m-phenylenebis(methylamine)

Endpoint summary

Description of key information

The significant toxic changes detected after repeated oral administration of the test material to rats were stomach membrane disorders. The changes in the digestive system are considered to be induced by the corrosive nature of the substance. The systemic LOAEL is 600 mg/kg bw/day.
The toxic changes detected after repeated inhalation administration of the test material to rats were minimal to mild bronchial epitelial degeneration, minimal bronchial squamous metaplasia and minimal to mild subacute inflamation in the lungs. The local changes in the inhalatory system are considered to be induced by the corrosive nature of the substance. The systemic NOAEC is assumed too be at 30 mg/m^3. The local NOAEC is determined at 5 mg/m^3.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, well documented and acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

not applicable

Principles of method if other than guideline:

Method for 28-day repeat dose toxicity testing of chemicals developed by the Research Institute for Animal Science in Biochemistry and Toxicology to Japanese guidelines

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Co Ltd
- Age at study initiation: Five weeks
- Weight at study initiation: 173-188 g (males); 139-162 g (females)
- Fasting period before study: Animals were not fasted prior to sampling.
- Housing: The animals were housed individually in metal wire cages placed in a breeding room
- Diet: Free access to pellet food (Japan Nosan Kogyo K.K. Labo MR stock)
- Water (e.g. ad libitum): The animals were allowed free access to water
- Acclimation period: Twelve to thirteen days


ENVIRONMENTAL CONDITIONS
- Temperature: 22˚C ± 3 degrees Centigrade
- Humidity: 55 ± 10 %
- Air changes: At least ten air changes per hour.
- Photoperiod: Lighting was controlled to give twelve hours continuous light (06:00 to 18:00)

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The dosing solution was formulated by dissolution of the test material in JP purified water (Kyoei Pharmaceutical Co Ltd) and administered by gavage.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

Dose range finding test: 14 days
Main test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days per week

Remarks:

Doses / Concentrations:
Dose range finding test
Basis:
other: 0, 30, 80, 200 and 500 mg/kg/day

Remarks:

Doses / Concentrations:
Main test
Basis:
other: 0, 10, 40, 150 and 600 mg/kg/day

No. of animals per sex per dose:

Dose range finding test
Male and Female: 3 animals per sex at 0 mg/kg/day
Male and Female: 3 animals per sex at 30 mg/kg/day
Male and Female: 3 animals per sex at 80 mg/kg/day
Male and Female: 3 animals per sex at 200 mg/kg/day
Male and Female: 3 animals per sex at 500 mg/kg/day

Main test
Male and Female: 6 animals per sex at 0 mg/kg/day
Male and Female: 6 animals per sex at 10 mg/kg/day
Male and Female: 6 animals per sex at 40 mg/kg/day
Male and Female: 6 animals per sex at 150 mg/kg/day
Male and Female: 6 animals per sex at 600 mg/kg/day

Main test 14 day recovery groups
Male and Female: 6 animals per sex at 0 mg/kg/day
Male and Female: 6 animals per sex at 600 mg/kg/day

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The LD50 of 1,3-bis(aminomethyl)benzene in a single oral administration to the rat had been reported as 930 mg/kg and this was used as the starting point for a dose range finding test.
- Rationale for animal assignment (if not random): The rat was selected for this study as it is a readily available rodent species rodent historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
- Rationale for selecting satellite groups: A group of three males and three females was established specifically for the purpose of documenting post treatment effects
- Post-exposure recovery period in satellite groups: 14 days:

Positive control:

No data

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed for mortality, appearance, behaviour etc every day throughout the treatment and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed before dosing every day during the treatment period and once weekly during the treatment free period.

FOOD CONSUMPTION AND COMPOUND INTAKE: Twenty four hour food consumption was recorded for each cage group at weekly intervals.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at the time of killing animals (Day 29 for treatment and control groups, Day 43 for treatment recovery group and control group)
- Anaesthetic used for blood collection: No data:
- Animals fasted: fasting took place from 5:00 pm on the day before blood collection but normal conditions of hydration were maintained
- How many animals: 41 males and 38 females
- Parameters examined: erythrocyte count, hemoglobin level, hematocrit level, mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration (theoretical), total leucocyte count, platelet count, reticulocyte count, differential leucocyte count, prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected at the time of killing animals (Day 29 for treatment and control groups, Day 43 for treatment recovery group and control group)
- Animals fasted: fasting took place from 5:00 pm on the day before blood collection but normal conditions of hydration were maintained
- How many animals: 41 males and 38 females
- Parameters examined: total protein, albumin, theoretical A/G ratio, blood sugar, triglyceride, total cholesterol, total bilirubin, urea nitrogen, creatinine, GOT, GTP, gamma-GTP, alkaliphosphatase, calcium, inorganic phosphorus, sodium, potassium and chloride

URINALYSIS: Yes
- Time schedule for collection of urine: Animals were forced to urinate via stimulation of the dorsolumbar part on day 25 (all animals) and day 39 (recovery and control groups)
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: appearance, pH, blood, protein, glucose, ketones, bilirubin and urobilinogen

NEUROBEHAVIOURAL EXAMINATION: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Animals were necropsied after blood collection and the weights of brain, liver, kidneys, adrenals, and testes/ovaries were measured.

