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Repeated dose toxicity: inhalation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Iodomethane
EC Number:
200-819-5
EC Name:
Iodomethane
Cas Number:
74-88-4
Molecular formula:
CH3I
IUPAC Name:
iodomethane
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Iodomethane
- Physical state: liquid
- Analytical purity: 99.7%
- Impurities (identity and concentrations): 0.2 % water and <0.1 % methanol
- Composition of test material, percentage of components: not stated
- Isomers composition: not stated

Test animals

Species:
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

Administration / exposure

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)
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.

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