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EC number: 216-032-5 | CAS number: 1477-55-0
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Repeated dose toxicity: inhalation
Administrative data
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- from 2013-02-14 to 2013-
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline conforming GLP study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 013
- Report date:
- 2013
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
Test material
- Reference substance name:
- m-phenylenebis(methylamine)
- EC Number:
- 216-032-5
- EC Name:
- m-phenylenebis(methylamine)
- Cas Number:
- 1477-55-0
- Molecular formula:
- C8H12N2
- IUPAC Name:
- 1-[3-(aminomethyl)phenyl]methanamine
- Test material form:
- liquid
- Details on test material:
- - Name of test material (as cited in study report): MXDA (meta-xylenediamine)
- Physical state: clear, colorless liquid
- Storage condition of test material: at ambient temperature in a sealed container with headspace purged with nitrogen
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 60 days old
- Weight at study initiation: 250 - 307 g (males), 166 - 217 g (females)
- Fasting period before study: no
- Housing: individually in clean, stainless steel, wire-mesh cages suspended above cage-board
- Diet (e.g. ad libitum): ad libitum, but withheld during each daily exposure period
- Water (e.g. ad libitum): ad libitum, but withheld during each daily exposure period
- Acclimation period: 15 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 - 22.2
- Humidity (%): 46.8 - 68.4
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Administration / exposure
- Route of administration:
- inhalation: aerosol
- Type of inhalation exposure:
- nose only
- Vehicle:
- clean air
- Remarks on MMAD:
- MMAD / GSD: Mean MMAD (Mean GSD), µm:
Group 2: 2.1 (2.65)
Group 3: 2.2 (3.04)
Group 4: 2.3 (2.99) - Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 7.9-L and 14.1-L stainless steel conventional nose-only exposure systems with synthetic rubber grommets in exposure ports to engage animal holding tubes
- Method of holding animals in test chamber: animal holding tubes
- Source and rate of air: in-house supply air system
- System of generating particulates/aerosols: 1-jet Collision nebulizer (BGI, Inc., Waltham, MA)
- Temperature, humidity, pressure in air chamber: 20.4 - 23.4 °C, 53.5 - 57.0 %, slight negative pressure
- Air flow rate: 45.4 - 49.7 LPM
- Air change rate: 10 per h
- Method of particle size determination: using a 7-stage stainless steel cascade impactor
- Treatment of exhaust air: with Solberg filter
TEST ATMOSPHERE
- Brief description of analytical method used: standard gravimetric method
- Samples taken from breathing zone: yes
VEHICLE (if applicable)
- Concentration of test material in vehicle: 0, 0.6, 5.0, 30 mg/m^3 - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Actual exposure concentrations were determined using standard gravimetric methods. Atmosphere samples were collected on pre-weighed, 25-mm glass-fiber filters held in open-faced filter holders positioned in the approximate animal breathing zones of the nose-only exposure systems. Following sample collection, the filter was re-weighed and the mass concentration (mg/m3) of the test substance was calculated as the filter weight difference divided by the sample volume.
- Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- 6 hours per day, 5 days per week
Doses / concentrations
- Remarks:
- Doses / Concentrations:
0, 0.64, 5.1, 31 mg/m^3
Basis:
analytical conc.
- No. of animals per sex per dose:
- Groups 1 and 4 each consisted of 20 animals/sex and Groups 2 and 3 each consisted of 10 animals/sex.
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: In a previous range-finding inhalation study, rats were exposed to MXDA for 6-hours per day, 5 days per week, for a period of 4 weeks (up to 20 total exposures), target concentrations were 10, 40, and 200 mg/m^3. Three males in the 200 mg/m^3 group were euthanized in extremis. Test
substance-related microscopic findings were observed in the lungs of the 10, 40, and 200 mg/m^3 group males and females. Findings included minimal to mild mucous cell hyperplasia noted in the bronchi and medium-sized bronchioles of the lungs in the 10, 40, and 200 mg/m^3 group males and females; and minimal to mild epithelial degeneration of the bronchial and bronchiolar epithelium noted in the 10, 40, and 200 mg/m^3 group males and females. No other test substance-related findings were noted. Based on the data, the high exposure concentration for this 13-week toxicity study was set at 30 mg/m^3.
- Post-exposure recovery period in satellite groups: 4-weeks - Positive control:
- Not applicable
Examinations
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and moribundity. Clinical examinations were performed daily, prior to exposure, at the approximate midpoint for visible clinical signs while animals were in the nose-only restraint tubes, and 0-1 hour (+0.25 hour) following the end of exposure (designated as 1 hour post-exposure for report presentation purposes).
