to blood collection while in metabolism cages for urine collection. Blood was collected from the jugular vein into tubes containing potassium EDTA (haematology) or sodium citrate (coagulation parameters) as the anticoagulant. Samples for serum chemistry were collected without anticoagulants.

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

CLINICAL CHEMISTRY PARAMETERS: 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: Triiodothyronine (Total T3), Thyroxide (Total T4), Thyroid Stimulating Hormone (TSH) and Reverse T3.

URINALYSIS PARAMETERS: 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.
- Clinical findings that were verified at necropsy were designated as CEO (correlates with externally observed). 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, Brain (Cerebrum level 1, Cerebrum level 2 and Cerebellum with medulla/pons), Epididymides (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: Adrenal glands, 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 4 to 8 microns, mounted on glass microscope slides and stained with haematoxylin and eosin. Microscopic examination was performed on all tissues listed in gross pathology from all animals at the scheduled necropsy and from all animals euthanized in extremis. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, not in plane of section or other reasons as appropriate.

Statistics:

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. 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 statistically significant (p < 0.05) intergroup variance, 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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- One 6.0 mg/kg/day group male and one 12.0 mg/kg/day group female were euthanised in extremis on study days 297 and 290, respectively. Clinical signs noted in these animals prior to euthanasia included clear material around the mouth, decreased defecation, dermal atonia, emesis, excessive drooling, hypoactivity, injected sclera, soft faeces, thinness and/or white foamy material around the mouth. These deaths were attributed to test material administration.
- All other animals survived to the scheduled necropsy. Test material-related clinical observations were noted throughout the study in the 6.0 and 12.0 mg/kg/day group males and females and included decreased defecation, diarrhoea, emesis, evidence of excessive salivation (clear material around the mouth and excessive drooling), head shaking, hypoactivity, increased incidences of injected sclera and green ocular discharge and white foamy material around the mouth. Excessive salivation extended to the 1.5 mg/kg/day group males and females.
- There were no other test material-related clinical observations. All other observations were noted with similar incidence in the control group, were limited to single animals, were not observed in a dose-related manner and/or were common findings for laboratory dogs of this age and breed.

Mortality:

mortality observed, treatment-related

Description (incidence):

One 6.0 mg/kg/day group male and one 12.0 mg/kg/day group female were euthanised in extremis on study days 297 and 290, respectively.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test material-related effects on body weights. However, several statistically significant differences from the control group were noted. Mean body weight change was significantly (p < 0.05) higher than the control group in the 1.5 mg/kg/day group males during study week 1 to 2, the 1.5 mg/kg/day group females during study week 12 to 13 and the 12.0 mg/kg/day group females during study week 16 to 17. Mean body weight change was significantly (p < 0.05 or p < 0.01) lower than the control group in the 1.5 mg/kg/day group males during study week 40 to 41, the 1.5 and 6.0 mg/kg/day group males during study week 21 to 22, the 12.0 mg/kg/day group males during study week 9 to 10 and the 12.0 mg/kg/day group females during study week 0 to 1. In addition, mean cumulative body weight change was significantly (p < 0.05 or p < 0.01) lower than the control group in the 6.0 mg/kg/day group males during study weeks 0 to 23 and in the 12.0 mg/kg/day group females during study weeks 0 to 2. Due to the sporadic nature of these changes and/or the lack of a dose response, these changes in body weights were not considered test material-related.

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 food consumption. However, mean food consumption in the 1.5 mg/kg/day group males during study week 29 to 30 and in the 1.5 mg/kg/day group females during study week 22 to 23 was significantly (p < 0.05) higher than the control group. Due to the lack of a dose response, these changes in food consumption were not considered test material-related.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmic lesions indicative of toxicity were observed in any of the test material-treated groups. All findings observed were typical in prevalence and appearance for laboratory dogs of this age and breed.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related higher mean platelet counts were noted in the 6.0 and 12.0 mg/kg/day groups. Mean platelet counts in the 6.0 and 12.0 mg/kg/day group males and females were higher than the control group at both the study week 25 and 52 evaluations; these changes were statistically significant (p < 0.05 or p < 0.01) in the 6.0 mg/kg/day group females at study week 52 and in the 12.0 mg/kg/day group males and females at study weeks 25 and 52.
- There were no other test material-related changes in haematology parameters. Mean red blood cell count in the 12.0 mg/kg/day group males and mean corpuscular haemoglobin concentration (MCHC) in the 1.5, 6.0 and 12.0 mg/kg/day group males were significantly (p<0.05) lower than the control group at the study week 25 evaluation. When compared to pre-test values, and in the absence of a dose response and similar changes in the opposite sex, these changes were not considered test material-related. At the study week 52 evaluation, mean activated partial thromboplastin time (APTT) in the 6.0 mg/kg/day group females was significantly (p < 0.05) lower than the control group. In the absence of a dose response and similar changes in the opposite sex, these alterations were considered to be incidental in nature and unrelated to test material administration.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related lower mean albumin levels were noted in the 12.0 mg/kg/day group at the study week 25 and 52 evaluations. Mean albumin levels in the 12.0 mg/kg/day group males and females were significantly (p < 0.05 or p < 0.01) lower than the control group at the study week 25 evaluation; the lower values persisted in the females at the study week 52 evaluation. As a result, mean albumin/globulin ratio (A/G) at study week 25 and mean total protein and serum calcium at study week 52 were significantly (p < 0.05 or p < 0.01) lower in the 12.0 mg/kg/day group females.
- Mean serum cholesterol in the 6.0 and 12.0 mg/kg/day group males and females was generally higher (occasionally statistically significant at p < 0.05) than the control group throughout the study. The toxicological significance of these alterations was unclear.
There were no other test material-related changes in serum chemistry parameters. Mean albumin level in the 6.0 mg/kg/day group females at study week 52 and mean total bilirubin level in the 12.0 mg/kg/day group females at study week 25 were significantly (p < 0.05 or p < 0.01) lower than the control group. Mean serum chloride in the 1.5 and 12.0 mg/kg/day group females was significantly (p < 0.05 or p < 0.01) higher than the control group at study week 52. When compared to pre-test values and in the absence of a dose response and similar changes in the opposite sex, these alterations were considered to be incidental in nature and unrelated to test material administration.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis parameters were unaffected by test material administration. There were no statistically significant differences when the control and test material-treated groups were compared.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related changes in organ weights were noted in the liver and thyroid/parathyroid glands in the 12.0 mg/kg/day group. Mean relative (to final body and/or brain weight) liver weights in the 12.0 mg/kg/day group males and/or females were significantly (p < 0.05) higher than the control group. The higher liver weights in both males and females were due to an increased incidence and severity of microscopically observed hepatocellular vacuolation (suggestive of increased glycogen deposition) in the 12.0 mg/kg/day group. In addition, mean absolute and relative (to final body and brain weight) thyroid/parathyroid weights in the 12.0 mg/kg/day group were lower than the control group; mean relative (to final body weight) thyroid weight in the 12.0 mg/kg/day group females was statistically significant (p < 0.05). These reductions were attributed to the colloid depletion observed microscopically in one male and one female in the 12.0 mg/kg/day group. There were no other test material-related changes in organ weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related macroscopic findings in the 6.0 mg/kg/day group male and 12.0 mg/kg/day group female euthanised in extremis were noted in the oesophagus (thickened and dark red or green raised areas), mandibular salivary gland (firm, reddened and/or enlarged) and/or the stomach (thickened and dark red discoloration).
- All other animals survived to the scheduled necropsy. Test material-related macroscopic findings were observed in the oesophagus, stomach and mandibular salivary gland of one 12.0 mg/kg/day group male. Thickened and discoloured areas in the oesophagus, firmness and enlargement of the mandibular salivary gland and thickening of the stomach were noted in this animal.
- There were no other test material-related macroscopic findings. Red discoloration throughout the gastrointestinal tract was noted in the 1.5, 6.0 and 12.0 mg/kg/day groups; however, these findings tended to correlate to minimal haemorrhage occurring at or near the time of necropsy and were not attributed to test material administration. Atrioventricular valve thickening was noted in two males in the 6.0 mg/kg/day group and one male in the 12.0 mg/kg/day group. This finding was attributed to valvular endocardiosis, a common finding in dogs and was not considered test material-related. Red discoloration of the trachea was also noted in one female in the 1.5 mg/kg/day group and one male in the 12.0 mg/kg/day group. Due to the lack of corresponding microscopic findings, this change was not considered test material-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- One male in the 6.0 mg/kg/day group and one female in the 12.0 mg/kg/day group were euthanised in extremis during the study. Both dogs were noted with extensive ulceration of the oesophagus; these findings were considered secondary to the irritative nature of the test material rather than a systemic effect. However, both deaths were considered test material-related. The same male was also noted with severe suppurative inflammation with necrosis and haemorrhage in the lungs. These changes contributed to the debilitation of the dog and most likely resulted from aspiration of the test material or ingesta. Other test material-related microscopic findings noted in the male and female that were euthanized in extremis included mucous cell hypertrophy and/or decreased secretion of the mandibular salivary gland and hyperplasia and/or decreased secretion of the stomach. Degeneration of the seminiferous tubules in the testes was also observed in the 6.0 mg/kg/day group male. All other microscopic findings were considered incidental or spontaneous and unrelated to test material administration.
- In the surviving animals, test material-related microscopic changes were noted in the thyroid gland and pituitary gland of the 12.0 mg/kg/day groups and the liver, testes, mandibular salivary gland, oesophagus and stomach of the 6.0 and 12.0 mg/kg/day groups. In the thyroid gland, severe colloid depletion and moderate follicular cell hypertrophy were observed in one 12.0 mg/kg/day group male, and luminal debris and mild colloid depletion were observed in one 12.0 mg/kg/day group female. These same dogs, as well as one control group female, were also noted with minimal to mild hyperplasia of basophilic cells within the pars distalis of the pituitary gland.
- In the testes, minimal to mild bilateral seminiferous tubule degeneration was noted in 2 of 4 males in the 12.0 mg/kg/day group. Minimal unilateral degeneration was observed in one dog each in the 1.5 and 6.0 mg/kg/day groups. In addition, one of these males in the 12.0 mg/kg/day group was observed to have minimal single cell necrosis of germ cells. Degeneration of the seminiferous tubules was characterised by a paucity of germ cells with or without vacuolation of Sertoli cells. Although seminiferous tubule degeneration occurred in a dose-related manner, the generally minimal severity, particularly following a 12-month dosing period, small group size, absence of corresponding testes weight decreases and the fairly common occurrence of these changes in young beagle dogs makes the toxicological significance of these changes uncertain. Oesophageal ulceration, mucous cell hypertrophy of the mandibular salivary gland, hyperplasia of the mucous neck and surface cells of the fundic stomach and mucous gland hyperplasia of the cardiac stomach and oesophagus were noted in one 12.0 mg/kg/day group male. These changes were considered test material-related.
- In the liver, vacuolar change was noted at an increased incidence and severity in the 12.0 mg/kg/day group. Vacuolar change was characterised by an increase in cell size due to an abundant amount of cleared cytoplasm consistent with glycogen accumulation. It is not uncommon to see small amounts of hepatocellular glycogen as a normal background change; however, in a few cases, including a control group male and female, the accumulation appeared excessive. Since glycogen content of the liver varies depending on the physiological condition of the animal, it is probable that vacuolar change was secondary to stress or anorexia and not a direct effect of test material administration.
- All other microscopic findings observed were consistent with normal background lesions in clinically normal dogs of the age and breed used on this study and were considered spontaneous and/or incidental in nature and unrelated to test material administration.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

SERUM HORMONES: Higher mean thyroid stimulating hormone (TSH) levels noted in the 12.0 mg/kg/day group males and females were considered test material-related, but these values were not statistically significant. These alterations were largely the result of two animals. The male had decreased T3 and T4 levels while the female had slightly lower T4 and reverse T3 levels. There were no other test material-related changes in serum hormone levels.

Details on results:

CONCLUSIONS
- There were no test material-related effects on body weights and food consumption. Urinalysis and ophthalmic parameters were unaffected by test material administration. One male in the 6.0 mg/kg/day group and one female in the 12.0 mg/kg/day group were euthanised in extremis during the study. All other animals survived to the scheduled necropsy. Test material-related clinical observations were noted in the 6.0 and 12.0 mg/kg/day group males and females throughout the study and included decreased defecation, diarrhoea, emesis, evidence of excessive salivation (clear material around the mouth and excessive drooling), head shaking, hypoactivity, soft faeces, thinness and white foamy material around the mouth. Excessive salivation extended into the 1.5 mg/kg/day group males and females.
- Higher mean platelet counts were noted in the 6.0 and 12.0 mg/kg/day groups at both the study week 25 and 52 evaluations. Lower mean serum albumin, total protein and serum calcium levels and albumin/globulin ratio were observed in the 12.0 mg/kg/day group at the study week 25 and/or 52 evaluations. Mean serum cholesterol in the 6.0 and 12.0 mg/kg/day group males and females tended to be higher than the control group throughout the study; the toxicological significance of these changes was unclear. Higher mean thyroid stimulating hormone (TSH) levels were noted in the 12.0 mg/kg/day group males and females; these changes were largely attributed to one male and one female.
- Test material-related macroscopic findings were noted in the 6.0 and 12.0 mg/kg/day group animals euthanized in extremis and in one 12.0 mg/kg/day group male that survived to the scheduled necropsy. These findings included thickening and discoloration of the oesophagus, firmness and enlargement of the mandibular salivary gland and thickening of the stomach. Microscopically, extensive ulceration of the oesophagus, mucous cell hypertrophy and/or decreased secretion of the mandibular salivary gland and hyperplasia and/or decreased secretion of the stomach were noted in these same animals. These effects were considered secondary to the irritative nature of the test material. Mild to severe colloid depletion and moderate follicular cell hypertrophy of the thyroid gland and minimal to mild hyperplasia of basophilic cells within the pars distalis of the pituitary gland were noted in one male and one female of the 12.0 mg/kg/day group. These animals were considered to have corresponding elevated TSH levels. Bilateral seminiferous tubule degeneration in the testes, and increased incidence and severity of vacuolar change in the liver were also noted in the 12.0 mg/kg/day group. Minimal unilateral seminiferous tubule degeneration was noted in one male each in the 1.5 and 6.0 mg/kg/day groups, although the toxicological significance is unknown. Higher mean relative (to final body and/or brain weight) liver weights and lower mean absolute and relative thyroid/parathyroid weights were noted in the 12.0 mg/kg/day group.
- When administered at 6.0 and 12.0 mg/kg/day, the irritative properties of the test material resulted in secondary effects of oesophageal ulceration and associated microscopic changes in the salivary glands and stomach. The local effect of ulceration was the cause of death for the 6.0 mg/kg/day group male and 12.0 mg/kg/day group female euthanised in extremis. Additional clinical pathology findings were noted at 6.0 and 12.0 mg/kg/day, as well as systemic microscopic findings in the liver and thyroid in the 12.0 mg/kg/day group.
- Based on the clinical findings of excessive salivation, as well as the potentially test material-related microscopic findings of seminiferous tubule degeneration of the testes extending to the 1.5 mg/kg/day group, the no-observed-adverse-effect level (NOAEL) for oral (capsule) administration of the test material to dogs for 52 consecutive weeks was 1.5 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Effect level:

1.5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

mortality

Critical effects observed:

not specified

Table 1: Summary of selected haematology, clinical chemistry clinical signs of toxicity and pathology findings

Dose (mg/kg/day)		0	1.5	6	15	0	1.5	6	15
Sex		Male				Female
No. in group		4	4	4	4	4	4	4	4
Haematology and clinical chemistry (mean values)
Platelet (thous/µL)	Week 25	280	270	342	387*	281	307	349	411*
	Week 52	312	330	374	478	314	347	422*	480**
Mean albumin (g/dL)	Week 25	3.5	3.6	3.2	3.0	3.6	3.6	3.3	2.9**
	Week 52	3.5	3.5	3.4	3.3	3.7	3.6	3.3**	2.7**
Total protein (g/dL)	Week 52	6.0	6.4	5.9	6.0	6.2	6.0	5.7	5.0**
TSH (ng/mL)	Week 52	0.1	0.2	0.1	4.2	0.3	0.3	0.2	2.7
Clinical signs of toxicity
Injected sclera (incidence expressed as % ±SD)	Pre-dose	11.2 ± 7.8	14.4 ± 14.4	7.7 ± 5.2	5.2 ± 2.5	1.7 ± 1.3	6.5 ± 4.6	21.6 ± 22.6	11.7 ± 15.2
	Post-dose	10.3 ± 8.4	8.3 ± 11.3	5.6 ± 5.0	3.9 ± 0.8	1.7 ± 1.5	3.7 ± 1.7	17.6 ± 17.1	10.6 ± 13.0
	Weekly	11.1 ± 9.3	7.0 ± 4.8	6.8 ± 4.4	10.3 ± 3.3	2.4 ± 1.7	3.8 ± 3.2	21.9 ± 23.5	6.6 ± 12.1
Organ Weights (relative to final body weights)
Thyroid/Parathyroid	-	0.009	0.008	0.009	0.007	0.01	0.007	0.009	0.005*
Histopathology (incidence)
Pituitary:	Hyperplasia, pars distalis	0 (4)	0 (4)	0 (3)	1 (4)	1 (4)	0 (4)	0 (4)	1 (3)
Thyroid:	Depleted colloid	0 (4)	0 (4)	0 (3)	1 (4)	0 (4)	0 (4)	0 (4)	1 (3)
	Hypertrophy follicular cell	0 (4)	0 (4)	0 (3)	1 (4)	0 (4)	0 (4)	0 (4)	1 (3)
	Infiltrate, lymphocyte	0 (4)	0 (4)	1 (3)	1 (4)	0 (4)	0 (4)	0 (4)	1 (3)
	Luminal debris	0 (4)	0 (4)	0 (3)	1 (4)	0 (4)	0 (4)	0 (4)	1 (3)

*Significant (p < 0.05) vs control;**Significant (p < 0.01) vs control

Conclusions:

Under the conditions of this study the no-observed-adverse-effect level (NOAEL) for oral (capsule) administration of the test material to dogs for 52 consecutive weeks was 1.5 mg/kg/day.