HISTOPATHOLOGY: Yes
- Collected organs were fixed in a neutral buffered 10% formalin solution. In the 600 mg/kg and associated control groups, preserved tissues of heart, liver, spleen, kidneys, adrenals, bone marrow, stomach, intestines (duodenum, jejunum, ileum, caecum, colon, rectum) and thymus were then separated, prepared as paraffin sections and stained with hematoxylin and eosin (H-E) for subsequent microscopic examination. For the 10, 40, and 150 mg/kg/day and recovery group animals the same procedure was used to examine adrenals, stomach and bone marrow.

Other examinations:

MORTALITY DATA

All animals were observed for mortality, appearance, behaviour etc every day throughout the treatment and recovery periods.

ORGAN WEIGHTS

Animals were necropsied after blood collection and the weights of brain, liver, kidneys, adrenals, and testes/ovaries were measured.

Statistics:

The mean values or frequencies obtained were analysed using Dunnett's or Scheffe's (when the groups are different in size) multiple comparison test. The values obtained for the recovery group were analysed by the "t" or "u" test.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

No treatment-related changes were observed in any observation or investigation parameters of the 10, 40 and 150 mg/kg/day groups.

Clinical observations and mortality
Among the 12 males and 12 females of the 600 mg/kg dose group, ten males and eight females showed increased salivation, three males and seven females decreased spontaneous locomotion, and one male and four females died (on day 15 to 19). No abnormalities were detected during the treatment free period.

Bodyweight and food consumption
Males treated with 600 mg/kg/day tended to show a reduction in body weight gain from day 2. The difference from the control group tended to increase with the progress of treatment and was significant after day 4 except day 7. However, the body weight gain of the males tended to recover during the treatment free period. Body weight changes are shown in Figure 1 (attached).

Food consumption of the males of the same dose group was also smaller than that of the control group throughout the treatment period, being significantly smaller during week 4. In the treatment free period, however, there was no difference in food consumption between males treated with 600 mg/kg/day and those of the control group.

Urinalysis
Urine protein of high dose males increased significantly. The urine protein value of many males from the 600 mg/kg dose was ++(100 mg/dl) while the value of many of the control animals was +(30 mg/dl). No significant changes were observed in any investigation parameter.

Hematology
Males from the 600 mg/kg/day dose group showed significant reductions in hemoglobin and hematocrit levels, an increase in prothrombin time, a reduction in activated partial thromboplastin time, an increase in segmented neutrophil ratio in differential leukocyte count, and a reduction in lymphocyte ratio. Details are shown in Table 1 (attached).

Females from the same dose group also tended to show a reduction in hematocrit level and an increase in leukocyte count primarily due to neutrophilia although these changes were not statistically significant. The recovery group animals showed no significant changes in any investigation parameter. Details are shown in Table 2 (attached).

Blood chemistry
A significant reduction in total protein and an increase in inorganic phosphorus were detected in males from the 600 mg/kg/day dose group and an increase in triglyceride in females from the same dose group. The recovery group showed none of these changes, but significant increases in total cholesterol and A/G ration and a decrease in chloride were detected in males and a decrease in albumin in females, apart from the changes seen in the animals killed after completion of the treatment period. Details are shown in Tables 3 and 4 (attached).

Necropsy
In the 600 mg/kg/day dose group, hyperplasia and ulcer of the anterior stomach and typhlectasis due to an increase in the contents of caecum were seen in all male and female rats and adrenocortical hypertrophy in half of the females. One male rat and four female rats from the same dose group which died during the treatment period showed changes in anterior stomach similar to those seen in animals that survived - reddened mucous membrane and ulcer of the grandular stomach, and expanded stomach and intestines due to gas retention. In addition, some of the females showed red stigma on the cecal mucous membrane, reddened adrenals, and atrophy of the thymus and spleen. Thickened anterior stomach wall was also seen in almost all of the animals in the recovery group, but the changes were slighter than those seen in the animals killed after completion of the treatment period.

Organ weight

Details of organ weight investigations are shown in Tables 5 and 6 (attached).

Relative adrenal weight of males treated with 600 mg/kg/day and absolute and relative adrenal weights of females treated with the same dose increased significantly.

Male animals from the 600 mg/kg/day dose group showed a significant reduction in absolute liver weight and a significant increase in relative brain weight following the reduction in body weight gain. However, there were no differences in the the relative liver weight and absolute brain weight between the group and the controls. No significant changes were detected in any of the organ weights of the recovery group animals.

Histopathology

Details of histopathological changes are shown in Tables 7 and 8 (attached).