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: within 4 days of receipt, 1 week prior to randomization (± 2 days), on the day of animal selection for randomization, and weekly (± 2 days) during the study period, and on the day of the scheduled necropsies.
BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded within 4 days of receipt, 1 week prior to randomization (± 2 days), on the day of randomization, study day 0 (prior to exposure), twice weekly (± 1 day) during the first 4 weeks and weekly (± 2 days) during the exposure period, weekly (± 1 day) during the recovery period, and on the day prior to the first day of the scheduled necropsies (non-fasted).
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ocular examinations were conducted on all animals during acclimation and near the end of the exposure period.
- Dose groups that were examined: all animals
HAEMATOLOGY: Yes
- Time schedule for collection of blood: on days 91 and 92
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals scheduled for the primary necropsy
- The following parameters were examined: Total leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLATELET), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count Percent (RETIC), Absolute (RETIC ABSOLUTE), Mean Platelet Volume (MPV), Red cell distribution width (RDW), Hemoglobin Distribution Width (HDW), Differential leukocyte count - Percent and absolute: Neutrophil (NEU), Lymphocyte (LYMPH), Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC); Platelet estimate, Red cell morphology (RBC Morphology).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on days 91 and 92
- Animals fasted: Yes
- How many animals: all animals scheduled for the primary necropsy
- The following parameters were examined: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio), Total bilirubin (Total Bili), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyltransferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium Chloride, Phosphorus Potassium, Sodium Sorbitol dehydrogenase (SDH), Triglycerides (Triglyceride), Appearance.
URINALYSIS: Yes
- Time schedule for collection of urine: on days 91 and 92
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- The following parameters were examined: Specific gravity (SG), pH, Urobilinogen (URO), Total volume (TVOL), Color (COL), Clarity (CLA), Protein (PRO), Glucose (GLU), Ketones (KET), Bilirubin (BIL), Occult blood (BLD), Leukocytes (LEU), Nitrites (NIT), Microscopy of sediment.
NEUROBEHAVIOURAL EXAMINATION: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
The following tissues and organs were collected, in addition, microscopic examination was performed: Adrenals (2), Aorta, Bone with marrow, Femur with joint, Sternum, Bone marrow smear (from femur), Brain, Cervix, Epididymides (2), Eyes with optic nerves (2), Gastrointestinal tract, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Harderian glands (2), Heart, Kidneys (2), Lacrimal gland (exorbital [2]), Larynx, Liver (sections of 2 lobes), Lungs (including bronchi, fixed by constant pressure inflation with fixative), Lymph nodes, Axillary (2), Mediastinal and bronchial (if visible), Mesenteric, mandibular (2), Mammary gland (females only), Nasal cavity with turbinates, Ovaries with oviducts (2), Pancreas, Peripheral nerve (sciatic), Peyer’s patches, Pharynx, Pituitary, Prostate, Salivary glands (mandibular [2]), Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin, Spinal cord (cervical, thoracic, lumbar), Spleen, Teeth, Testes (2), Thymus, Thyroid (with parathyroids [2]), Tongue, Trachea, Ureters (2), Urethra, Urinary bladder, Uterus, Vagina, Gross lesions (when possible). - Statistics:
- Each mean was presented with the standard deviation (SD), standard error (SE), and the number of animals (N) used to calculate the mean. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1 % and 5 %, comparing each test substance-treated group to the control group by sex. Body weight, body weight change, food consumption, clinical pathology, and organ weight data were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group.
Results and discussion
Results of examinations
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- incidental dead of one male animal in the Group 3 (5.0 mg/m^3).
- Mortality:
- no mortality observed
- Description (incidence):
- incidental dead of one male animal in the Group 3 (5.0 mg/m^3).
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not specified
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- please see Details on results
- Histopathological findings: neoplastic:
- not specified
- Details on results:
- CLINICAL SIGNS AND MORTALITY
Incidental dead of one male animal in the Group 3 (5.0 mg/m^3).
BODY WEIGHT AND WEIGHT GAIN
Statistically significant higher mean body weight gains were noted in the 5.0 mg/m^3 group males from study day 56 through 63. The difference in body weight gain was considered to be due to biological variability and was not considered related to test substance exposure due to lack of a dose-response trend.