Executive summary:

The repeated dose toxicity of the test material was investigated in accordance with the standardised guidelines OECD 452, EPA OPPTS 870.4100 and JMAFF 12 NouSan No.8147, under GLP conditions.

The objective of this study was to evaluate the toxicity potential of the test material in corn oil when administered orally via capsule to dogs for a minimum of 12 months (52 weeks, 364 days). The test material in the vehicle, corn oil, was administered orally via capsules once daily, 7 days per week, for a minimum of 52 weeks at dosage levels of 1.5, 6.0 and 12.0 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 hormone and urinalysis) were performed prior to the initiation of dose administration (study week -1) and during study weeks 25 and 52 (last week of the dosing period). Ophthalmic examinations were performed during study weeks -1 and 51. Complete necropsies were performed on all dogs, and selected organs were weighed at the scheduled necropsy. Selected tissues were examined microscopically from all animals.

There were no test material-related effects on body weights and food consumption. Urinalysis and ophthalmic parameters were unaffected by test material administration. One male in the 6.0 mg/kg/day group and one female in the 12.0 mg/kg/day group were euthanised in extremis during the study. All other animals survived to the scheduled necropsy. Test material-related clinical observations were noted in the 6.0 and 12.0 mg/kg/day group males and females throughout the study and included decreased defecation, diarrhoea, emesis, evidence of excessive salivation (clear material around the mouth and excessive drooling), head shaking, hypoactivity, soft faeces, thinness and white foamy material around the mouth. Excessive salivation extended into the 1.5 mg/kg/day group males and females.

Higher mean platelet counts were noted in the 6.0 and 12.0 mg/kg/day groups at both the study week 25 and 52 evaluations. Lower mean serum albumin, total protein and serum calcium levels and albumin/globulin ratio were observed in the 12.0 mg/kg/day group at the study week 25 and/or 52 evaluations. Mean serum cholesterol in the 6.0 and 12.0 mg/kg/day group males and females tended to be higher than the control group throughout the study; the toxicological significance of these changes was unclear. Higher mean thyroid stimulating hormone (TSH) levels were noted in the 12.0 mg/kg/day group males and females; these changes were largely attributed to one male and one female.

Test material-related macroscopic findings were noted in the 6.0 and 12.0 mg/kg/day group animals euthanized in extremis and in one 12.0 mg/kg/day group male that survived to the scheduled necropsy. These findings included thickening and discoloration of the oesophagus, firmness and enlargement of the mandibular salivary gland and thickening of the stomach. Microscopically, extensive ulceration of the oesophagus, mucous cell hypertrophy and/or decreased secretion of the mandibular salivary gland and hyperplasia and/or decreased secretion of the stomach were noted in these same animals. These effects were considered secondary to the irritative nature of the test material. Mild to severe colloid depletion and moderate follicular cell hypertrophy of the thyroid gland and minimal to mild hyperplasia of basophilic cells within the pars distalis of the pituitary gland were noted in one male and one female of the 12.0 mg/kg/day group. These animals were considered to have corresponding elevated TSH levels. Bilateral seminiferous tubule degeneration in the testes, and increased incidence and severity of vacuolar change in the liver were also noted in the 12.0 mg/kg/day group. Minimal unilateral seminiferous tubule degeneration was noted in one male each in the 1.5 and 6.0 mg/kg/day groups, although the toxicological significance is unknown. Higher mean relative (to final body and/or brain weight) liver weights and lower mean absolute and relative thyroid/parathyroid weights were noted in the 12.0 mg/kg/day group.

When administered at 6.0 and 12.0 mg/kg/day, the irritative properties of the test material resulted in secondary effects of oesophageal ulceration and associated microscopic changes in the salivary glands and stomach. The local effect of ulceration was the cause of death for the 6.0 mg/kg/day group male and 12.0 mg/kg/day group female euthanised in extremis. Additional clinical pathology findings were noted at 6.0 and 12.0 mg/kg/day, as well as systemic microscopic findings in the liver and thyroid in the 12.0 mg/kg/day group.

Based on the clinical findings of excessive salivation, as well as the potentially test material-related microscopic findings of seminiferous tubule degeneration of the testes extending to the 1.5 mg/kg/day group, the no-observed-adverse-effect level (NOAEL) for oral (capsule) administration of the test material to dogs for 52 consecutive weeks was 1.5 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1.5 mg/kg bw/day

Study duration:

chronic

Species:

dog

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 June 2001 to 23 December 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 221 to 282 g for males and 162 to 213 g for females
- Housing: all animals were housed individually in clean, wire-mesh cages suspended above cage-board
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY: No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.4 to 22.6 °C
- Humidity: 40.4 to 63.1 %
- Photoperiod: 12-hour light/12-hour dark photoperiod

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: All animals were exposed simultaneously in four 2.0 m^3 stainless steel and glass exposure chambers. One chamber was dedicated for each group for the duration of exposures. Animals were individually caged in one to three cage batteries that were rotated around the cage rack positions within the chamber on a daily basis to minimize the effects of any potential variation due to chamber position. The control group was exposed to filtered air under conditions identical to those used for the test material exposure groups. The rats were removed from their home cages, placed in exposure caging in the animal room and transported to the exposure chambers for exposure to the test material. The animals were exposed for the requisite duration and then returned to their home cages. Food and water were withheld during each daily exposure period.
- Temperature, relative humidity, chamber ventilation rate, and negative pressure within the chambers were continually monitored and generally recorded every 35 minutes. Temperature: 22 to 25 °C, Humidity: 46 to 61 % and Chamber ventilation: 453 to 456 SLPM.
- Vapour atmospheres of the test material were generated using an ambient temperature bubbler-type vaporisation system, in which carrier gas (air) is dispersed (bubbled) through the liquid test material. In this process, the carrier gas picks up and vaporises the test material as the fine air bubbles pass through the liquid and into the vapour phase above the liquid. Glass gas washing bottles were used as bubblers. A 250 mL bottle with a 25 mm fritted glass cylinder for gas dispersion was used for chamber 2 and 250 mL bottles with a 50 mm fritted glass disc for gas dispersion was used for chambers 3 and 4. Liquid test material was added to the bubblers prior to daily exposures, as needed, and the bubblers were wrapped with aluminium foil to protect the test material from light. Regulated compressed air controlled by Swagelok® S-series metering needle valves was dispersed by the fritted disc and carried the test material vapours to the chamber inlet through ¼” Teflon delivery tubing. The generator airflow rate was monitored by compact rotameters; 1-280 mL/min rotameter for Chambers 2 and 3; and a 20 - 2100 mL/min rotameter for Chamber 4. These rotameters had been pre-calibrated prior to animal exposures using a mini Buck Flow Calibrator. The concentrated vapours were piped from the gas washing bottles to a 2” I.D. glass chamber inlet, where the concentration was diluted to the target level by the chamber ventilation air flow. Compressed air was also metered to the control chamber to maintain consistency between the control and exposure system.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

NOMINAL EXPOSURE CONCENTRATIONS
- A nominal exposure concentration was calculated for each daily exposure for each chamber from the total amount of test material used during the exposure and the total volume of air passed through the chamber during that day's exposure. The amount of test material used was obtained by weighing the gas-washing bottle containing the test material for each chamber prior to and after each daily exposure. The total volume of air passed through each chamber was calculated from the daily average chamber ventilation flow rate in litres per minute (LPM) and the exposure duration. The nominal concentration was calculated as follows:
ppm Iodomethane = (Wt. Iodomethane · Mol. Vol. · 10^6) / (MW · Ch. Flow · Exp. Dur.)
Where:
Wt. Iodomethane = weight of test material in grams
Mol. Vol. = Molar volume at 730 mmHg and 21 °C, 25.11 L/mole
10^6 = ppm conversion factor
MW = Iodomethane molecular weight, 141.94 g/mole
Ch. Flow = Daily average chamber flowrate for a given day, in LPM.
Exp. Dur. = Duration of a given day's exposure, in minutes

ACTUAL EXPOSURE CONCENTRATIONS
- Actual exposure concentrations were measured using a gas chromatograph (GC). Samples of the exposure atmospheres from each chamber were automatically collected at approximately 35-minute intervals using a sample loop and computer-controlled gas-sampling and multiposition valve. The following table summarises the GC conditions:
Instrument: Hewlett Packard 5890 Series II with a 3396 Series II integrator
Detector: Flame ionisation (FID)
Column: J & W DB-5, 30 m x 0.32 mm I.D., 0.25- micron film thickness
Gases: (Pressure (psig) Flow Rate (mL/min.)): Carrier - Helium 35 8.7, Fuel - Hydrogen 20 30, Air 40 300
Temperatures (°C): Injector 100, Column 60 and Detector 100
Injection volume (mL) 0.25
Retention time (min.) Approximately 0.84 min.
Integrator Run Parameters: Chart Zero Offset 0, Chart Attenuation 0, Chart Speed 1.5 cm/min, Peak Area Rejection Value 1200, Peak Threshold 0 and Peak Width 0.28
- During the animal exposure on 7/22/01 the gas chromatograph malfunctioned during the fourth round of sampling. Following the animal exposure, the gas chromatograph was replaced with a similar one using the original DB-5 column. However, analytical problems continued on 7/23/01 and two samples were collected from chambers 2, 3 and 5 in Tedlar gas sample bags to permit later determination of the exposure concentrations. Following the 7/23/01 exposure, the DB-5 column was replaced with a DB-Wax column and the gas chromatograph was calibrated by preparation of a prime calibration curve based on a single set of gas standards. Using this prime calibration curve, the samples collected on 7/23/01 were analysed. During the animal exposure period on 07/24/01, analytical problems continued due to retention time shifts that resulted in improper integration of multiple room air and chamber samples during the LabVIEW-controlled automated sampling rounds. It was again necessary to collect and analyse chamber samples collected in Tedlar gas sample bags. After conditioning the DB-Wax column and adjustment of specific GC run parameters, a complete prime calibration curve was created using three sets of standards prepared and analysed on 7/25/01 and 7/26/01. This prime curve was put in place starting on 7/27/01. For the animal exposures on 7/25/01 and 7/26/01, the chamber concentrations were calculated using calibration curves based on one set of gas standards and two sets of gas standards, respectively. Specifics concerning these changes and documentation of manual samples and gas chromatograph parameters appear in the study records. After making adjustments to the run parameters the final GC parameters are the following:
Instrument: Hewlett Packard 5890 Series II with a 3396 Series II integrator
Detector: Flame ionization (FID)
Column: J & W DB-Wax, 30 m x 0.25 mm I.D., 0.25-micron film thickness
Gases: (Pressure (psig) Flow Rate (mL/min.)): Carrier - Helium 35 8.7, Fuel - Hydrogen 20 30, Air 40 300
Temperatures (°C): Injector 65, Column 40 and Detector 75
Injection volume (mL) 0.25
Retention time (min.) Approximately 0.84 min.
Integrator Run Parameters: Chart Zero Offset 0, Chart Attenuation 0, Chart Speed 1.0 cm/min, Peak Area Rejection Value 0, Peak Threshold 0 and Peak Width 0.04.
- The chromatograph was standardised using 40-liter Tedlar® gas bags prepared to contain known concentrations of the test material. The standard bags were prepared by injecting known volumes of test material into a 500 mL glass vaporisation bulb. A continuous flow of air carried the vaporised test material to a 40 L bag. The total volume of air was measured by a dry test meter (Model DTM-200A, American Meter Co., Nebraska City, PA). Concentrations of the gas-phase standards were calculated as follows: Concentration = (VOL · R · T · D · 10^-3 · 10^6)
L · GMW · P
Where:
Conc. is in ppm
VOL = volume of test material vaporised into bag in μL
R = universal gas constant, 62.36 L mmHg/mole K
T = nominal laboratory temperature in K (273 + 21 °C = 294 K)
D = density of the test material, 2.280 g/mL
L = volume of air used to prepare bag, 32 L
GMW = gram molecular weight, 141.94 g/mole
P = nominal laboratory barometric pressure, 730 mmHg
10^-3 = μL to mL conversion factor
10^6 = conversion factor to ppm
- Standards prepared for this study: 3.8 ppm: 0.3 µL test material and 32 L air, 19 ppm: 1.5 µL test material and 32 air, 39 ppm: 3.1 µL test material and 32 L air, 58 ppm: 4.6 µL test material and 32 L air and 78 ppm: 6.2 µL test material and 32 L air. Each standard was prepared in triplicate prior to the exposure period and analysed with the GC. A least-squares line was fitted to the resulting peak areas. Concentrations were then calculated using the slope and intercept of this prime calibration curve. On a daily basis, the integrity of the prime calibration curve was checked by analysing one freshly prepared standard. On a rotational basis, a different one of the five standards was used each day. If the analysed concentration were within ± 10 % of the known concentration, the GC was considered within calibration specifications.

DETERMINATION OF HOMOGENEITY OF EXPOSURE ATMOSPHERES
- Evaluation of the homogeneity of exposure concentrations was accomplished during the method development phase of the study prior to animal exposures. Four test locations and a reference location were used for these determinations. The test locations were Right Lower Front, Right Upper Rear, Left Lower Rear, Left Upper Front identified as 1, 2, 3 and 4, respectively. Samples were collected as rapidly as possible always collecting a sample from the reference location and then from one of the four test locations. For each test location, the measured concentration was calculated as a percent difference from the reference location. The homogeneity determination was performed in triplicate for chamber 5, 70 ppm, exposure chamber.
- Results indicated that homogeneity of exposure atmospheres were adequate for the purpose of this study. Maximum mean % from reference was – 3.2 %.

RESULTS
- Nominal Exposure Concentrations: The overall mean nominal concentrations for the 4 week period were 7.8 ppm, 25 ppm, and 78 ppm for the 5 ppm, 20 ppm, and 70 ppm groups, respectively. The overall mean nominal concentrations for the 13 week period were 7.1 ppm, 25 ppm and 78 ppm for the 5 ppm, 20 ppm and 70 ppm groups, respectively.
- Actual Exposure Concentrations: The overall mean concentrations for the 4 week period were 5 ppm, 20 ppm, and 70 ppm for the 5 ppm, 20 ppm, and 70 ppm groups, respectively. The overall mean concentrations for the 13 week period were 5 ppm, 21 ppm and 70 ppm for the 5 ppm, 20 ppm and 70 ppm groups, respectively.

Duration of treatment / exposure:

5 days/week for either 4 (minimum of 20 exposures) or 13 weeks (minimum of 65 exposures)

Frequency of treatment:

daily (6hours/day)

Dose / conc.:

5 ppm (analytical)

Remarks:

4 week and 13 week periods

Dose / conc.:

20 ppm (analytical)

Remarks:

4 week period

Dose / conc.:

21 ppm (analytical)

Remarks:

13 week period

Dose / conc.:

70 ppm (analytical)

Remarks:

4 week and 13 week periods

No. of animals per sex per dose:

20 animals/sex/group (10 animals/sex/group were euthanised and necropsied following four weeks of exposure; the remaining 10 animals/sex/group were euthanised and necropsied following 13 weeks of exposure).

Control animals:

yes

Details on study design:

- Dose selection rationale: The exposure levels used on this study were chosen based upon the results of a previous inhalation study of Iodomethane in rats

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.
- Clinical examinations were performed twice daily, prior to exposure and following exposure (up to two hours following exposure). Pre-exposure clinical examinations were not performed on days when detailed physical examinations were conducted. On non-dosing days, the animals were observed once daily. All significant findings were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Detailed physical examinations were conducted on all animals weekly, beginning one week prior to the initiation of test material exposure and prior to the scheduled necropsies. For detailed examinations, the animals were removed from their home cages and placed in a standard arena for observations. Observations were detailed and carefully noted. When appropriate, explicitly defined scoring systems were used if, in the opinion of the study director, doing so would increase the utility of the data. Signs noted included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling, as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behaviour (e.g., self-mutilation, walking backwards) were recorded.

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

FOOD CONSUMPTION:
- Individual food consumption was recorded weekly, beginning one week prior to test material exposure (study week -1). Food intake was calculated as g/animal/day for the corresponding body weight intervals. When food consumption could not be measured for a given interval (due to spillage, weighing error, obvious erroneous value, etc.), the appropriate interval was footnoted as "NA" (Not Applicable) on the individual tables.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Ocular examinations were conducted on all animals prior to the initiation of dosing (week -1), prior to the interim necropsy (study week 4) and during the last week of dosing (study week 12). All ocular examinations were conducted using an indirect ophthalmoscope (or other suitable equivalent equipment), preceded by pupillary dilation with an appropriate mydriatic agent.

CLINICAL PATHOLOGY
- Blood samples for clinical pathology evaluations (haematology and serum chemistry) were collected from all animals at the scheduled necropsies (study weeks 4 and 13). The animals were fasted overnight prior to the collection of blood samples. Blood was collected from the vena cava at the time of necropsy. Blood for haematology assessment was collected into tubes containing EDTA as the anticoagulant; sodium citrate was used as the anticoagulant for clotting parameters.

- HAEMATOLOGY PARAMETERS: 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 and Basophil, Platelet Estimate and Red Cell Morphology (RBC Morphology).

- SERUM CHEMISTRY PARAMETERS: 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 (Aspartate Transfer), Gamma Glutamyltransferase (Glutamyl Transfer), Glucose, Total Cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium and Sodium.

Sacrifice and pathology:

GROSS PATHOLOGY
- A complete necropsy was conducted on all animals. Animals were euthanised by isoflurane inhalation 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 viscera.
- The following tissues and organs were collected and placed in 10 % neutral buffered formalin (except as noted): Adrenals glands, Aorta, Bone with marrow (Femur and Sternebrae), Bone marrow smear(femur), Brain (forebrain, midbrain, hindbrain), Exorbital lacrimal gland, Eyes with optic nerve (Fixed in Davidson’s solution), Gastrointestinal tract (Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum), Harderian glands, Heart, Kidneys, Larynx, Liver (sections of two lobes), Lungs (including bronchi, fixed by inflation with fixative), Lymph nodes, Mediastinal, Tracheobronchial, Mammary gland (females only), Nasal tissues, Ovaries with oviducts, Pancreas, Parathyroids, Peripheral nerve (sciatic), Pituitary, Prostate, Salivary glands (mandibular), Seminal vesicles, Skeletal muscle (rectus femoris), Skin, Spinal cord (cervical, thoracic, lumbar), Spleen, Testes with epididymides (Fixed in Bouin’s solution), Thymus, Thyroids, Trachea, Urinary bladder, Uterus with vagina and Gross lesions.

ORGAN WEIGHTS
- The following organs were weighed from all animals at the scheduled necropsies: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Lung (prior to inflation with fixative), Ovaries with oviducts, Spleen, Testes, Thymus, Thyroid with parathyroids and Uterus (Only weighed at the study week 13 necropsy).
- The thyroids with parathyroids were weighed after fixation. Paired organs were weighed together. Organ to final body weight ratios were calculated.

SLIDE PREPARATION AND MICROSCOPIC EXAMINATION
- After fixation, protocol-specified tissues were trimmed according to standard operating procedures and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned at five to eight microns, mounted on glass microscope slides and stained with haematoxylin and eosin. Following collection of the protocol-specified tissues, the entire head was removed and preserved. Following decalcification, six cross-sections of the nasal cavities were prepared for microscopic examination.
- Microscopic examination was performed on all tissues listed above from all animals in the control and 70 ppm groups at the scheduled necropsies. The kidneys, larynx, liver, lungs, nasal tissues, trachea and gross lesions were examined from all animals in the 5 and 20 ppm groups at the scheduled necropsies.

Statistics:

- All analyses were conducted using two-tailed tests 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.
- All statistical tests were performed using appropriate computing devices or programs. 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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- An increased incidence of wet yellow material on the urogenital area was noted for the 70 ppm group males and females following exposure.
- There were no other test material-related clinical observations. All findings noted in the test material-exposed groups were observed with similar incidence in the control group, were limited to single animals, were not observed in a dose-related manner and/or were common findings for laboratory rats of this age and strain.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled necropsies.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related effects on mean body weights and body weight gains were observed in the 70 ppm group males and females.
- For males, significantly (p<0.01) lower mean body weight gains were observed for study weeks 0 to 1, 1 to 2 and 4 to 5. As a result of these reductions, mean body weights were significantly (p<0.05 or p<0.01) lower in the 70 ppm group males during study weeks 2, 3, 5 and 6. The maximum decrease in mean body weight was observed during study week 6 (13 % relative to control). At the end of the study (study week 13), mean cumulative body weight gain was 15 % lower and mean body weight was 8 % lower when compared to the control group.
-For females, significantly (p<0.05 or p<0.01) reduced mean body weight gains were observed for study weeks 0 to 1, 4 to 5 and 5 to 6. The maximum decrease for females in mean body weight, although not statistically significant, was observed during study week 6 (9 %). At the end of the study (study week 13), mean cumulative body weight gain was 17 % lower and mean body weight was 7 % lower than the control group.
- There were no other test material-related effects on body weight data. Several other statistically significant (p<0.05 or p<0.01) differences from the control group were noted in mean body weight gains in the various groups. However, no trends were apparent to attribute the changes to test material exposure.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Slight, statistically significant (p<0.01) reductions in mean food consumption were observed in the 70 ppm group males for study weeks 0 to 1 and 4 to 5.
- These reductions were attributed to the test material since they corresponded with periods of lower body weight gains. There were no other test material-related effects on food consumption.

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 ophthalmic lesions attributed to test material exposure.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test material-related effects on haematology parameters.
- Mean prothrombin time in the 70 ppm group males was significantly (statistically at p<0.01) lower than the control group at study week 4. Mean activated partial thromboplastin time (APTT) in the 20 and 70 ppm group males was also significantly (p<0.05) lower than the control group at study week 4. However, decreases in prothrombin time and APTT are usually not considered toxicologically significant; therefore, it is unlikely that these changes were the result of test material exposure.
- There were no other remarkable changes in haematology parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- The only test material-related effect on serum chemistry parameters was increased total cholesterol in the 70 ppm group males and females. Mean cholesterol was significantly (p<0.01) higher than the control group in the 70 ppm group males and females at both study week 4 (55 and 67 %, respectively) and study week 13 (56 and 43 %, respectively). There were no other test material-related effects on serum chemistry parameters.
- At the study week 4 evaluation, mean total bilirubin in the 5 ppm group females was significantly (p<0.01) higher than the control group and mean sodium in the 70 ppm group females was significantly (p<0.05) higher than the control group. These changes were slight and/or not observed in a dose-related manner; therefore, they were considered spontaneous and unrelated to test material exposure.

Urinalysis findings:

not examined

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 adverse test material-related effects on organ weight data. Mean liver weights relative to final body weights were increased in a dose-related manner in males and females at study week 13 when compared to the control group. For males, the increases in relative liver weight were 3, 8 and 15 % in the 5, 20 and 70 ppm groups, respectively. For females, the increases in relative liver weight were 3, 11 and 22 % in these same groups, respectively. The differences from the control group were statistically significant (p<0.05 or p<0.01) in the 20 ppm group females and the 70 ppm group males and females. At study week 4, a significantly (p<0.01) higher mean liver weight relative to final body weight was noted for the 70 ppm group females (8 %). Increased liver weights are commonly observed following treatment with chemical agents and are not considered to be adverse without the presence of correlating histopathological findings.
- No other test material-related effects on organ weights were observed. However, several statistically significant (p<0.05 or p<0.01) differences from the control group were noted. Most of these differences were a result of the lower final body weights (not statistically significant) in the 70 ppm group and consisted of lower mean absolute heart weight in the 70 ppm group males and higher mean relative heart and lower mean absolute adrenal gland weights in the 70 ppm group females at study week 13. In addition, mean absolute heart weight was decreased in the 20 ppm group males at the study week 13 necropsy. These differences were not attributed to test material exposure since there were no microscopic correlates or treatment-related effects in these organs at the 70 ppm exposure level and since the changes noted for absolute weights were not present for relative organ weights. There were no other remarkable differences from the control group in organ weight data.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- There were no test material-related macroscopic findings at the interim or primary necropsies. Those findings observed were noted with similar incidence in the control group, were limited to single animals in various groups and/or were findings commonly observed in laboratory rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the study week 4 interim necropsy, test material-related alterations were observed in the nose of the 70 ppm group. Minimal to mild degeneration of the olfactory epithelium, primarily in the dorsal meatus and on the dorsal septum and upper turbinates, was observed in the 70 ppm group males and females. Degeneration included disruption of the normal epithelial architecture (loss of dendritic processes, disorganisation of the nuclear layers and/or presence of enlarged ducts of Bowman’s gland in the nuclear layer), and either decreased or increased cellularity. Consistent with previous findings with this compound, degeneration was diagnosed even when regeneration was obvious because the degeneration clearly preceded the regenerative response. Degeneration/regeneration was observed in the 70 ppm group males at nasal levels 3-6 and in the 70 ppm group females at nasal levels 2-5. Respiratory epithelial metaplasia, the presence of single-layered ciliated epithelium admixed with olfactory epithelium, was noted at nasal level 2 in both sexes of the 70 ppm group and was also considered a test material-related effect. Respiratory epithelial metaplasia has been reported as a regenerative response following injury and loss of the olfactory epithelium6. At the study week 13 primary necropsy, test material-related alterations were again confined to the nose and consisted of degeneration/regeneration of the olfactory mucosa at nasal levels 2 through 6 in 70 ppm group males and females. Incidence and/or severity of degeneration at nasal levels 3, 4, and 5 were increased compared to the study week 4 interim necropsy. Two males and one female in the 70 ppm group were also observed to have metaplasia of the respiratory epithelium at nasal level 2 and this alteration was also considered related to test material exposure. No other test material-related findings were observed at study week 13. Degeneration was noted in one 20 ppm group female at nasal levels 4 and 5 but because of the occurrence of this lesion in control group animals at the study week 4 interim necropsy, and because similar lesions were not observed in the 20 ppm group males, this incidence was not considered related to treatment. A slightly increased incidence of intra-alveolar macrophages and subacute inflammation in the lungs was observed in the 5 ppm group males. However, the incidence of these alterations was not increased in the females, and there was no dose-relationship. Thus, these alterations were not considered related to test material exposure.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEC

Effect level:

20 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

histopathology: non-neoplastic

Critical effects observed:

not specified

Table 1: Body weights during the study

Week	Mean Average Bodyweight (g)
	0 ppm		5 ppm		20 ppm		70 ppm
	M	F	M	F	M	F	M	F
-1	199	167	202	165	201	163	202	163
0	247	182	247	182	248	182	247	182
1	290	207	281	204	284	205	276	198
2	315	222	306	215	306	217	292**	209
3	338	231	329	226	331	227	314*	218
4	356	236	350	236	352	238	335	228
5	389	251	369	246	364	246	348**	235
6	418	266	392	250	376	252	365**	242
7	420	260	402	254	396	258	379	243
8	435	266	411	266	405	266	397	253
9	444	271	426	269	422	272	406	256
10	458	275	437	272	435	274	422	260
11	474	278	449	276	443	276	425	260
12	484	288	459	284	449	284	442	264
13	484	286	465	285	459	283	445	267

* = Significantly different from the control group at 0.05 using Dunnett’s test

** = Significantly different from the control group at 0.01 using Dunnett’s test

 

Table 2: Weekly food consumption during the study (g/animal/day)

Week	Mean Average Food Consumption (g/animal/day)
	0 ppm		5 ppm		20 ppm		70 ppm
	M	F	M	F	M	F	M	F
-1 - 0	21	17	20	16	22	16	21	15
0 – 1	22	17	21	16	21	17	19**	16
1 – 2	22	18	21	17	21	18	22	18
2 – 3	22	18	22	18	22	18	22	18
3 – 4	23	18	22	17	23	18	22	18
4 – 5	25	18	23	17	24	17	22**	18
5 - 6	26	18	24	18	23	19	23	18
6 – 7	26	19	25	19	24	20	24	19
7 – 8	25	18	23	17	23	19	23	18
8 – 9	26	18	24	18	23	19	24	19
9 – 10	26	19	25	18	24	19	23	18
10 – 11	26	18	24	18	24	19	24	20
11 - 12	24	17	23	18	22	18	22	16
12 – 13	24	18	25	17	23	18	25	18

** = Significantly different from the control group at 0.01 using Dunnett’s test

 

Table 3: Clinical Chemistry Cholesterol Levels

Week	Mean Average Cholesterol Level (mg/dL)
	0 ppm		5 ppm		20 ppm		70 ppm
	M	F	M	F	M	F	M	F
4	44	49	55	62	51	62	68**	82**
13	48	70	50	73	55	83	75**	100**

** = Significantly different from the control group at 0.01 using Dunnett’s test

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for whole-body inhalation exposure to the test material in rats for four or 13 weeks was 20 ppm.

Executive summary:

The repeated dose inhalation toxicity of the test material was investigated in accordance with the standardised guideline OPPTS 870.3465, under GLP conditions.

The test material was administered six hours/day, five days/week via whole-body inhalation exposure for either four (minimum of 20 exposures) or 13 weeks (minimum of 65 exposures) to three groups (Groups 2-4) of male and female Crl:CD(SD)IGS BR rats. Dosage levels were 5, 20 and 70 ppm. A concurrent control group (Group 1) received filtered air on a comparable regimen. Groups 1-4 each consisted of 20 animals/sex/group. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded weekly. Clinical pathology evaluations (haematology and serum chemistry) were performed at the scheduled necropsies. Ophthalmic examinations were performed during study weeks -1, 4 and 12. Following four weeks of exposure, 10 rats/sex/group were euthanized (interim necropsy). The remaining 10 rats/sex/group were euthanized after 13 weeks of exposure (primary necropsy). Complete necropsies were conducted on all animals, and selected organs were weighed. Selected tissues were examined microscopically.

All animals survived to the scheduled necropsies. There were no test material-related effects on haematology parameters. No test material-related ophthalmic findings were observed. There were no test material- related macroscopic findings at either necropsy. No adverse test material-related changes were noted in the 5 and 20 ppm groups. Mean relative to final body weight liver weight was increased in the 20 ppm group but was not considered adverse due to the absence of correlating histopathological findings. Test material-related effects noted in the 70 ppm group consisted of: An increased incidence of wet yellow material on the urogenital area for males and females following exposure. Lower mean body weight gains periodically during the exposure period resulting in mean cumulative body weight gains that were 15 and 17 % lower than the control group for males and females, respectively, at study week 13. In addition, mean body weights were 8 and 7 % lower for males and females, respectively, by the end of the study. Lower mean food consumption periodically (study weeks 0 to 1and 4 to 5) for males. Increased mean serum cholesterol levels for both males and females at study weeks 4 and 13. The toxicological significance of these cholesterol changes was unknown. Higher mean liver weight relative to final body weight (15 and 22% for males and females, respectively) were observed at study week 13. These changes were not considered adverse since there were no correlating microscopic changes in the liver. Microscopic changes consisted of degeneration/regeneration of the olfactory epithelium in males at nasal levels 3-6 and in females at nasal levels 2-5 at study week 4 and at nasal levels 2-6 in males and females at study week 13. Respiratory epithelial metaplasia was also noted at nasal level 2 in both sexes at study weeks 4 and 13.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for whole-body inhalation exposure to the test material in rats for four or 13 weeks was 20 ppm.

Endpoint conclusion

Dose descriptor:

NOAEC

116 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 June 2001 to 23 December 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 221 to 282 g for males and 162 to 213 g for females
- Housing: all animals were housed individually in clean, wire-mesh cages suspended above cage-board
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY: No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.4 to 22.6 °C
- Humidity: 40.4 to 63.1 %
- Photoperiod: 12-hour light/12-hour dark photoperiod

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: All animals were exposed simultaneously in four 2.0 m^3 stainless steel and glass exposure chambers. One chamber was dedicated for each group for the duration of exposures. Animals were individually caged in one to three cage batteries that were rotated around the cage rack positions within the chamber on a daily basis to minimize the effects of any potential variation due to chamber position. The control group was exposed to filtered air under conditions identical to those used for the test material exposure groups. The rats were removed from their home cages, placed in exposure caging in the animal room and transported to the exposure chambers for exposure to the test material. The animals were exposed for the requisite duration and then returned to their home cages. Food and water were withheld during each daily exposure period.
- Temperature, relative humidity, chamber ventilation rate, and negative pressure within the chambers were continually monitored and generally recorded every 35 minutes. Temperature: 22 to 25 °C, Humidity: 46 to 61 % and Chamber ventilation: 453 to 456 SLPM.
- Vapour atmospheres of the test material were generated using an ambient temperature bubbler-type vaporisation system, in which carrier gas (air) is dispersed (bubbled) through the liquid test material. In this process, the carrier gas picks up and vaporises the test material as the fine air bubbles pass through the liquid and into the vapour phase above the liquid. Glass gas washing bottles were used as bubblers. A 250 mL bottle with a 25 mm fritted glass cylinder for gas dispersion was used for chamber 2 and 250 mL bottles with a 50 mm fritted glass disc for gas dispersion was used for chambers 3 and 4. Liquid test material was added to the bubblers prior to daily exposures, as needed, and the bubblers were wrapped with aluminium foil to protect the test material from light. Regulated compressed air controlled by Swagelok® S-series metering needle valves was dispersed by the fritted disc and carried the test material vapours to the chamber inlet through ¼” Teflon delivery tubing. The generator airflow rate was monitored by compact rotameters; 1-280 mL/min rotameter for Chambers 2 and 3; and a 20 - 2100 mL/min rotameter for Chamber 4. These rotameters had been pre-calibrated prior to animal exposures using a mini Buck Flow Calibrator. The concentrated vapours were piped from the gas washing bottles to a 2” I.D. glass chamber inlet, where the concentration was diluted to the target level by the chamber ventilation air flow. Compressed air was also metered to the control chamber to maintain consistency between the control and exposure system.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

NOMINAL EXPOSURE CONCENTRATIONS
- A nominal exposure concentration was calculated for each daily exposure for each chamber from the total amount of test material used during the exposure and the total volume of air passed through the chamber during that day's exposure. The amount of test material used was obtained by weighing the gas-washing bottle containing the test material for each chamber prior to and after each daily exposure. The total volume of air passed through each chamber was calculated from the daily average chamber ventilation flow rate in litres per minute (LPM) and the exposure duration. The nominal concentration was calculated as follows:
ppm Iodomethane = (Wt. Iodomethane · Mol. Vol. · 10^6) / (MW · Ch. Flow · Exp. Dur.)
Where:
Wt. Iodomethane = weight of test material in grams
Mol. Vol. = Molar volume at 730 mmHg and 21 °C, 25.11 L/mole
10^6 = ppm conversion factor
MW = Iodomethane molecular weight, 141.94 g/mole
Ch. Flow = Daily average chamber flowrate for a given day, in LPM.
Exp. Dur. = Duration of a given day's exposure, in minutes