FOOD CONSUMPTION
Statistically significantly lower mean food consumption was noted in the 30 mg/m^3 group males during study week 16 to 16’ compared to the control group value. This difference in food consumption was considered to be due to biological variability and was not considered related to test substance exposure due to the single occurrence.
OPHTHALMOSCOPIC EXAMINATION
No effects
HAEMATOLOGY
Higher mean prothrombin time in the 0.6 mg/m^3 group males and higher mean absolute basophil count in the 0.6 mg/m^3 group females were noted at study week 13; however, these changes did not show a dose-related response. Thus, these changes were not considered to be test substance-related.
CLINICAL CHEMISTRY
No effects
URINALYSIS
No effects
NEUROBEHAVIOUR
Not examined
ORGAN WEIGHTS
Higher mean thyroids/parathyroids weight (absolute, relative to final body weight, and relative to brain weight) was noted in the 0.6 and 30 mg/m^3 group males at the study week 13 primary necropsy; however, this change did not show a clear dose-related response. Lower mean liver weight relative to final body weight was noted in the 30 mg/m^3 group males at the study week 17 recovery necropsy; however, all individual animal absolute liver weight values in the 30 mg/m^3 group males were within the range of individual animal values in the concurrent control group. Thus, these changes were not considered to be test substance-related.
GROSS PATHOLOGY
No effects
HISTOPATHOLOGY: NON-NEOPLASTIC
Test substance-related microscopic findings were noted in the lungs of the 5.0 and 30 mg/m^3 groups at the primary necropsy (study days 91 and 92). Degeneration of the bronchial epithelium (4/9 (m) and 3/10 (f) in 5.0 mg/m^3 group, and 10/10 (m) and 9/10 (f) in 30 mg/m^3 group) was characterized by loss of cilia, attenuation of the epithelium, increased cytoplasmic basophilia, and/or pyknotic nuclei. Degeneration was most pronounced at bronchial branch points and over regions of bronchus-associated lymphoid tissue (BALT). Squamous metaplasia was observed in association with regions of epithelial degeneration in 1 high-dose group male and was characterized by layers of flattened epithelial cells with visible cellular borders and eosinophilic cytoplasm. Subacute inflammation in the lungs was characterized by multifocal areas of infiltration of the alveoli and interstitium by mononuclear and polymorphonuclear inflammatory cells, thickening of alveolar septae, and/or pneumocyte hyperplasia. An increase in the
incidence of pulmonary subacute inflammation was noted in the 30 mg/m^3 group males compared to the control group males at the primary necropsy. Test substance-related histologic changes in the lungs were not observed at the recovery necropsy (study day 119). In the lungs, bronchial epithelial degeneration and squamous metaplasia were not present. Subacute inflammation was present, but without any increase in incidence or severity in the high-dose groups compared to the concurrent control groups. Thus, it was not considered to be test substance-related. There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of
experimental manipulation other than exposure to the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. Subacute inflammation in the larynx was characterized by infiltration of the laryngeal
submucosa and occasionally the laryngeal epithelium by mixed inflammatory cells. At the primary necropsy, the incidence and severity of laryngeal inflammation in the 30 mg/m^3 group males and females was higher than that observed in the control group animals. Based on this observation, the larynx was evaluated microscopically from the low- and mid-dose group animals at the primary necropsy and the control and high-dose group animals at the recovery necropsy. Subacute inflammation in the larynx was present in all dose groups examined with equivocal differences in the incidence and severity of this finding between dose groups. Differences observed were attributed to individual animal biological variation and were not considered to be test substance-related.
HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No data
HISTORICAL CONTROL DATA (if applicable)
Available
Effect levels
open allclose all
- Dose descriptor:
- NOEC
- Effect level:
- 0.6 mg/m³ air
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Test substance-related, minimal bronchial epithelial degeneration was observed in the 5.0 mg/m^3 group euthanized at the primary necropsy.
- Dose descriptor:
- NOAEC
- Effect level:
- 5 mg/m³ air
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: see 'Remark'
Target system / organ toxicity
- Critical effects observed:
- not specified
Applicant's summary and conclusion
- Conclusions:
- The no-observed-ffect concentration (NOEC) is 0.6 mg/m^3. The no-observed-adverse-effect target concentration is 5.0 mg/m^3.