ACTUAL EXPOSURE CONCENTRATIONS
- Actual exposure concentrations were measured using a gas chromatograph (GC). Samples of the exposure atmospheres from each chamber were automatically collected at approximately 35-minute intervals using a sample loop and computer-controlled gas-sampling and multiposition valve. The following table summarises the GC conditions:
Instrument: Hewlett Packard 5890 Series II with a 3396 Series II integrator
Detector: Flame ionisation (FID)
Column: J & W DB-5, 30 m x 0.32 mm I.D., 0.25- micron film thickness
Gases: (Pressure (psig) Flow Rate (mL/min.)): Carrier - Helium 35 8.7, Fuel - Hydrogen 20 30, Air 40 300
Temperatures (°C): Injector 100, Column 60 and Detector 100
Injection volume (mL) 0.25
Retention time (min.) Approximately 0.84 min.
Integrator Run Parameters: Chart Zero Offset 0, Chart Attenuation 0, Chart Speed 1.5 cm/min, Peak Area Rejection Value 1200, Peak Threshold 0 and Peak Width 0.28
- During the animal exposure on 7/22/01 the gas chromatograph malfunctioned during the fourth round of sampling. Following the animal exposure, the gas chromatograph was replaced with a similar one using the original DB-5 column. However, analytical problems continued on 7/23/01 and two samples were collected from chambers 2, 3 and 5 in Tedlar gas sample bags to permit later determination of the exposure concentrations. Following the 7/23/01 exposure, the DB-5 column was replaced with a DB-Wax column and the gas chromatograph was calibrated by preparation of a prime calibration curve based on a single set of gas standards. Using this prime calibration curve, the samples collected on 7/23/01 were analysed. During the animal exposure period on 07/24/01, analytical problems continued due to retention time shifts that resulted in improper integration of multiple room air and chamber samples during the LabVIEW-controlled automated sampling rounds. It was again necessary to collect and analyse chamber samples collected in Tedlar gas sample bags. After conditioning the DB-Wax column and adjustment of specific GC run parameters, a complete prime calibration curve was created using three sets of standards prepared and analysed on 7/25/01 and 7/26/01. This prime curve was put in place starting on 7/27/01. For the animal exposures on 7/25/01 and 7/26/01, the chamber concentrations were calculated using calibration curves based on one set of gas standards and two sets of gas standards, respectively. Specifics concerning these changes and documentation of manual samples and gas chromatograph parameters appear in the study records. After making adjustments to the run parameters the final GC parameters are the following:
Instrument: Hewlett Packard 5890 Series II with a 3396 Series II integrator
Detector: Flame ionization (FID)
Column: J & W DB-Wax, 30 m x 0.25 mm I.D., 0.25-micron film thickness
Gases: (Pressure (psig) Flow Rate (mL/min.)): Carrier - Helium 35 8.7, Fuel - Hydrogen 20 30, Air 40 300
Temperatures (°C): Injector 65, Column 40 and Detector 75
Injection volume (mL) 0.25
Retention time (min.) Approximately 0.84 min.
Integrator Run Parameters: Chart Zero Offset 0, Chart Attenuation 0, Chart Speed 1.0 cm/min, Peak Area Rejection Value 0, Peak Threshold 0 and Peak Width 0.04.
- The chromatograph was standardised using 40-liter Tedlar® gas bags prepared to contain known concentrations of the test material. The standard bags were prepared by injecting known volumes of test material into a 500 mL glass vaporisation bulb. A continuous flow of air carried the vaporised test material to a 40 L bag. The total volume of air was measured by a dry test meter (Model DTM-200A, American Meter Co., Nebraska City, PA). Concentrations of the gas-phase standards were calculated as follows: Concentration = (VOL · R · T · D · 10^-3 · 10^6)
L · GMW · P
Where:
Conc. is in ppm
VOL = volume of test material vaporised into bag in μL
R = universal gas constant, 62.36 L mmHg/mole K
T = nominal laboratory temperature in K (273 + 21 °C = 294 K)
D = density of the test material, 2.280 g/mL
L = volume of air used to prepare bag, 32 L
GMW = gram molecular weight, 141.94 g/mole
P = nominal laboratory barometric pressure, 730 mmHg
10^-3 = μL to mL conversion factor
10^6 = conversion factor to ppm
- Standards prepared for this study: 3.8 ppm: 0.3 µL test material and 32 L air, 19 ppm: 1.5 µL test material and 32 air, 39 ppm: 3.1 µL test material and 32 L air, 58 ppm: 4.6 µL test material and 32 L air and 78 ppm: 6.2 µL test material and 32 L air. Each standard was prepared in triplicate prior to the exposure period and analysed with the GC. A least-squares line was fitted to the resulting peak areas. Concentrations were then calculated using the slope and intercept of this prime calibration curve. On a daily basis, the integrity of the prime calibration curve was checked by analysing one freshly prepared standard. On a rotational basis, a different one of the five standards was used each day. If the analysed concentration were within ± 10 % of the known concentration, the GC was considered within calibration specifications.

DETERMINATION OF HOMOGENEITY OF EXPOSURE ATMOSPHERES
- Evaluation of the homogeneity of exposure concentrations was accomplished during the method development phase of the study prior to animal exposures. Four test locations and a reference location were used for these determinations. The test locations were Right Lower Front, Right Upper Rear, Left Lower Rear, Left Upper Front identified as 1, 2, 3 and 4, respectively. Samples were collected as rapidly as possible always collecting a sample from the reference location and then from one of the four test locations. For each test location, the measured concentration was calculated as a percent difference from the reference location. The homogeneity determination was performed in triplicate for chamber 5, 70 ppm, exposure chamber.
- Results indicated that homogeneity of exposure atmospheres were adequate for the purpose of this study. Maximum mean % from reference was – 3.2 %.

RESULTS
- Nominal Exposure Concentrations: The overall mean nominal concentrations for the 4 week period were 7.8 ppm, 25 ppm, and 78 ppm for the 5 ppm, 20 ppm, and 70 ppm groups, respectively. The overall mean nominal concentrations for the 13 week period were 7.1 ppm, 25 ppm and 78 ppm for the 5 ppm, 20 ppm and 70 ppm groups, respectively.
- Actual Exposure Concentrations: The overall mean concentrations for the 4 week period were 5 ppm, 20 ppm, and 70 ppm for the 5 ppm, 20 ppm, and 70 ppm groups, respectively. The overall mean concentrations for the 13 week period were 5 ppm, 21 ppm and 70 ppm for the 5 ppm, 20 ppm and 70 ppm groups, respectively.

Duration of treatment / exposure:

5 days/week for either 4 (minimum of 20 exposures) or 13 weeks (minimum of 65 exposures)

Frequency of treatment:

daily (6hours/day)

Dose / conc.:

5 ppm (analytical)

Remarks:

4 week and 13 week periods

Dose / conc.:

20 ppm (analytical)

Remarks:

4 week period

Dose / conc.:

21 ppm (analytical)

Remarks:

13 week period

Dose / conc.:

70 ppm (analytical)

Remarks:

4 week and 13 week periods

No. of animals per sex per dose:

20 animals/sex/group (10 animals/sex/group were euthanised and necropsied following four weeks of exposure; the remaining 10 animals/sex/group were euthanised and necropsied following 13 weeks of exposure).

Control animals:

yes

Details on study design:

- Dose selection rationale: The exposure levels used on this study were chosen based upon the results of a previous inhalation study of Iodomethane in rats

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.
- Clinical examinations were performed twice daily, prior to exposure and following exposure (up to two hours following exposure). Pre-exposure clinical examinations were not performed on days when detailed physical examinations were conducted. On non-dosing days, the animals were observed once daily. All significant findings were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Detailed physical examinations were conducted on all animals weekly, beginning one week prior to the initiation of test material exposure and prior to the scheduled necropsies. For detailed examinations, the animals were removed from their home cages and placed in a standard arena for observations. Observations were detailed and carefully noted. When appropriate, explicitly defined scoring systems were used if, in the opinion of the study director, doing so would increase the utility of the data. Signs noted included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling, as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behaviour (e.g., self-mutilation, walking backwards) were recorded.

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

FOOD CONSUMPTION:
- Individual food consumption was recorded weekly, beginning one week prior to test material exposure (study week -1). Food intake was calculated as g/animal/day for the corresponding body weight intervals. When food consumption could not be measured for a given interval (due to spillage, weighing error, obvious erroneous value, etc.), the appropriate interval was footnoted as "NA" (Not Applicable) on the individual tables.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Ocular examinations were conducted on all animals prior to the initiation of dosing (week -1), prior to the interim necropsy (study week 4) and during the last week of dosing (study week 12). All ocular examinations were conducted using an indirect ophthalmoscope (or other suitable equivalent equipment), preceded by pupillary dilation with an appropriate mydriatic agent.

CLINICAL PATHOLOGY
- Blood samples for clinical pathology evaluations (haematology and serum chemistry) were collected from all animals at the scheduled necropsies (study weeks 4 and 13). The animals were fasted overnight prior to the collection of blood samples. Blood was collected from the vena cava at the time of necropsy. Blood for haematology assessment was collected into tubes containing EDTA as the anticoagulant; sodium citrate was used as the anticoagulant for clotting parameters.

- HAEMATOLOGY PARAMETERS: 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 and Basophil, Platelet Estimate and Red Cell Morphology (RBC Morphology).

- SERUM CHEMISTRY PARAMETERS: 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 (Aspartate Transfer), Gamma Glutamyltransferase (Glutamyl Transfer), Glucose, Total Cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium and Sodium.

Sacrifice and pathology:

GROSS PATHOLOGY
- A complete necropsy was conducted on all animals. Animals were euthanised by isoflurane inhalation 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 viscera.
- The following tissues and organs were collected and placed in 10 % neutral buffered formalin (except as noted): Adrenals glands, Aorta, Bone with marrow (Femur and Sternebrae), Bone marrow smear(femur), Brain (forebrain, midbrain, hindbrain), Exorbital lacrimal gland, Eyes with optic nerve (Fixed in Davidson’s solution), Gastrointestinal tract (Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum), Harderian glands, Heart, Kidneys, Larynx, Liver (sections of two lobes), Lungs (including bronchi, fixed by inflation with fixative), Lymph nodes, Mediastinal, Tracheobronchial, Mammary gland (females only), Nasal tissues, Ovaries with oviducts, Pancreas, Parathyroids, Peripheral nerve (sciatic), Pituitary, Prostate, Salivary glands (mandibular), Seminal vesicles, Skeletal muscle (rectus femoris), Skin, Spinal cord (cervical, thoracic, lumbar), Spleen, Testes with epididymides (Fixed in Bouin’s solution), Thymus, Thyroids, Trachea, Urinary bladder, Uterus with vagina and Gross lesions.

ORGAN WEIGHTS
- The following organs were weighed from all animals at the scheduled necropsies: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Lung (prior to inflation with fixative), Ovaries with oviducts, Spleen, Testes, Thymus, Thyroid with parathyroids and Uterus (Only weighed at the study week 13 necropsy).
- The thyroids with parathyroids were weighed after fixation. Paired organs were weighed together. Organ to final body weight ratios were calculated.

SLIDE PREPARATION AND MICROSCOPIC EXAMINATION
- After fixation, protocol-specified tissues were trimmed according to standard operating procedures and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned at five to eight microns, mounted on glass microscope slides and stained with haematoxylin and eosin. Following collection of the protocol-specified tissues, the entire head was removed and preserved. Following decalcification, six cross-sections of the nasal cavities were prepared for microscopic examination.
- Microscopic examination was performed on all tissues listed above from all animals in the control and 70 ppm groups at the scheduled necropsies. The kidneys, larynx, liver, lungs, nasal tissues, trachea and gross lesions were examined from all animals in the 5 and 20 ppm groups at the scheduled necropsies.

Statistics:

- All analyses were conducted using two-tailed tests 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.
- All statistical tests were performed using appropriate computing devices or programs. 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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- An increased incidence of wet yellow material on the urogenital area was noted for the 70 ppm group males and females following exposure.
- There were no other test material-related clinical observations. All findings noted in the test material-exposed groups were observed with similar incidence in the control group, were limited to single animals, were not observed in a dose-related manner and/or were common findings for laboratory rats of this age and strain.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the scheduled necropsies.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related effects on mean body weights and body weight gains were observed in the 70 ppm group males and females.
- For males, significantly (p<0.01) lower mean body weight gains were observed for study weeks 0 to 1, 1 to 2 and 4 to 5. As a result of these reductions, mean body weights were significantly (p<0.05 or p<0.01) lower in the 70 ppm group males during study weeks 2, 3, 5 and 6. The maximum decrease in mean body weight was observed during study week 6 (13 % relative to control). At the end of the study (study week 13), mean cumulative body weight gain was 15 % lower and mean body weight was 8 % lower when compared to the control group.
-For females, significantly (p<0.05 or p<0.01) reduced mean body weight gains were observed for study weeks 0 to 1, 4 to 5 and 5 to 6. The maximum decrease for females in mean body weight, although not statistically significant, was observed during study week 6 (9 %). At the end of the study (study week 13), mean cumulative body weight gain was 17 % lower and mean body weight was 7 % lower than the control group.
- There were no other test material-related effects on body weight data. Several other statistically significant (p<0.05 or p<0.01) differences from the control group were noted in mean body weight gains in the various groups. However, no trends were apparent to attribute the changes to test material exposure.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Slight, statistically significant (p<0.01) reductions in mean food consumption were observed in the 70 ppm group males for study weeks 0 to 1 and 4 to 5.
- These reductions were attributed to the test material since they corresponded with periods of lower body weight gains. There were no other test material-related effects on food consumption.

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 ophthalmic lesions attributed to test material exposure.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test material-related effects on haematology parameters.
- Mean prothrombin time in the 70 ppm group males was significantly (statistically at p<0.01) lower than the control group at study week 4. Mean activated partial thromboplastin time (APTT) in the 20 and 70 ppm group males was also significantly (p<0.05) lower than the control group at study week 4. However, decreases in prothrombin time and APTT are usually not considered toxicologically significant; therefore, it is unlikely that these changes were the result of test material exposure.
- There were no other remarkable changes in haematology parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- The only test material-related effect on serum chemistry parameters was increased total cholesterol in the 70 ppm group males and females. Mean cholesterol was significantly (p<0.01) higher than the control group in the 70 ppm group males and females at both study week 4 (55 and 67 %, respectively) and study week 13 (56 and 43 %, respectively). There were no other test material-related effects on serum chemistry parameters.
- At the study week 4 evaluation, mean total bilirubin in the 5 ppm group females was significantly (p<0.01) higher than the control group and mean sodium in the 70 ppm group females was significantly (p<0.05) higher than the control group. These changes were slight and/or not observed in a dose-related manner; therefore, they were considered spontaneous and unrelated to test material exposure.

Urinalysis findings:

not examined

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 adverse test material-related effects on organ weight data. Mean liver weights relative to final body weights were increased in a dose-related manner in males and females at study week 13 when compared to the control group. For males, the increases in relative liver weight were 3, 8 and 15 % in the 5, 20 and 70 ppm groups, respectively. For females, the increases in relative liver weight were 3, 11 and 22 % in these same groups, respectively. The differences from the control group were statistically significant (p<0.05 or p<0.01) in the 20 ppm group females and the 70 ppm group males and females. At study week 4, a significantly (p<0.01) higher mean liver weight relative to final body weight was noted for the 70 ppm group females (8 %). Increased liver weights are commonly observed following treatment with chemical agents and are not considered to be adverse without the presence of correlating histopathological findings.
- No other test material-related effects on organ weights were observed. However, several statistically significant (p<0.05 or p<0.01) differences from the control group were noted. Most of these differences were a result of the lower final body weights (not statistically significant) in the 70 ppm group and consisted of lower mean absolute heart weight in the 70 ppm group males and higher mean relative heart and lower mean absolute adrenal gland weights in the 70 ppm group females at study week 13. In addition, mean absolute heart weight was decreased in the 20 ppm group males at the study week 13 necropsy. These differences were not attributed to test material exposure since there were no microscopic correlates or treatment-related effects in these organs at the 70 ppm exposure level and since the changes noted for absolute weights were not present for relative organ weights. There were no other remarkable differences from the control group in organ weight data.

Gross pathological findings:

no effects observed

Description (incidence and severity):

- There were no test material-related macroscopic findings at the interim or primary necropsies. Those findings observed were noted with similar incidence in the control group, were limited to single animals in various groups and/or were findings commonly observed in laboratory rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the study week 4 interim necropsy, test material-related alterations were observed in the nose of the 70 ppm group. Minimal to mild degeneration of the olfactory epithelium, primarily in the dorsal meatus and on the dorsal septum and upper turbinates, was observed in the 70 ppm group males and females. Degeneration included disruption of the normal epithelial architecture (loss of dendritic processes, disorganisation of the nuclear layers and/or presence of enlarged ducts of Bowman’s gland in the nuclear layer), and either decreased or increased cellularity. Consistent with previous findings with this compound, degeneration was diagnosed even when regeneration was obvious because the degeneration clearly preceded the regenerative response. Degeneration/regeneration was observed in the 70 ppm group males at nasal levels 3-6 and in the 70 ppm group females at nasal levels 2-5. Respiratory epithelial metaplasia, the presence of single-layered ciliated epithelium admixed with olfactory epithelium, was noted at nasal level 2 in both sexes of the 70 ppm group and was also considered a test material-related effect. Respiratory epithelial metaplasia has been reported as a regenerative response following injury and loss of the olfactory epithelium6. At the study week 13 primary necropsy, test material-related alterations were again confined to the nose and consisted of degeneration/regeneration of the olfactory mucosa at nasal levels 2 through 6 in 70 ppm group males and females. Incidence and/or severity of degeneration at nasal levels 3, 4, and 5 were increased compared to the study week 4 interim necropsy. Two males and one female in the 70 ppm group were also observed to have metaplasia of the respiratory epithelium at nasal level 2 and this alteration was also considered related to test material exposure. No other test material-related findings were observed at study week 13. Degeneration was noted in one 20 ppm group female at nasal levels 4 and 5 but because of the occurrence of this lesion in control group animals at the study week 4 interim necropsy, and because similar lesions were not observed in the 20 ppm group males, this incidence was not considered related to treatment. A slightly increased incidence of intra-alveolar macrophages and subacute inflammation in the lungs was observed in the 5 ppm group males. However, the incidence of these alterations was not increased in the females, and there was no dose-relationship. Thus, these alterations were not considered related to test material exposure.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEC

Effect level:

20 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

histopathology: non-neoplastic

Critical effects observed:

not specified

Table 1: Body weights during the study

Week	Mean Average Bodyweight (g)
	0 ppm		5 ppm		20 ppm		70 ppm
	M	F	M	F	M	F	M	F
-1	199	167	202	165	201	163	202	163
0	247	182	247	182	248	182	247	182
1	290	207	281	204	284	205	276	198
2	315	222	306	215	306	217	292**	209
3	338	231	329	226	331	227	314*	218
4	356	236	350	236	352	238	335	228
5	389	251	369	246	364	246	348**	235
6	418	266	392	250	376	252	365**	242
7	420	260	402	254	396	258	379	243
8	435	266	411	266	405	266	397	253
9	444	271	426	269	422	272	406	256
10	458	275	437	272	435	274	422	260
11	474	278	449	276	443	276	425	260
12	484	288	459	284	449	284	442	264
13	484	286	465	285	459	283	445	267

* = Significantly different from the control group at 0.05 using Dunnett’s test

** = Significantly different from the control group at 0.01 using Dunnett’s test

 

Table 2: Weekly food consumption during the study (g/animal/day)

Week	Mean Average Food Consumption (g/animal/day)
	0 ppm		5 ppm		20 ppm		70 ppm
	M	F	M	F	M	F	M	F
-1 - 0	21	17	20	16	22	16	21	15
0 – 1	22	17	21	16	21	17	19**	16
1 – 2	22	18	21	17	21	18	22	18
2 – 3	22	18	22	18	22	18	22	18
3 – 4	23	18	22	17	23	18	22	18
4 – 5	25	18	23	17	24	17	22**	18
5 - 6	26	18	24	18	23	19	23	18
6 – 7	26	19	25	19	24	20	24	19
7 – 8	25	18	23	17	23	19	23	18
8 – 9	26	18	24	18	23	19	24	19
9 – 10	26	19	25	18	24	19	23	18
10 – 11	26	18	24	18	24	19	24	20
11 - 12	24	17	23	18	22	18	22	16
12 – 13	24	18	25	17	23	18	25	18

** = Significantly different from the control group at 0.01 using Dunnett’s test

 

Table 3: Clinical Chemistry Cholesterol Levels

Week	Mean Average Cholesterol Level (mg/dL)
	0 ppm		5 ppm		20 ppm		70 ppm
	M	F	M	F	M	F	M	F
4	44	49	55	62	51	62	68**	82**
13	48	70	50	73	55	83	75**	100**

** = Significantly different from the control group at 0.01 using Dunnett’s test

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for whole-body inhalation exposure to the test material in rats for four or 13 weeks was 20 ppm.

Executive summary:

The repeated dose inhalation toxicity of the test material was investigated in accordance with the standardised guideline OPPTS 870.3465, under GLP conditions.

The test material was administered six hours/day, five days/week via whole-body inhalation exposure for either four (minimum of 20 exposures) or 13 weeks (minimum of 65 exposures) to three groups (Groups 2-4) of male and female Crl:CD(SD)IGS BR rats. Dosage levels were 5, 20 and 70 ppm. A concurrent control group (Group 1) received filtered air on a comparable regimen. Groups 1-4 each consisted of 20 animals/sex/group. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded weekly. Clinical pathology evaluations (haematology and serum chemistry) were performed at the scheduled necropsies. Ophthalmic examinations were performed during study weeks -1, 4 and 12. Following four weeks of exposure, 10 rats/sex/group were euthanized (interim necropsy). The remaining 10 rats/sex/group were euthanized after 13 weeks of exposure (primary necropsy). Complete necropsies were conducted on all animals, and selected organs were weighed. Selected tissues were examined microscopically.

All animals survived to the scheduled necropsies. There were no test material-related effects on haematology parameters. No test material-related ophthalmic findings were observed. There were no test material- related macroscopic findings at either necropsy. No adverse test material-related changes were noted in the 5 and 20 ppm groups. Mean relative to final body weight liver weight was increased in the 20 ppm group but was not considered adverse due to the absence of correlating histopathological findings. Test material-related effects noted in the 70 ppm group consisted of: An increased incidence of wet yellow material on the urogenital area for males and females following exposure. Lower mean body weight gains periodically during the exposure period resulting in mean cumulative body weight gains that were 15 and 17 % lower than the control group for males and females, respectively, at study week 13. In addition, mean body weights were 8 and 7 % lower for males and females, respectively, by the end of the study. Lower mean food consumption periodically (study weeks 0 to 1and 4 to 5) for males. Increased mean serum cholesterol levels for both males and females at study weeks 4 and 13. The toxicological significance of these cholesterol changes was unknown. Higher mean liver weight relative to final body weight (15 and 22% for males and females, respectively) were observed at study week 13. These changes were not considered adverse since there were no correlating microscopic changes in the liver. Microscopic changes consisted of degeneration/regeneration of the olfactory epithelium in males at nasal levels 3-6 and in females at nasal levels 2-5 at study week 4 and at nasal levels 2-6 in males and females at study week 13. Respiratory epithelial metaplasia was also noted at nasal level 2 in both sexes at study weeks 4 and 13.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for whole-body inhalation exposure to the test material in rats for four or 13 weeks was 20 ppm.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

116 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 March 2001 to 29 August 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9 weeks
- Weight at study initiation: males: 262 to 313 g and females: 182 to 232 g
- Housing: All animals were housed individually in clean, wire-mesh cages suspended above cage-board.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 16 days

DETAILS OF FOOD AND WATER QUALITY: Contaminants were not present in animal feed or water at levels expected to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- Temperature: 20.6 to 21.7 °C
- Humidity: 36 to 60 %

Type of coverage:

occlusive

Vehicle:

corn oil

Details on exposure:

TEST SITE
- On the day prior to dose administration the hair was clipped from the back of each animal, from the scapula (shoulder) to the wing of the ileum (hipbone) and halfway down the flank of each side of the animal.
- The control or appropriate test material was applied to the shaved, intact dorsal skin of each animal for six hours per day for 21 consecutive days. Control group animals were dosed with the vehicle at a volume equal to the dose volume administered to the high dose group. Doses were applied evenly by gentle inunction using a glass rod over the maximum area possible.
- The area of test material application was measured and recorded once per week for an arbitrarily selected animal of each sex in each group [Total body surface area (cm^2) = K x body weight (grams)(^2/3); K = 9 for rats]. The mean area of coverage during the study was approximately 24, 25, 20 and 21 % for males in the control, 30, 300 and 1000 mg/kg/day groups, respectively. The mean area of coverage during the study was approximately 24, 21, 19 and 17 % for the females in these same groups, respectively.
- The application site for each animal was covered with a wrap consisting of a gauze binder around the trunk, which was then covered with an impervious plastic wrap and secured with several overwraps of tape. The corners of the application site were marked with indelible ink to allow proper identification of the treated and untreated skin.

REMOVAL OF TEST SUBSTANCE
- At the end of the six-hour exposure period, the dressings were removed. The test sites were gently washed by disposable paper towels with tepid tap water to remove any residual test material, and then gently dried.

TEST MATERIAL
- Amount(s) applied: 2 mL/kg

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

- On the first day of dosing for the males, 5 mL samples were collected from the top, middle and bottom strata of each test material formulation for stability and homogeneity determinations.
- Subsequent concentration analyses were performed concurrently with administration of newly prepared dosing formulations periodically throughout the study.

- Gas Chromatography:
Instrument: Hewlett Packard 5890A (Series II) gas chromatograph equipped with an FID detector, a HP Headspace analyser
Column: J & W Scientific GS-GasPro, 30 m x 0.316 mm ID (0.25 μm film thickness)
Temperature (Program): 70 °C for 1.0 min, ramp at 40 °C/min to 230 °C, hold 2 min
Carrier gas: Helium set at 12psi (EPC constant flow on)
Injector temperature: 225 °C
Injection volume: 1 mL splitless
Detector: FID at 225 °C
Retention time: Approximately 4.7 min

- Headspace Parameters:
Zone temp: Oven 50 °C, Loop 100 °C and Transfer Line 100 °C
Event Times: GC cycle time 10 min, Vial EQ time 7 min, Pressuriation time 0.20 min, Loop fill time 0.20 min, Loop EQ time 0.05 min and Inject time 0.20 min.

- Preparation of Calibration Stock Solutions: The calibration stock solutions were prepared by transferring under the surface of the oil, the appropriate amount of test material into a 10 mL volumetric flask containing approximately 7 mL of corn oil. The contents were diluted to a final volume of 10 mL with corn oil. These stocks were prepared in the concentration range of 10 to 600 mg/mL.

- Preparation of Quality Control Samples: The quality control (QC) stock solutions were prepared as above. These stocks were prepared at the concentrations of 20, 100 and 400 mg/mL.
- Sample Processing: Calibration, QC’s, and formulation samples were prepared for analysis by transferring 30 μL of the sample, into a headspace vial containing 970 μL of corn oil.
- 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. 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
- Under the described chromatographic conditions, the retention time of the test material was approximately 4.7 minutes. The total analysis time required for each run was approximately 10 minutes. The validity of the assay procedure was established through a careful study of the calibration reproducibility, accuracy, precision, ruggedness, and stability of the test material in quality control stocks and dosing formulations.
- Specificity/Selectivity: The assay specificity/selectivity was confirmed when GC analysis of control formulations revealed that there were no significant peaks near the retention time for the test material.
- Calibration Reproducibility: During each of three validation sets, triplicate calibration samples at five concentration levels were prepared and analysed as described above. Single injections were made of each processed calibration sample. The resulting peak area-concentration data were fit to the ln- quadratic function using the least-squares regression analysis. The results of the regression analyses were used to back-calculate from the peak area data the corresponding concentrations. The reproducibility of the calibration curve data was considered valid when the inter-set variability (RSD) of the back-calculated concentrations at each concentration level was ≤ 15 %, except at the lowest concentration level where ≤ 20 % was acceptable; and the mean back-calculated concentrations at each concentration level were within 15 % of the theoretical values (% RE within ± 15 %), except at the lowest concentration level where % RE ≤ 20 % was acceptable. The inter-set variability (RSD) of the back-calculated concentrations at each level ranged from 2.4 to 6.3 %. The inter-set concentration means had % RE values ranging from -0.89 to 2.5 %. Based on these criteria and the resulting validation data, the reproducibility of the calibration data was acceptable.
- Precision and Accuracy: During each of four validation sets, triplicate quality control (QC) samples at three concentration levels were prepared and analysed as described above. Single injections were made of each processed QC sample. The results of the regression analyses were used to calculate from these QC peak area data the corresponding concentrations. The variability (RSD) of these calculated QC concentration data was used as a measure of assay precision. The precision of the method was considered acceptable when the inter-set RSD of the calculated concentrations at each QC concentration level is ≤ 10 %. The difference from theoretical of the calculated QC concentration means (% RE) was used as a measure of assay accuracy. The accuracy of the method was considered acceptable when the inter-set concentration means of the calculated concentrations at each QC concentration level had % RE values within ± 15 %. The inter-set variability (RSD) of the calculated concentrations at each level (precision) ranged from 2.4 to 9.0 %. The inter-set concentration means had % RE values (accuracy) ranging from –1.1 to 8.2 %. Based on the criteria mentioned above, the precision and accuracy of the test material assay were acceptable.
- Stability of QC Stock Solutions: The QC stock solution prepared on 3/17/01 and initially used in validation set #1 was stored refrigerated. After 4 days, it was used to prepare triplicate samples at each of the three QC concentrations. The analysed concentrations were 79.4, 103, and 101 % of the respective time-zero values. Stability of the refrigerated stock was demonstrated for the mid and high QC stock. The QC stock should be prepared fresh for each analysis.
- Assay Ruggedness: In this assay validation, ruggedness is referred to as the ability of another analyst to successfully perform the procedure as described. Assay ruggedness is demonstrated when two or more different analysts independently and successfully conduct at least one session of the required three validation sets. Assay ruggedness was successfully demonstrated for this procedure.
- Homogeneity of Dosing Formulations: A representative set of formulations was prepared by the pharmacy department on 3/15/01 for the evaluation of homogeneity and stability. Samples from the top, middle and bottom of the formulation batches were collected and analysed. Each group met the requirement for homogeneity, i.e., the RSD for the overall mean concentration was ≤ 10 % at a concentration that is within the acceptable limits (%RE ± 15 %); with the exception of group 2 RSD and the group 4 mean % of target.
- Stability of Dosing Formulations: Samples from the middle of the formulation batches were stored at room temperature for 6-hours. The mean concentrations ranged from 97.1 to 108 % of the time zero concentrations, and, therefore, the formulations were considered stable.
- Summary of Concentration Analysis: The analysed formulations; (3/23/01, 3/29/01, 4/05/01) met the requirements for concentration acceptability for suspension formulations, i.e., the analysed concentrations were within 15 % of the target dose concentrations. Formulations prepared on 3/19/01 (Group 2) and 3/20/01 (all groups) did not meet requirements for concentration acceptability.

Duration of treatment / exposure:

6 hours per day, for 21 consecutive days

Frequency of treatment:

Daily

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels were selected based on the results of a 5-day range-finding study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS
- The animals were observed twice daily, in the morning and afternoon, for mortality and moribundity. All animals received a clinical examination prior to application of the control or test material.

DETAILED CLINICAL OBSERVATIONS: Yes
- Each animal received a detailed physical examination once weekly, beginning approximately one week prior to the initiation of test material administration, and ending on the day of necropsy. During detailed physical examinations, the animals were removed from their home cages and placed in a standard arena for observation.

DERMAL IRRITATION: Yes
- Application sites were examined for erythema, oedema and other dermal findings once per week at the time of the detailed physical examination. Hair was clipped from the backs of the animals as necessary to facilitate dermal scoring. Erythema and oedema were evaluated in accordance with the method of Draize, based on a four-step grading system of very slight, slight, moderate, and severe. Other dermal findings, if present, were noted.

BODY WEIGHT: Yes
- Individual body weights were recorded weekly, beginning approximately one week prior to test material administration. Mean body weight changes were calculated for each corresponding interval. A final (fasted) body weight was collected for each animal on the day of scheduled necropsy.

FOOD CONSUMPTION:
- Individual food consumption was measured weekly, beginning approximately one week prior to test material administration. Food intake was calculated as g/animal/day for the corresponding body weight intervals.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Ocular examinations were conducted on all animals prior to initiation of test material administration (study week -1) and during study week 3. All ocular examinations were conducted using an indirect ophthalmoscope (or other suitable equipment), preceded by mydriasis.

CLINICAL PATHOLOGY: Blood and urine samples for clinical pathology evaluation were collected from all surviving animals at the time of the primary necropsy (study week 3). Blood was collected for hormone level analysis the day prior to necropsy from the lateral tail vein of the non-fasted, non-anesthetiSed animals. For other analyses, the animals were fasted overnight prior to collection of blood samples from the vena cava. Urine was collected overnight using metabolism cages.

- HAEMATOLOGY PARAMETERS: Total Leukocyte Count (White Cell), Erythrocyte Count (Red Cells), Hemoglobin, Hematocrit, Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (Platelet), Prothrombin Time, Activated Partial Thromboplastin Time (APTT), Reticulocyte Count (Percent (Reticulocyte) and Absolute (Retic Absolute)), Differential Leukocyte Count- (Percent and Absolute, -Neutrophil, -Lymphocyte, -Monocyte, -Eosinophil and –Basophil), Platelet Estimate and Red Cell Morphology (RBC Morphology).

- CLINICAL CHEMISTRY PARAMETERS: 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).

- URINALYSIS PARAMETERS: 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.

- SERUM HORMONE PARAMETERS: T3, TSH, T4 and T3/T4 Ratio

- NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- A complete necropsy was conducted on all animals found dead or euthanised in extremis and at the scheduled necropsies. All animals were euthanised by isoflurane anesthesia followed by exsanguination. The necropsy included examination of the external surface, all orifices, and the cranial, thoracic, abdominal and pelvic cavities including viscera. At the time of necropsy the following tissues and organs were collected and preserved in 10 % neutral buffered formalin: Adrenal glands (2), Aorta, Bone with marrow (Sternum and Femur), Bone marrow smear, Brain (Forebrain, Midbrain and Hindbrain), Epididymides (2, Placed in Bouin’s solution), Exorbital lacrimal glands (2), Eyes with optic nerve (2, placed in Davidson's solution), Gastrointestinal tract (Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon and Rectum), Harderian gland (2), Heart, Kidneys (2), Larynx, Liver (sections of two lobes), Lungs (including bronchi, fixed by inflation with fixative), Lymph node (Mandibular and Mesenteric), Mammary gland (females only), Nose, Ovaries with oviducts (2), Pancreas, Parathyroids (if present), Peripheral nerve (sciatic), Pharynx, Pituitary, Prostate, Salivary glands [mandibular (2)], Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin (treated and untreated), Spinal cord (Cervical, Thoracic and Lumbar), Spleen, Testes (2, Placed in Bouin’s solution), Thymus, Thyroid, 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, Ovaries (with oviducts), Spleen, Testes, Thymus, Thyroid 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, sectioned at 5-8 microns, mounted on glass microscope slides and stained with haematoxylin and eosin. All tissues listed in gross pathology were examined microscopically from all animals in the control and 1000 mg/kg/day groups euthanised at the scheduled necropsy and for all animals found dead or euthanised in extremis. The treated and untreated skin, liver, kidneys, adrenal cortex, bone marrow (sternum and femur), lymph node (mandibular and mesenteric), spleen, thymus, glandular stomach, prostate, seminal vesicles and gross lesions (when possible) were examined for all animals in the 30 and 300 mg/kg/day groups.

Statistics:

- All analyses were conducted using two-tailed tests for significance levels of 5 and 1 % comparing the treated groups to the control group by sex. All means are presented with standard deviations (S.D.) and the number of sampling units (N) used to calculate the means. Statistical analyses were not performed if the number of animals was two or less. All statistical tests were performed using appropriate computing devices or programs.
- Body weights, body weight changes, food consumption, clinical pathology values and absolute and relative organ weight data were subjected to a one-way analysis of variance (ANOVA), followed by Dunnett’s test if the ANOVA revealed statistical significance (p<0.05). Clinical laboratory values for cell types that occur at a low incidence (i.e. monocytes, eosinophils and basophils) were not subjected to statistical analysis.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related clinical observations were noted in the 300 and 1000 mg/kg/day group males and females. These findings included wet yellow staining in the urogenital and/or anogential areas. There were no other test material-related clinical findings.
- It should be noted that a 30 mg/kg/day group female was noted with a swollen facial area on study day 19. This animal was observed thrashing itself around the cage in order to get free of the wrapping material applied at dosing. The swollen facial area was most likely the result of the animal hitting its head on the feed jar and cage walls, and not a result of test material treatment.
- Additional clinical findings observed in the test material-treated groups were noted similarly in the control group, were not present in a dose-related manner, were noted at a low incidence, typically in single animals and/or were clinical signs commonly observed in rats of this strain and age.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related dermal findings were noted in the 30, 300 and 1000 mg/kg/day group males and females. These findings included very slight to severe erythema and oedema, fissuring, more pronounced desquamation, eschar, exfoliation, atonia, subcutaneous haemorrhage, ulceration and coriaceousness. These findings were noted as early as the first week of dosing. Very slight erythema was noted for 2/10 and 5/10 animals in the 30 mg/kg/day male and female groups, respectively, and desquamation was noted for 7/10 animals/sex at this dose level. The severity of erythema and oedema tended to increase over the course of the study for the 300 and 1000 mg/kg/day groups. Severe erythema was noted for the 300 and 1000 mg/kg/day group males and females primarily during the last two weeks of the study. The severity of oedema tended to increase with dose, as moderate oedema was generally noted in the 300 mg/kg/day group males and females and severe oedema was primarily noted in the 1000 mg/kg/day group males and females. Desquamation, eschar and exfoliation were noted for all animals in the 300 and 1000 mg/kg/day groups. Atonia was noted for all 1000 mg/kg/day group animals and for 9/10 and 9/10 animals in the 300 mg/kg/day male and female groups, respectively. Fissuring and coriaceousness were common in both the 300 and 1000 mg/kg/day group males and females. Subcutaneous haemorrhage was noted for one 300 mg/kg/day group female after the second week of dosing.
- There were no other definite test material-related dermal findings. Several occurrences of desquamation were also noted in the control group during the study.