- Executive summary:
The objective of the study was to evaluate potential toxic effects of aerosolized MXDA when administered via nose-only inhalation to Sprague Dawley rats for 6 hours per day on a 5 day per week basis for 13 weeks (minimum of 65 exposures for each animal). In
addition, a 4-week recovery period (minimum of 27 days) was included as appropriate to assess the persistence, reversibility, and/or delayed occurrence of potential test substance-related effects according to the OECD TG 413. Standard toxicity endpoints included clinical observations, body weights, hematology and serum chemistry evaluations, urinalysis, macroscopic examinations and organ weight determinations at necropsy, and microscopic examinations of tissues. The test substance, MXDA (meta-xylenediamine), is an intermediate in the production of epoxy curing agents, polyamides, and polyurethanes.
MXDA was administered via nose-only inhalation exposure for 6 hours per day, 5 days per week for a period of 13 weeks (minimum of 65 total exposures), to 3 groups (Groups 2-4) of Crl:CD(SD) rats. To accommodate a staggered necropsy schedule, the females received an additional exposure during the final week (66 total exposures). Target exposure concentrations were 0.6, 5.0, and 30 mg/m^3 for Groups 2, 3, and 4, respectively. Overall mean measured exposure concentrations were 0.64, 5.1, and 31 mg/m^3 for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) was exposed to filtered air on a comparable regimen. Groups 1 and 4 each consisted of 20 animals/sex and Groups 2 and 3 each consisted of 10 animals/sex. Following 13 weeks of exposure, up to 10 rats/sex/group were euthanized; the remaining ≤10 rats/sex in the control and high-dose groups were euthanized following a 4-week non-exposure (recovery) period. 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 twice weekly for the first 4 weeks and weekly thereafter. Individual food weights were recorded weekly. Ophthalmic
examinations were performed on study days -8 and 86. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals at the primary necropsy (study day 91 or 92). Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals in the control and high-concentration groups at the primary necropsy. The lungs, larynx, and gross lesions were examined from all animals in the low- and mid-concentration groups at the primary necropsy and from all animals at the recovery necropsy.
One male in the 5.0 mg/m^3 group was found dead on study day 77 with no prior clinical observations. The cause of death of this animal was undetermined; however, based on the occurrence of this single unscheduled death in the mid-dose group and the lack of
unscheduled deaths in high-dose group animals, the death of this animal was not considered test substance-related. All other animals survived until the scheduled primary or recovery necropsy. There were no test substance-related clinical observations or effects on body weights or food consumption. There were no alterations in hematology, coagulation, serum chemistry, or urinalysis parameters that were associated with test substance-exposure. There were no test substance-related ophthalmic findings. In addition, there were no test substance-related macroscopic or organ weight findings at the scheduled necropsies. Test substance-related microscopic findings were noted in the lungs of the 5.0 and 30 mg/m^3 groups at the primary necropsy. Degeneration of the bronchial epithelium was characterized by loss of cilia, attenuation of the epithelium, increased cytoplasmic basophilia, and/or pyknotic nuclei.Degeneration was most pronounced at the bronchial branch points and over regions of the bronchus-associated lymphoid tissue (BALT). Squamous metaplasia was observed in association with regions of epithelial degeneration in 1 high-dose group male and was characterized by layers of flattened epithelial cells with visible cellular borders and eosinophilic cytoplasm. In addition, subacute
inflammation in the lungs was characterized by multifocal areas of infiltration of the alveoli and interstitium by mononuclear and polymorphonuclear inflammatory cells, thickening of alveolar septae, and/or pneumocyte hyperplasia. An increase in the incidence of pulmonary subacute inflammation was noted in the 30 mg/m^3 group males compared to the control group males at the primary necropsy. Test substance-related histologic changes in the lungs were not observed at the recovery necropsy.
Exposure of Crl:CD(SD) rats to MXDA via nose-only inhalation, 6 hours per day for 5 days per week over a period of 13 weeks (65 or 66 total exposures) at target exposure concentrations of 0.6, 5.0, and 30 mg/m^3 was well tolerated. However, test substance-related, minimal bronchial epithelial degeneration was observed in the 5.0 mg/m^3 group euthanized at the primary necropsy. Therefore, the no-observed-effect concentration (NOEC) was 0.6 mg/m^3. In the 30 mg/m^3 group, findings from the primary necropsy consisted of test substance-related minimal to mild bronchial epithelial degeneration, minimal bronchial squamous metaplasia, and minimal to mild subacute inflammation in the lungs. Concurrence of the histopathological findings of bronchial epithelial degeneration and bronchial squamous metaplasia at 30 mg/m^3 was considered to be adverse. Therefore, the no-observed-adverse-effect target concentration was 5.0 mg/m^3. In addition, following the 4-week recovery period there were no test substance-related effects.
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