Mortality:

mortality observed, treatment-related

Description (incidence):

- Four males were found dead or euthanized in extremis during the study.
- One 300 mg/kg/day group male was found dead on study day 18. Two 1000 mg/kg/day group males were euthanised in extremis on study days 11 and 10, respectively. Another 1000 mg/kg/day group male was found dead on study day 4, this animal was noted with tremors, dried red material around the nose and wet red material on the urogenital area prior to death. The cause of death or moribundity for three animals was urinary tract obstruction. Each of these animals had hydronephrosis, a condition that can be acquired or secondary to an obstructive lesion of the urinary tract distal to the kidney. The cause of death for the other animal could not be determined. The condition that lead to the death of these animals was most likely test material-related.
- All other animals survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related decreases in mean body weights and body weight gains were noted in the 300 and 1000 mg/kg/day group males. These decreases were statistically significant (p < 0.05 or p < 0.01) throughout the study. A test material-related transient decrease (statistically significant at p < 0.05) in mean body weight gains was also noted in the 1000 mg/kg/day group females during study week 0 to 1.
- There were no other test material-related changes noted during the study. No other remarkable differences were noted when the control and treated groups were compared.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Test material-related decreases in mean food consumption (often statistically significant at p < 0.01) were noted in the 300 and 1000 mg/kg/day group males throughout the study. A test material-related transient decrease in mean food consumption (statistically significant at p<0.01) was also noted in the 1000 mg/kg/day group females during study week 0 to 1.
- There were no other test material-related changes in mean food consumption noted during the study. There were no other remarkable differences noted when the control and treated groups were compared.

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 oculopathic lesions indicative of a toxic effect were observed.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related changes in haematology parameters were noted in the 300 and 1000 mg/kg/day group males and/or females and consisted of lower red blood counts, haemoglobin, haematocrit, APTT, white blood cell counts and lymphocyte counts and higher neutrophil counts, MCV, MCH and platelet counts.
- Statistically significant decreases (p<0.05 or p<0.01) were noted in mean red blood cell counts, haemoglobin and haematocrit in the 300 and 1000 mg/kg/day group males and females. Mean white blood cell counts were decreased in the 300 and 1000 mg/kg/day group males. Statistically significant (p<0.05 or p<0.01) increases and decreases were also noted in mean absolute and/or relative neutrophil and lymphocyte counts, respectively, for these groups. Necrosis of the treated skin and the subsequent inflammatory response seen microscopically were the direct cause of the white blood cell variations and most of the variations were typical of an acute inflammatory response. Also, the dose-related decrease in white cell counts in treated males and the absence of a definitive leukocytosis in the females suggests that the demand for leukocytes in the skin exceeded marrow production. Additional test material-related changes included decreased APTT and increased platelets in the 300 and 1000 mg/kg/day group males. MCV and MCH were increased in the 1000 mg/kg/day group females due to decreased red blood cell counts.
- There were no other test material-related changes in haematology parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related changes in serum chemistry parameters were noted in the 300 and 1000 mg/kg/day group males and females and consisted of higher globulin, urea nitrogen, alkaline phosphatase, chloride, sodium, aspartate aminotransferase, alanine aminotransferase and gamma glutamyltransferase and lower albumin, A/G ratios, triglycerides and calcium.
- Mean globulin, urea nitrogen, alkaline phosphatase and chloride were increased and statistically significant (p<0.05 or p<0.01) in the 300 and 1000 mg/kg/day group males and females. Mean albumin and A/G ratio values were decreased in these groups. Mean sodium and aspartate aminotransferase were increased in the 300 mg/kg/day group males and females and the 1000 mg/kg/day group females. Mean triglycerides were decreased and alanine aminotransferase was increased in the 300 and 1000 mg/kg/day group females. Mean gamma glutamyltransferase levels were increased in the 1000 mg/kg/day group males and females. Mean calcium was decreased in the 300 and/or 1000 mg/kg/day group females. The decrease in calcium values was considered to be an effect of hypoalbuminemia and not an accurate reflection of true serum calcium concentration.
- There were no other test material-related changes in serum chemistry parameters noted during the study. However, many statistically significant (p < 0.05 or p < 0.01) differences were noted when the control and treated groups were compared. Mean potassium was increased and mean total protein was decreased in the 300 mg/kg/day group males and females, respectively. Mean creatinine was decreased in the 300 and 1000 mg/kg/day group females. Mean cholesterol levels were increased in the 1000 mg/kg/day group males. Mean glucose was increased in the 300 mg/kg/day group females. These changes were not considered test material-related as the alterations were slight, occurred without apparent dose responses and/or were inconsistent between sexes. There were no other remarkable changes when the control and treated groups were compared.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Test material-related changes in urinalysis parameters were noted in the 300 and 1000 mg/kg/day group males and consisted of higher specific gravity values. These changes were statistically significant (p < 0.01) in the 300 and 1000 mg/kg/day group males. This change most likely represented a physiological response to fluid or electrolyte disturbances induced by inflammation and necrosis of the skin or by decreased water consumption that most likely accompanied lower food consumption values. Thus, the differences in specific gravity values were not a direct test material-related effect and were not considered adverse.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related changes in mean absolute and/or relative organ weights (statistically significant at p < 0.05 or p < 0.01) were noted in the brain, spleen, thymus and adrenal glands of the 300 and 1000 mg/kg/day group males and/or females. In the spleen, the inconsistent trends in organ weights were best explained by the effects of lymphoid depletion/necrosis (tendency for lower weights) and extramedullary haematopoiesis (tendency for increased weights) noted microscopically. Both these microscopic changes were considered test material-related; therefore, decreased mean absolute spleen weights in the 300 mg/kg/day group males and increased mean absolute and relative spleen weights in the 1000 mg/kg/day group females were considered test material-related.
- Absolute brain weights in the 300 and 1000 mg/kg/day groups were lower than control group values. These lower values were not related to lower body weights in these groups because brain weights remain relatively unaffected by body weight fluctuations. Furthermore, the gender difference in body weight effects was not present in the brain weight changes. Therefore, the brain weight differences were considered test material-related. However, there were no microscopic changes to explain these lower brain weights.
- In the thymus, decreased absolute and relative weights were noted in the 300 and 1000 mg/kg/day group males and females. These weight changes correlated with lymphoid necrosis/depletion noted microscopically in these groups.
- Increased mean absolute and/or relative adrenal gland weights were noted for males and females in the 300 and 1000 mg/kg/day groups. These weight changes corresponded with vacuolar change and/or extramedullary haematopoiesis noted microscopically in the adrenal cortex of treated animals.
- There were no other test material-related changes in organ weights. However, many statistically significant (p < 0.05 or p < 0.01) differences were noted when the control and treated groups were compared. Mean kidney and heart weights were decreased in the 300 and 1000 mg/kg/day group males. Epididymides and testes weights were decreased in the 1000 mg/kg/day group males and ovary and uterus weights were decreased in the 300 and/or 1000 mg/kg/day group females. Due to the low final body and brain weights in the 300 and 1000 mg/kg/day group males and females, numerous organ weights relative to final body or brain weight were statistically significant when compared to control group values. In the absence of microscopic changes to support these mean absolute weight decreases, these changes were most likely related to lower terminal body weights and not a direct test material effect.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related macroscopic findings were noted in the treated skin, thymus, stomach and seminal vesicles of the 300 and 1000 mg/kg/day group males and/or females. Slough skin was noted for 8/9 males and 10/10 females in the 300 mg/kg/day group and 7/7 males and 9/10 females in the 1000 mg/kg/day group. Scabbing and thickening of the skin were also noted at increased incidences in the 300 and 1000 mg/kg/day group males and females. Microscopic findings of necrosis and exfoliation, both of the epidermis, commonly correlated with the findings noted above.
- Test material-related changes were seen affecting the skin at treated and untreated sites in the skin. Reddened, thickened, slough and/or scabbing of treated skin were seen in all animals in the 300 and 1000 mg/kg/day groups. Single instances of scabbing were noted in both sexes at 30 mg/kg/day and in one control group male. Similar changes were noted at lower incidences in untreated skin primarily in the 1000 mg/kg/day groups. These findings often correlated with microscopic findings of necrosis and exfoliation noted in both treated and untreated skin. Macroscopic changes in the untreated skin were not noted in the 30 mg/kg/day or control groups.
- A small thymus was noted for 2/10 males and 3/10 females in the 300 mg/kg/day group and for 8/10 females in the 1000 mg/kg/day group. This change correlated with the microscopic finding of lymphoid necrosis or depletion noted in these groups.
- In the stomach, dark red areas were noted in 3/10 females in the 1000 mg/kg/day group. Dark red gastric contents were noted for a few males and females at dose levels of 300 and 1000 mg/kg/day. Dark red areas correlated with microscopic findings of erosion in the glandular stomach. Gastric erosion was considered the source of dark red contents throughout the gastrointestinal tract.
- Small and/or soft seminal vesicles were noted for 1/10 and 4/10 males in the 300 and 1000 mg/kg/day groups, respectively. This change correlated with decreased glandular secretion noted microscopically.
- There were no other remarkable macroscopic findings noted at the scheduled necropsy. Findings noted for treated animals were observed similarly in the control group and/or were noted at low incidence, typically single animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- For the unscheduled deaths, urinary tract obstruction, often accompanied by decreased glandular secretion, inflammation and/or oedema of the seminal vesicles or prostate, was a common cause of death or moribundity in the 300 and 1000 mg/kg/day groups. This conclusion was supported by the condition of hydronephrosis in three of the four unscheduled deaths.
- Test material-related microscopic changes were noted at the scheduled necropsy in the skin, adrenal cortex, bone marrow, lymph nodes, spleen, thymus, glandular stomach, prostate and seminal vesicles in the 300 and 1000 mg/kg/day groups.
- The most extensive changes, and the only findings directly related to the test material, were noted in the skin and included exfoliation, necrosis, epithelial hyperplasia, subacute inflammation, oedema and intraepithelial vesicle. Exfoliation was the term used to describe the full-thickness loss of a necrotic epidermis, often including hair follicles, and a small portion of the superficial dermis. This finding correlated with slough noted macroscopically. Histologically, there was an underlying regenerative epithelium that was often hyperplastic. In contrast, necrosis was used to describe a necrotic but otherwise intact epidermis and commonly correlated with the macroscopic findings of scabbing and/or thickened skin. Epidermal necrosis/ulceration commonly has dermal inflammation, oedema and epithelial hyperplasia as associated changes. In this study, subacute inflammation, oedema and epithelial hyperplasia were diagnosed only when they were not associated with epidermal necrosis. However, these three changes were often associated and commonly were subjacent to regions of exfoliation. Epithelial hyperplasia and hyperkeratosis, generally of minimal to mild severity, were commonly seen in the control and low-dose groups and were attributed to mechanical irritation of test material administration. The incidence and severity of hyperplasia increased with dose. Intra-epithelial vesicles were considered to represent oedema within the epidermis. These findings were seen in a small number of animals in the 30 mg/kg/day group, but all males and females in the 300 and 1000 mg/kg/day groups had varying combinations of these test material-related changes.
- In the skin, generally from the ventral abdominal region, necropsy findings similar to those seen in the treated skin were observed. One female from the 300 mg/kg/day group and 3/10 males and 9/10 females in the 1000 mg/kg/day group were noted with test material-related exfoliation and/or necrosis in regions other than the application site. The incidence of hyperkeratosis also tended to increase in a dose-related manner in treated females. Finding treatment-related skin lesions in areas other than the application site probably represents mechanical transfer of the test material between sites on the animal rather than systemic involvement of the skin. In the lymphoid tissues (spleen, gut associated lymphoid tissue, lymph nodes and thymus), lymphoid necrosis and/or depletion was seen in one or more tissues from 8/10 males and all females in the 300 mg/kg/day group and all animals in the 1000 mg/kg/day group. The thymus was the lymphoid tissue most commonly affected, but none of these findings were seen in the control or 30 mg/kg/day groups. Lymphoid depletion and necrosis were considered to be secondary to the stress associated with the treatment-related skin changes.
- In the bone marrow, hypercellularity was seen in 8/10 males and 6/10 females in the 300 mg/kg/day group and 9/10 males and 6/10 females in the 1000 mg/kg/day groups. Increased production of hematopoietic cells was also seen at lower incidences at sites of extramedullary haematopoiesis (spleen, liver and adrenal cortex). These changes were in response to the treatment-related inflammation and necrosis seen in the skin that resulted in the decreased red cell count and increased demand for neutrophils and were not a direct effect of test material treatment.
- In the adrenal cortex, cytoplasmic vacuolation was seen in 1/9 males from the 300 mg/kg/day group and 8/10 males and 5/10 females in the 1000 mg/kg/day group. The vacuoles were clear, round, well-defined and morphologically consistent with lipid. This change was considered to be secondary to stress and not a direct effect of treatment. Extramedullary haematopoiesis (EMH) was seen only in one male and seven females in the 1000 mg/kg/day group. This change was a manifestation of increased EMH in other tissues as well as bone marrow hypercellularity in response to the treatment-related skin changes and not a direct effect of the test material.
- In the glandular stomach, erosion was seen in 2/10 males and 1/10 females in the 300 mg/kg/day group and 3/10 females in the 1000 mg/kg/day group. This change was considered to be secondary to stress and not a direct effect of test material treatment.
- In the males, urinary tract obstruction was a common cause of early death or debilitation, accounting for 3 of 4 cases in the 300 and 1000 mg/kg/day groups. The animals commonly had decreased glandular secretion, inflammation, and/or oedema of the seminal vesicles or prostate that may have contributed to the urinary obstruction. When all animals were considered, 2/10 from the 300 mg/kg/day group and 3/10 from the 1000 mg/kg/day group had acute or chronic active inflammation of the prostate with inflammation extending to the seminal vesicles in one of the 1000 mg/kg/day animals. Since a control group male also had minimal chronic active inflammation of the prostate, the significance of the slightly increased inflammation in treated animals was uncertain. Similarly it was unclear if decreased secretion within the seminal vesicles, noted in 1/10 and 4/10 males in the 300 and 1000 mg/kg/day groups, respectively, was related to inflammation, general debilitation, or a direct effect of test material treatment.
- There were no other test material-related microscopic findings. Other findings noted in test material-treated animals occurred similarly in the control group, were considered spontaneous and/or were common changes observed in laboratory animals.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

SERUM HORMONES:
- Test material-related changes in serum hormones were noted in the 300 mg/kg/day group males and the 30 and 1000 mg/kg/day group females and consisted of decreased mean T4 values and increased mean T3 values during the study. These changes were statistically significant (p < 0.01) when the control and treated groups were compared. Mean T4 was decreased in the 300 mg/kg/day group males and the 1000 mg/kg/day group females. It is unclear if these decreases represented a reduction in thyroid function or were secondary to inflammation or hypoalbuminemia. Mean T3 was increased in the 30 mg/kg/day group females. The absence of a dose response and the inconsistency between sexes suggests that this change in T3 may be incidental and not test material-related. There were no other remarkable differences noted when the control and treated groups were compared.

Details on results:

CONCLUSIONS

- No oculopathic changes indicative of a test material-related effect were noted. One 300 mg/kg/day group male and three 1000 mg/kg/day group males were found dead or euthanized in extremis during the study. The cause of death or moribundity for three of these animals was attributed to possible urinary tract obstruction. The cause of death for the fourth animal (1000 mg/kg/day group male) could not be determined. The condition that lead to the death of these animals was most likely test material-related.
- Test material-related clinical findings were noted in the 300 and 1000 mg/kg/day group males and females and included wet yellow staining in the urogenital and/or anogenital areas.
- Test material-related decreases in mean body weights, body weight gains and food consumption were noted in the 300 and 1000 mg/kg/day group males throughout the study.
- Test material-related dermal findings were noted in the 30, 300 and 1000 mg/kg/day group males and females and included very slight to severe erythema and oedema, fissuring, desquamation, eschar, exfoliation, atonia, subcutaneous haemorrhage, ulceration and/or coriaceousness. These findings were noted as early as the first week of dosing. The severity of erythema and oedema tended to increase with dose and over the course of the study for the 300 and 1000 mg/kg/day groups.
- Test material-related changes in haematology parameters were noted in the 300 and 1000 mg/kg/day group males and females. Decreases were noted in mean red blood cell counts, haemoglobin, haematocrit, APTT and white blood cell counts in the 300 and 1000 mg/kg/day group males and/or females. Increases and decreases were also noted in mean absolute and/or relative neutrophil and lymphocyte counts, respectively, for these groups. Necrosis of the treated skin and the subsequent inflammatory response seen microscopically were the direct cause of the white blood cell variations and most of the variations were typical of an acute inflammatory response. Also, the dose-related decrease in white cell counts in treated males and the absence of a definitive leukocytosis in the females suggests that the demand for leukocytes in the skin exceeded marrow production. Increased platelets were noted in the 300 and 1000 mg/kg/day group males. MCV and MCH were also increased in the 1000 mg/kg/day group females due to decreased red blood cell counts.
- Mean globulin, urea nitrogen, alkaline phosphatase, sodium, aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase and chloride were increased in the 300 and/or 1000 mg/kg/day group males and/or females. Mean albumin, triglycerides, calcium and A/G ratio values were decreased in these groups. The decrease in calcium values was considered to be an effect of hypoalbuminemia and not an accurate reflection of true serum calcium concentration.
- Test material-related changes in serum hormones were noted in the 300 mg/kg/day group males and the 30 and 1000 mg/kg/day group females and consisted of decreased mean T4 and increased T3 values.
- Test material-related changes in urinalysis parameters were noted in the 300 and 1000 mg/kg/day group males and consisted of higher specific gravity values.
- The most extensive microscopic changes were noted in the skin and included exfoliation, necrosis, epithelial hyperplasia, subacute inflammation, oedema and intraepithelial vesicle. Exfoliation correlated with slough noted macroscopically. Necrosis was commonly correlated with the macroscopic findings of scabbing and/or thickened skin. Epithelial hyperplasia and hyperkeratosis, generally of minimal to mild severity, were commonly seen in the control and low-dose groups and were attributed to mechanical irritation of test material administration. The incidence and severity of hyperplasia increased with dose as there was a regenerative and often hyperplastic epithelium underlying the exfoliated (sloughed) epidermis. Intra-epithelial vesicles were considered to represent oedema within the epidermis. These findings were seen in a small number of animals in the 30 mg/kg/day group, but all males and females in the 300 and 1000 mg/kg/day groups had varying combinations of these test material-related changes.
- In the lymphoid tissues, test material-related changes were noted for organ weights, macroscopic and/or microscopic examinations in the spleen and thymus of the 300 and 1000 mg/kg/day group males and females. Test material-related changes in organ weights were noted in the spleen and thymus of the 300 and 1000 mg/kg/day group males and females. A small thymus was noted for males and females at necropsy, correlating with decreased absolute and relative thymus weights and the histologic finding of lymphoid necrosis or depletion noted in these groups. The thymus was the lymphoid tissue most commonly affected with necrosis and depletion. In the spleen, the inconsistent trends in organ weights were best explained by the effects of lymphoid depletion/necrosis (tendency for lower weights) and extramedullary haematopoiesis (tendency for increased weights) noted microscopically. Since both these microscopic changes were related to treatment, decreased mean absolute spleen weights in the 300 mg/kg/day group males and increased mean absolute and relative spleen weights in the 1000 mg/kg/day group females were considered test material-related.
- Lymphoid necrosis and/or depletion was also noted in the gut associated lymphoid tissue and/or lymph nodes in the 300 and 1000 mg/kg/day groups. Lymphoid depletion and necrosis in all affected tissues were considered to be secondary to the stress associated with the treatment-related skin changes. Splenic extramedullary haematopoiesis (EMH) was considered to be secondary to the treatment-related inflammation and necrosis seen in the skin.
- Increased mean absolute and/or relative adrenal gland weights were noted for males and females in the 300 and 1000 mg/kg/day groups. These weight changes corresponded with cytoplasmic vacuolation of the adrenal cortex in males and/or females from the 300 and 1000 mg/kg/day groups. The vacuoles were clear, round, well-defined and morphologically consistent with lipid. This change was considered to be secondary to stress and not a direct effect of treatment. Extramedullary haematopoiesis (EMH) was seen only in one male and seven females in the 1000 mg/kg/day group. This change was a manifestation of increased EMH in other tissues as well as bone marrow hypercellularity in response to the treatment-related skin changes and not a direct effect of the test material.
- Test material-related decreases in mean absolute brain weights were noted in the 300 and 1000 mg/kg/day groups.
- In the stomach, dark red areas were noted in the 1000 mg/kg/day group females, correlating with microscopic findings of erosion in the glandular stomach of the males and/or females in the 300 and 1000 mg/kg/day groups. Dark red gastric contents were noted for a few males and females at dose levels higher than 300 mg/kg/day. Gastric erosion was considered the source of dark red contents throughout the gastrointestinal tract. Erosion of the glandular stomach was considered to be secondary to stress and not a direct effect of test material treatment.
- Other test material-related microscopic changes were noted in the bone marrow, prostate and seminal vesicles in the 300 and 1000 mg/kg/day groups. In the bone marrow, hypercellularity was seen in males and females in the 300 and 1000 mg/kg/day groups. Increased production of hematopoietic cells was also seen at lower incidences at sites of extramedullary hematopoiesis (spleen, liver and adrenal cortex). These changes were in response to the treatment-related inflammation and necrosis seen in the skin that resulted in the decreased red cell count and increased demand for neutrophils and were not a direct effect of test material treatment. In the males, urinary tract obstruction was a common cause of early death or debilitation, accounting for 3 of 4 cases in the 300 and 1000 mg/kg/day groups. The animals commonly had decreased glandular secretion (correlating with small and/or soft seminal vesicles noted macroscopically), inflammation, and/or oedema of the seminal vesicles or prostate that may have contributed to the urinary obstruction. Since a control group male also had minimal chronic active inflammation of the prostate, the significance of the slightly increased inflammation in treated animals was uncertain. Similarly it was unclear if decreased secretion within the seminal vesicles, noted in the 300 and 1000 mg/kg/day groups, was related to inflammation, general debilitation, or a direct effect of test material treatment.
- Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity of the test material when administered dermally to rats for 21 consecutive days was 30 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

haematology

histopathology: neoplastic

mortality

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Table 1: Summary of selected clinical chemistry and pathology findings (at termination)

Dose (mg/kg/day)		0	30	300	1000	0	30	300	1000
Sex		Male				Female
No. in group		10	10	10	10	10	10	10	10
Haematology and clinical chemistry (mean values)
Red cells (mil/µL)		7.19	7.18	6.16**	5.70**	6.11	5.66	5.08**	4.60**
White cells (thous/µL)		10.2	8.8	7.0*	6.4*	8.0	7.9	7.5	9.0
Haemoglobin (g/dL)		14.2	14.2	12.3**	11.4**	12.2	11.4	10.4**	9.9**
Haemocrit (%)		38.8	39.0	33.6**	31.7**	33.2	31.1	28.4	27.0
Globulin (g/dL)		2.1	2.21	3.1*	3.3**	2.3	2.3	3.0*	3.4**
Albumin/ Globulin ratio		1.84	1.77	0.94**	0.86**	1.79	1.77	1.01**	0.87**
Urea nitrogen (mg/dL)		14.0	15.1	32.4**	31.1**	19.4	19.5	34.2**	35.9**
Chloride (mEq/L)		101	100	107**	105**	100	101	108**	106**
Histopathology (incidence), Skin:
Hyperplasia (epithelial)		-	-	1 (1)	2 (3)	-	-	1 (10)	5 (10)
Necrosis		1 (10)	1 (10)	9 (9)	7 (7)	-	-	1 (10)	9 (10)
Exfoliation		-	-	1 (1)	2 (3)	-	-	1 (10)	4 (10)
Sub-acute inflammation		-	-	1 (1)	3 (3)	0 (10)	1 (10)	10 (10)	10 (10)

*Significant (p < 0.05) vs control;**Significant (p < 0.01) vs control

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity of the test material when administered dermally to rats for 21 consecutive days was 30 mg/kg/day.

Executive summary:

The repeated dose dermal toxicity of the test material was investigated in accordance with the standardised guideline OPPTS 870.3200, under GLP conditions.

The possible toxic effects of the test material were evaluated in this 21-day dermal study in Crl:CD®(SD)IGS BR rats. The test material in the vehicle, corn oil, was applied seven days per week for three weeks to the shaved intact dorsal skin of each rat. The application sites were covered with gauze, occluded with plastic wrap and secured with non-irritating tape for a period of six hours per exposure. Following the exposure period, the application sites were gently washed with water and disposable paper towels to remove residual test material. Each of the three test material groups consisted of 10 males and 10 females. Dosage levels were 30, 300 and 1000 mg/kg/day. A concurrent control group received the vehicle on a comparable regimen. The animals were examined twice daily for mortality and moribundity. Clinical observations were performed once daily prior to exposure. Detailed physical examinations were performed and dermal observations were recorded weekly. Ophthalmological examinations were performed prior to dosing and during study week 3. Clinical pathology evaluations (haematology, serum chemistry, serum hormones and urinalysis) were performed prior to the scheduled necropsy. Complete necropsies were performed for all animals and selected organs weighed. Selected tissues were examined microscopically.

No oculopathic changes indicative of a test material-related effect were noted. One 300 mg/kg/day group male and three 1000 mg/kg/day group males were found dead or euthanized in extremis during the study. These deaths were attributed to test material administration.

Test material-related clinical findings were noted in the 300 and 1000 mg/kg/day group males and females and included wet yellow staining in the urogenital and/or anogenital areas.

Test material-related decreases in mean body weights, body weight gains and food consumption were noted in the 300 and 1000 mg/kg/day group males throughout the study.

Test material-related dermal findings were noted in the 30, 300 and 1000 mg/kg/day group males and females and included very slight to severe erythema and oedema, fissuring, desquamation, eschar, exfoliation, atonia, subcutaneous haemorrhage, ulceration and/or coriaceousness. The severity of the dermal reactions tended to increase with dose and/or over the course of the study. These findings were often accompanied by microscopic findings of exfoliation, necrosis, epithelial hyperplasia, subacute inflammation, oedema and intraepithelial vesicles.

Several haematology, organ weight and histological changes were noted in the 300 and 1000 mg/kg/day groups as secondary effects of treatment with the test material. Necrosis of the treated skin and the subsequent inflammatory response seen microscopically were the direct cause of the white blood cell variations. Most other variations in haematology parameters (red blood cell parameters and APTT) were typical of an acute inflammatory response. Increased urine specific gravity was also considered secondary to test material-related changes in the skin due to possible fluid or electrolyte imbalances. In the lymphoid tissues (spleen, gut associated lymphoid tissue, lymph nodes and thymus), necrosis and/or depletion of these tissues and/or changes in organ weights were considered secondary to stress associated with treatment-related skin changes. Additionally, changes in adrenal gland weights, cytoplasmic vacuolation of the adrenal cortex, erosion of the glandular stomach and hypercellularity of the bone marrow were also attributed to stress and considered secondary to treatment.

Test material-related changes were noted in serum chemistry and serum hormone parameters. Mean globulin, urea nitrogen, alkaline phosphatase, sodium, aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase and chloride were increased in the 300 and/or 1000 mg/kg/day group males and/or females. Mean albumin, triglycerides, calcium and A/G ratio values were decreased in these groups. Changes in serum hormones noted in the 300 mg/kg/day group males and the 30 and 1000 mg/kg/day group females consisted of decreased mean T4 and increased mean T3 values.

Additional test material-related changes included decreased mean absolute brain weights in the 300 and 1000 mg/kg/day group males and females and microscopic changes in the prostate and seminal vesicles in the 300 and 1000 mg/kg/day group males. In the males, urinary tract obstruction was a common cause of early death or debilitation. The animals often had decreased glandular secretion (correlating with small and/or soft seminal vesicles noted macroscopically), inflammation, and/or oedema of the seminal vesicles or prostate that may have contributed to the urinary tract obstruction. The significance of the slightly increased inflammation of the prostate in treated animals was uncertain. Similarly, it was unclear if decreased secretion within the seminal vesicles, noted in the 300 and 1000 mg/kg/day groups, was related to inflammation, general debilitation, or a direct effect of test material treatment.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity of the test material when administered dermally to rats for 21 consecutive days was 30 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

ORAL

90 Day dog study

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 study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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.

90 day rat

The repeated dose oral toxicity of the test material was investigated in accordance with the standardised guidelines EPA OPPTS 870.3100 and JMAFF 12 NouSan No. 8147, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was administered orally to 10 male and 10 female Crj : CD (SD) IGS rats per group once daily for 90 days at dose levels of 0 (control), 5, 10, 25 and 50 mg/kg to evaluate the toxicity potential of the test material in corn oil. Ten males and 10 females were allocated to the control and 25 and 50 mg/kg groups to assess reversibility during a 28-day recovery period. The control group received corn oil in the same manner as the test material.

Four females died at 50 mg/kg on Days 81, 83, and 86. In the animals that died, salivation was sporadically observed from Day 5, and it was accompanied by prone position, a trace of reddish rhinorrhoea, reddish eye gum, lacrimation, soft stool, dirty around anus and a decrease in spontaneous activity in some animals. A decrease in food consumption, suppression of body weight gain, and a decrease in body weight were also observed. In gross pathology, adhesion of the abdominal organs in the abdominal cavity, reddening or black foci of the mucosa in the glandular stomach, thickening of the mucosa in the forestomach, ascites in the abdominal cavity, and reddening or dark reddening of the thymus were observed in the animals that died. Histopathology revealed that these changes were hyperkeratosis, hyperplasia, necrosis, ulceration, inflammatory cell infiltration in the squamous epithelium in the forestomach, necrosis of the lymphocyte, haemorrhage, a decrease in the number of the lymphocytes in the thymus, decreases in the number of myeloid cells and the megakaryocytes in the femoral and sternal bone marrow, a decrease or necrosis of the lymphocytes in the white pulp in the spleen, and mesenteric and submandibular lymph nodes, an increase in the number of the macrophages in the red pulp in the spleen, and squamous metaplasia of the ductal epithelium in the submandibular glands.

In the surviving animals at 50 mg/kg, salivation was sporadically observed in all males and 15 females from Day 3 to the final dosing day. A trace of reddish rhinorrhoea, reddish eye gum, dirty lower belly by urine, and a decrease in spontaneous activity were observed in 2 females between Days 82 and 86. Suppression of body weight gain and a decrease in body weight were observed in males from Week 1, and a decrease in food consumption was observed at Weeks 2 and 4. No abnormalities were observed in males or females in body weight or food consumption during the recovery period. In blood chemistry, high alkaline phosphatase, total bilirubin, total protein, albumin, phospholipid, calcium, and sodium in males, high a2-globulin ratio, 13-globulin ratio, and low A/G ratio in females were observed in the examination at the end of the dosing period. In gross pathology at the end of the dosing period, adhesion in the abdominal organs in males and females, thickening and attaching of the greyish white material of the mucosa in the forestomach, enlargement of the liver, yellowish brown focus of the liver, reddening and red focus in the stomach in females were observed. Adhesion in the abdominal organs and thickening of the mucosa in the forestomach were observed in only 1 female at the end of recovery period. In histopathology at the end of the dosing period, hyperkeratosis, hyperplasia, necrosis/ulceration of the squamous epithelium in the stomach, haemorrhage in the forestomach, oedema in the submucosa of the glandular stomach, fibrosis of the submucosa in the forestomach, inflammatory cell infiltration in the submucosa in the forestomach and glandular stomach, lymphocyte infiltration in the submucosa in the glandular stomach, haemorrhage in the forestomach, and adhesion in the abdominal organs were observed. Focal necrosis of the hepatocyte, focal haemorrhage and fibrosis, bile duct proliferation, and giant cell granuloma in the liver were observed. In the submandibular glands, a decrease in the granules in the granular ducts, squamous metaplasia and neutrophil infiltration of the ductal epithelium, and neutrophil accumulation were observed, and squamous metaplasia of the ductal epithelium in the sublingual gland was also observed. A decrease in the lymphocytes in the cortex in the thymus was observed. Adhesion in the abdominal organs and thickening in the mucosa in the forestomach were observed in 1 female at the end of the recovery period.

Salivation was sporadically observed in 18 males and 19 females at 25 mg/kg from Day 6 to the end of the final dosing day. In blood chemistry at the end of the dosing period, high total bilirubin, total protein calcium, and sodium were observed in males. Adhesion in the gastrointestinal tracts was observed in females in gross pathology at the end of the dosing period. Hyperkeratosis, hyperplasia, and necrosis/ulceration of the squamous epithelium in the forestomach, inflammatory cell infiltration and oedema of the submucosa in the forestomach, and adhesion in the abdominal organs were observed in histopathology at the end of the dosing period. Squamous metaplasia and neutrophil infiltration in the ductal epithelium in the submandibular gland, focal necrosis of the hepatocyte and a decrease in the granules in the granular ducts were observed in 1 male. Focal haemorrhage and necrosis of the hepatocyte were observed in 1 male at the end of the recovery period.

Salivation was sporadically observed in 5 males and 4 females at 10 mg/kg from Day 7 to the final dosing day. Reddish eye gum was observed in 1 male on Days 67 and 68. Hyperkeratosis and hyperplasia of the squamous epithelium in the stomach, focal haemorrhage and necrosis in the hepatocyte in males and squamous metaplasia of the ductal epithelium in the submandibular or sublingual gland in males and females were observed in histopathology at the end of the dosing period.

Focal haemorrhage and necrosis of the hepatocyte were observed in 1 male at 5 mg/kg in histopathology at the end of the dosing period. No other abnormalities were observed in any other examination parameter.

From these results, test material-related effects on the liver and digestive system were observed in males and females at 10 mg/kg and above in this study. Effects on the liver were observed in only one male at 5 mg/kg. These changes were almost recovered during the recovery period. Accordingly, it was considered that under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) of the test material was 5 mg/kg/day in males, and the No Observed Effect Level (NOEL) of the test material was 5 mg/kg/day in females.

90 day mouse dietary study

The repeated dose oral toxicity of the test material was investigated in accordance with the standardised guidelines OECD 408, EPA OPPTS 870.3100 and JMAFF 12 NouSan No. 8147, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objectives of the study were to evaluate the toxicity potential of the test material when administered orally via the diet to mice for 90 days and to assist in the selection of doses for a carcinogenicity study.

The test material was administered continuously in the diet to three groups mice for a minimum of 90 days. Dosage levels were 133, 400 and 1200 ppm. A concurrent control group received the control material, Placebo Microcapsules, on a comparable regimen. All four groups consisted of 10 animals/sex/group. All 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 and food consumption was recorded every other day. Clinical pathology evaluations (haematology, serum chemistry and urinalysis) were performed at the time of the scheduled necropsy (study week 13). Ophthalmic examinations were performed during study weeks -1 and 12. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues were examined microscopically from all animals.

Haematology, serum chemistry and urinalysis parameters were unaffected by test material administration. No test material-related ophthalmic findings were noted, and no test material-related macroscopic changes were observed at the scheduled necropsy.

Test material-related effects noted in the 1200 ppm group consisted of: Death of one male and one female. Clinical signs of decreased defecation and faeces smaller than normal during study weeks 1-5. Decreased mean body weight and mean cumulative body weight gains; mean body weight was 11 to 17 % lower than the control group by study week 13. Decreased mean food consumption throughout the study (most severe during study weeks 1 to 5). Increased mean absolute and relative thyroid/parathyroid weights. Microscopically observed increased accumulations of follicular colloid with corresponding attenuation of follicular lining cells in the thyroid gland and hyperkeratosis in the oesophagus.

Test material-related effects noted in the 400 ppm group consisted of: Clinical signs of decreased defecation and faeces smaller than normal during study weeks 1-5. Decreased mean body weight and mean cumulative body weight gains; mean body weight was 6 to 8 % lower than the control group by study week 13. Decreased mean food consumption throughout the study (most severe during study weeks 1 to 5). Increased mean absolute and relative thyroid/parathyroid weights. Microscopically observed increased accumulations of follicular colloid with corresponding attenuation of follicular lining cells in the thyroid gland and hyperkeratosis in the oesophagus.

Test material-related effects noted in the 133 ppm group consisted of: Increased mean absolute and relative thyroid/parathyroid weights. Microscopically observed increased accumulations of follicular colloid with corresponding attenuation of follicular lining cells in the thyroid gland and hyperkeratosis in the oesophagus.

Based on the increase in thyroid weights with correlating accumulation of follicular colloid and hyperkeratosis in the oesophagus in the 133 ppm group, the no-observed-effect level (NOEL) for oral (diet) administration of the test material to mice for at least 90 days was less than 133 ppm. The no-observed-adverse-effect level (NOAEL) was 400 ppm. The NOAEL is based on the reductions in body weight of less than 10 %, with corresponding decreased food consumption and clinical signs. The thyroid changes were most likely an adaptive response and therefore, not considered adverse. Hyperkeratosis in the oesophagus is likely a result of local irritation and therefore, not considered adverse.

1 year dog

The repeated dose toxicity of the test material was investigated in accordance with the standardised guidelines OECD 452, EPA OPPTS 870.4100 and JMAFF 12 NouSan No.8147, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to evaluate the toxicity potential of the test material in corn oil when administered orally via capsule to dogs for a minimum of 12 months (52 weeks, 364 days). The test material in the vehicle, corn oil, was administered orally via capsules once daily, 7 days per week, for a minimum of 52 weeks at dosage levels of 1.5, 6.0 and 12.0 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 hormone and urinalysis) were performed prior to the initiation of dose administration (study week -1) and during study weeks 25 and 52 (last week of the dosing period). Ophthalmic examinations were performed during study weeks -1 and 51. Complete necropsies were performed on all dogs, and selected organs were weighed at the scheduled necropsy. Selected tissues were examined microscopically from all animals.

There were no test material-related effects on body weights and food consumption. Urinalysis and ophthalmic parameters were unaffected by test material administration. One male in the 6.0 mg/kg/day group and one female in the 12.0 mg/kg/day group were euthanised in extremis during the study. All other animals survived to the scheduled necropsy. Test material-related clinical observations were noted in the 6.0 and 12.0 mg/kg/day group males and females throughout the study and included decreased defecation, diarrhoea, emesis, evidence of excessive salivation (clear material around the mouth and excessive drooling), head shaking, hypoactivity, soft faeces, thinness and white foamy material around the mouth. Excessive salivation extended into the 1.5 mg/kg/day group males and females.

Higher mean platelet counts were noted in the 6.0 and 12.0 mg/kg/day groups at both the study week 25 and 52 evaluations. Lower mean serum albumin, total protein and serum calcium levels and albumin/globulin ratio were observed in the 12.0 mg/kg/day group at the study week 25 and/or 52 evaluations. Mean serum cholesterol in the 6.0 and 12.0 mg/kg/day group males and females tended to be higher than the control group throughout the study; the toxicological significance of these changes was unclear. Higher mean thyroid stimulating hormone (TSH) levels were noted in the 12.0 mg/kg/day group males and females; these changes were largely attributed to one male and one female.

Test material-related macroscopic findings were noted in the 6.0 and 12.0 mg/kg/day group animals euthanized in extremis and in one 12.0 mg/kg/day group male that survived to the scheduled necropsy. These findings included thickening and discoloration of the oesophagus, firmness and enlargement of the mandibular salivary gland and thickening of the stomach. Microscopically, extensive ulceration of the oesophagus, mucous cell hypertrophy and/or decreased secretion of the mandibular salivary gland and hyperplasia and/or decreased secretion of the stomach were noted in these same animals. These effects were considered secondary to the irritative nature of the test material. Mild to severe colloid depletion and moderate follicular cell hypertrophy of the thyroid gland and minimal to mild hyperplasia of basophilic cells within the pars distalis of the pituitary gland were noted in one male and one female of the 12.0 mg/kg/day group. These animals were considered to have corresponding elevated TSH levels. Bilateral seminiferous tubule degeneration in the testes, and increased incidence and severity of vacuolar change in the liver were also noted in the 12.0 mg/kg/day group. Minimal unilateral seminiferous tubule degeneration was noted in one male each in the 1.5 and 6.0 mg/kg/day groups, although the toxicological significance is unknown. Higher mean relative (to final body and/or brain weight) liver weights and lower mean absolute and relative thyroid/parathyroid weights were noted in the 12.0 mg/kg/day group.

When administered at 6.0 and 12.0 mg/kg/day, the irritative properties of the test material resulted in secondary effects of oesophageal ulceration and associated microscopic changes in the salivary glands and stomach. The local effect of ulceration was the cause of death for the 6.0 mg/kg/day group male and 12.0 mg/kg/day group female euthanised in extremis. Additional clinical pathology findings were noted at 6.0 and 12.0 mg/kg/day, as well as systemic microscopic findings in the liver and thyroid in the 12.0 mg/kg/day group.

Based on the clinical findings of excessive salivation, as well as the potentially test material-related microscopic findings of seminiferous tubule degeneration of the testes extending to the 1.5 mg/kg/day group, the no-observed-adverse-effect level (NOAEL) for oral (capsule) administration of the test material to dogs for 52 consecutive weeks was 1.5 mg/kg/day.

DERMAL

21 day rat

The repeated dose dermal toxicity of the test material was investigated in accordance with the standardised guideline OPPTS 870.3200, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The possible toxic effects of the test material were evaluated in this 21-day dermal study in Crl:CD®(SD)IGS BR rats. The test material in the vehicle, corn oil, was applied seven days per week for three weeks to the shaved intact dorsal skin of each rat. The application sites were covered with gauze, occluded with plastic wrap and secured with non-irritating tape for a period of six hours per exposure. Following the exposure period, the application sites were gently washed with water and disposable paper towels to remove residual test material. Each of the three test material groups consisted of 10 males and 10 females. Dosage levels were 30, 300 and 1000 mg/kg/day. A concurrent control group received the vehicle on a comparable regimen. The animals were examined twice daily for mortality and moribundity. Clinical observations were performed once daily prior to exposure. Detailed physical examinations were performed and dermal observations were recorded weekly. Ophthalmological examinations were performed prior to dosing and during study week 3. Clinical pathology evaluations (haematology, serum chemistry, serum hormones and urinalysis) were performed prior to the scheduled necropsy. Complete necropsies were performed for all animals and selected organs weighed. Selected tissues were examined microscopically.

No oculopathic changes indicative of a test material-related effect were noted. One 300 mg/kg/day group male and three 1000 mg/kg/day group males were found dead or euthanized in extremis during the study. These deaths were attributed to test material administration.

Test material-related clinical findings were noted in the 300 and 1000 mg/kg/day group males and females and included wet yellow staining in the urogenital and/or anogenital areas.

Test material-related decreases in mean body weights, body weight gains and food consumption were noted in the 300 and 1000 mg/kg/day group males throughout the study.

Test material-related dermal findings were noted in the 30, 300 and 1000 mg/kg/day group males and females and included very slight to severe erythema and oedema, fissuring, desquamation, eschar, exfoliation, atonia, subcutaneous haemorrhage, ulceration and/or coriaceousness. The severity of the dermal reactions tended to increase with dose and/or over the course of the study. These findings were often accompanied by microscopic findings of exfoliation, necrosis, epithelial hyperplasia, subacute inflammation, oedema and intraepithelial vesicles.

Several haematology, organ weight and histological changes were noted in the 300 and 1000 mg/kg/day groups as secondary effects of treatment with the test material. Necrosis of the treated skin and the subsequent inflammatory response seen microscopically were the direct cause of the white blood cell variations. Most other variations in haematology parameters (red blood cell parameters and APTT) were typical of an acute inflammatory response. Increased urine specific gravity was also considered secondary to test material-related changes in the skin due to possible fluid or electrolyte imbalances. In the lymphoid tissues (spleen, gut associated lymphoid tissue, lymph nodes and thymus), necrosis and/or depletion of these tissues and/or changes in organ weights were considered secondary to stress associated with treatment-related skin changes. Additionally, changes in adrenal gland weights, cytoplasmic vacuolation of the adrenal cortex, erosion of the glandular stomach and hypercellularity of the bone marrow were also attributed to stress and considered secondary to treatment.

Test material-related changes were noted in serum chemistry and serum hormone parameters. Mean globulin, urea nitrogen, alkaline phosphatase, sodium, aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase and chloride were increased in the 300 and/or 1000 mg/kg/day group males and/or females. Mean albumin, triglycerides, calcium and A/G ratio values were decreased in these groups. Changes in serum hormones noted in the 300 mg/kg/day group males and the 30 and 1000 mg/kg/day group females consisted of decreased mean T4 and increased mean T3 values.

Additional test material-related changes included decreased mean absolute brain weights in the 300 and 1000 mg/kg/day group males and females and microscopic changes in the prostate and seminal vesicles in the 300 and 1000 mg/kg/day group males. In the males, urinary tract obstruction was a common cause of early death or debilitation. The animals often had decreased glandular secretion (correlating with small and/or soft seminal vesicles noted macroscopically), inflammation, and/or oedema of the seminal vesicles or prostate that may have contributed to the urinary tract obstruction. The significance of the slightly increased inflammation of the prostate in treated animals was uncertain. Similarly, it was unclear if decreased secretion within the seminal vesicles, noted in the 300 and 1000 mg/kg/day groups, was related to inflammation, general debilitation, or a direct effect of test material treatment.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity of the test material when administered dermally to rats for 21 consecutive days was 30 mg/kg/day.

INHALATION

90 Day Rat

The repeated dose inhalation toxicity of the test material was investigated in accordance with the standardised guideline OPPTS 870.3465, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was administered six hours/day, five days/week via whole-body inhalation exposure for either four (minimum of 20 exposures) or 13 weeks (minimum of 65 exposures) to three groups (Groups 2-4) of male and female Crl:CD(SD)IGS BR rats. Dosage levels were 5, 20 and 70 ppm. A concurrent control group (Group 1) received filtered air on a comparable regimen. Groups 1-4 each consisted of 20 animals/sex/group. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded weekly. Clinical pathology evaluations (haematology and serum chemistry) were performed at the scheduled necropsies. Ophthalmic examinations were performed during study weeks -1, 4 and 12. Following four weeks of exposure, 10 rats/sex/group were euthanized (interim necropsy). The remaining 10 rats/sex/group were euthanized after 13 weeks of exposure (primary necropsy). Complete necropsies were conducted on all animals, and selected organs were weighed. Selected tissues were examined microscopically.

All animals survived to the scheduled necropsies. There were no test material-related effects on haematology parameters. No test material-related ophthalmic findings were observed. There were no test material- related macroscopic findings at either necropsy. No adverse test material-related changes were noted in the 5 and 20 ppm groups. Mean relative to final body weight liver weight was increased in the 20 ppm group but was not considered adverse due to the absence of correlating histopathological findings. Test material-related effects noted in the 70 ppm group consisted of: An increased incidence of wet yellow material on the urogenital area for males and females following exposure. Lower mean body weight gains periodically during the exposure period resulting in mean cumulative body weight gains that were 15 and 17 % lower than the control group for males and females, respectively, at study week 13. In addition, mean body weights were 8 and 7 % lower for males and females, respectively, by the end of the study. Lower mean food consumption periodically (study weeks 0 to 1and 4 to 5) for males. Increased mean serum cholesterol levels for both males and females at study weeks 4 and 13. The toxicological significance of these cholesterol changes was unknown. Higher mean liver weight relative to final body weight (15 and 22% for males and females, respectively) were observed at study week 13. These changes were not considered adverse since there were no correlating microscopic changes in the liver. Microscopic changes consisted of degeneration/regeneration of the olfactory epithelium in males at nasal levels 3-6 and in females at nasal levels 2-5 at study week 4 and at nasal levels 2-6 in males and females at study week 13. Respiratory epithelial metaplasia was also noted at nasal level 2 in both sexes at study weeks 4 and 13.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for whole-body inhalation exposure to the test material in rats for four or 13 weeks was 20 ppm.

21 Day Rat

The repeated dose toxicity of the test material via the inhalation route was investigated under GLP conditions to determine the dose range for a subsequent 90-day study. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The subchronic toxicity study consisted of three groups, each composed of 10 male and 10 female rats, exposed to the test material for six hours daily for four weeks, five days per week (for a total exposure period of 20 or 21 days). Target exposure concentrations were 25, 75 and 100 ppm. The measured mean exposure concentrations were 25, 72 and 99 ppm. Clinical observations were conducted daily; detailed physical examinations were performed weekly. Body weights and food consumption were recorded weekly. Necropsies were performed on study days 28 and 29. Blood samples from fasted animals were collected from the vena cava (following anaesthesia) at the scheduled necropsy. Haematology and serum chemistry parameters were evaluated. Selected organs were weighed and tissues collected for histopathologic evaluation.

All animals survived to the scheduled necropsy; no test material-related internal findings were observed at necropsy. Arousal level was characterised as somewhat high for one male in the 100 ppm group at the week 2 and week 3 detailed observations and for one female in the 75 ppm group at the week 2 observation. One male in the 75 ppm group had circling at the week 3 examination. These findings were considered to be potentially related to test material exposure since similar findings were noted in other phases of this study.

Mean body weight gains in the 100 ppm group males and females were reduced (statistically significant) compared to the control group values during weeks 0-1. Mean body weight gains were statistically significantly reduced in the males and slightly reduced in the females (not statistically significant) compared to the control group values during weeks 1-2 and 2-3. Cumulative mean body weight gain in the 100 ppm group males and females were reduced throughout the study; the decrements in weight gain were generally statistically significant. Mean body weights were statistically significantly reduced by 8.3-14.8% in these males during weeks 2-4 and by 5.5-9.7% in the females during weeks 2-4. Mean body weight gains were reduced compared to the control group values in the 75 ppm group males (statistically significant) and females (not statistically significant) during weeks 0-1 and 1-2. Cumulative mean body weight gains in these males and females were reduced throughout the study; the majority of the differences were statistically significant. Compared to the control group values, mean body weights were 5.5-7.2% lower in the 75 ppm group males and mean body weights in the females were 5.0-8.1% lower during weeks 2-4. No test material-related effects on mean body weights, body weight gains and cumulative body weight gains were observed in the 25 ppm group males and females.

Food consumption, evaluated as g/animal/day and g/kg/day, was reduced in the 100 ppm group males and females and the 75 ppm group males during week 0-1. Food consumption (g/animal/day) was reduced (statistically significant) in the 100 ppm group males for the remainder of the study (weeks 1-2 through 3-4) compared to that in the control group. Food consumption in the 75 ppm group males and 100 ppm group females was similar to the control group values for the remainder of the study. Food consumption in the 75 ppm group females and in the 25 ppm group males and female was similar to that in the control group throughout the study.

No direct test material-related effects on haematology parameters were noted at any exposure level. Test material-related increases in mean albumin (males only), A/G ratio (females only), globulin and total protein were observed in the 100 ppm group animals. The differences from the control group values were statistically significant. Cholesterol was increased (statistically significant) in the 75 and 100 ppm group males and females compared to the control group values. Phosphorus in the 75 and 100 ppm group males and females was statistically significantly reduced compared to the control group values. No test material-related effects on serum chemistry were noted in the 25 ppm group males and females. Macroscopic examination of the males and females after four weeks of exposure revealed no test material-related effects.

Test material-related increases in mean absolute thyroid weights and thyroid weight relative to final body weights (statistically significant) were observed in the 100 ppm group males and females compared to the control group values. This increase in these animals correlated to thyroid follicular hyperplasia observed microscopically. The diagnosis of follicular hyperplasia consisted of an increase in number and size of cells lining the follicles as well as the formation of small cellular aggregates without a lumen or the presence of colloid. Degeneration of the thyroid follicle (characterized as colloid depletion, individual follicular cell degeneration and necrosis and an increased number/amount of cells and cellular debris admixed with colloid) was also often observed associated with areas of follicular cell hyperplasia in all animals in the 100 ppm group. In addition to thyroid follicular cell hyperplasia and degeneration of the thyroid follicle mentioned above, hypertrophy of the pituitary gland (pars distalis) was observed in the 100 ppm group males and females. This finding was characterised by diffuse to regionally extensive cellular enlargement. This change may be secondary to the thyroid alterations, since thyroid stimulating hormone (TSH) is produced by the basophilic cells of the pars distalis. Minimal to mild renal pelvic mineralisation was observed in the 100 ppm group females. The pathologic/toxicologic significance of this finding was unclear. Test material-related irritation was observed in the nasal tissues in the 100 ppm males and females. This was characterised primarily by minimal to moderate degeneration of the olfactory epithelium in nasal sections 3-6. No other test material-related microscopic findings were observed.

This study was designed as a dose range-finding study for a subsequent 90-day study and histopathological investigations were not conducted at the low and intermediate dose levels to enable a NOAEC to be defined. Based on the results of this study, exposure levels of 5, 20 and 60 ppm were selected for a definitive inhalation developmental toxicity study in rats, exposure levels of 5, 20 and 50 ppm were selected for the two-generation inhalation reproduction study in rats and exposure levels of 5, 20 and 70 ppm were selected for a 13-week inhalation subchronic toxicity study in rats.