Treatment-related changes were observed in stomach, adrenals and bone marrow. In all of the 600 mg/kg/day animals that survived the twenty-eight day exposure period (6 males and 4 females), deep ulcers ranging from the muscular tunics to the serous membrane were formed in the anterior gastric mucous membrane. In the submucosa, inflammatory changes were seen. In addition, hyperplasia of the stratified squamous epithelium and thickening due to hyperkeratosthenia were observed in the peripheral mucous membrane. Erosion was detected in the mucous membrane of the glandular stomach of one male and one female from the 600 mg/kg/day dose group, but the changes were smaller than those seen in the anterior stomach. Abnormalities were also detected in the adrenals, including vacuolation of the cortex, inparticular, the spongiocte in one female and one male treated with 600 mg/kg/day, and hypertrophy in two females. In the bone marrow of four males and two females of the same dose group, a slight increase in granulocytic hematopoietic cells was observed.

The one male and four female decedents from the 600 mg/kg/day dose group showed congestive or atrophic changes in various organs in addition to the changes seen in the animals killed after completion of the treatment period. Furthermore, necrosis, erosion and ulcer were observed on the glandular mucous membrane of the stomach. Vacuolation in the duodenal mucosal epithelium, hyperemia of cecal mucosa, dilation of renal distal tubulus, and degeneration/necrosis of adrenocortical cells were also noted in the deceased females. The recovery group animals recovered or tended to recover from the changes seen in the animals killed after completion of the treatment period. The stomach showed no mucosal coloboma, but only after submucosal fibrination and slight mucosal thickening in many cases. The adrenals and bone marrow showed no abnormalities.

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: body weight; food consumption

Critical effects observed:

not specified

The significant toxic changes detected after repeated oral administration of 1,3 -bis(aminomethyl)benzene to rats were stomach membrane disorders.

The animals treated with 10, 40 and 150 mg/kg/day showed no changes considered to be attributable to the administration of the test material.

Male and female animals from the 600 mg/kg/day dose group showed increased salivation, decreased spontaneous locomotion, pilo-erection, and distended abdomen. The males showed decreased food consumption and restrained body weight gain and one male and four females out of twelve of each sex died. Severe gastric disorders were observed both in the survived and deceased animals.

In necropsy there were observed ulceration and parietal thickening of the anterior stomach. The parietal thickening was caused by hyperplasia of the stratified squamous epithelium and hyperkeratosthenia, which is histopathologically considered to be a submucosal inflammatory reaction accompanying the ulceration and reactive hyperplasia to gastric disorders. The decedents showed mucosal necrosis, erosion, and ulcer not only in the anterior but also glandular stomach. It was, therefore, confimed that the leading cause of death was gastric disorder.

The slight anemia, increased neutrophil, increased hematopoietic granulocyte tendancy in the bone marrow, and decreased total serum protein observed in males and females treated with 600 mg/kg/day are also considered to be related to the inflammation and bleeding caused by the gastric disorders. It was also inferred that the increased prothrombin time and reduced activated partial thromboplastin time have some relation to gastric bleeding and inflammation although those effects are not constantly directed to the blood coagulant system, since no hepatic abnormalities were detected.

In addition to distended abdomen, caecal dilation due to increased contents in surviving animals, and gastric and intestinal dilations due to gas retention in deceased animals, was noted. Epithelial vacuolation in the duodenal musous membrane and hyperemia in the caecal mucous membrane were also seen in some of the decedents. Thus, toxic effects were observed throught the gastrointestinal tract.

The adrenals of the 600 mg/kg/day group increased in weight and histopathologically showed vacuolation and hypertrophy of the adrenocortical cells, in particular, in the zona fasciculate. It is inferred that these symptoms reflect the effect of stress caused by gastric disorders.

1,3 -bis(aminomethyl)benzene is very irritant to eyes and skin. It is , therefore, considered that the gastrointestinal disorders, and gastric disorders in particular, were caused by the test material inducing local irritation when administered orally.

The congestive and atrophic changes in various organs detected in the descedents were not seen in the animals that survived and, therefore, were not considered to be findings that suggested a direct effect of 1,3 -bis(aminomethyl)benzene.

Changes observed in animals killed after completion of the treatment period were not detected, or tended to be undetected, in animals killed after completion of a treatment-free period. Such changes were, therefore, confirmed as reversible. Reduce serum albumin level, which was not clear in the animals killed after completion of treatment, was detected in females killed after completion of a treatment-free period. The change is, however, considered to be due to delayed development of gastric disorder.

In addition to the changes discussed above, increases in urine protein level and serum inorganic phosphorus were observed in male 600 mg/kg/day animals killed after completion of the treatment period and an increase in triglyceride in females from the same dose group killed at the same time. An increase in total cholesterol and a reduction in chloride were also seen in male animals from the same dose group killed after completion of the treatment-free period. However, no renal or hepatic changes were observed pathologically and all of the above changes were slight.

Conclusions:

The leading toxic change caused by 28 -day repeated oral administration of 1,3 -bis(aminomethyl)benzene was gastric disorder and the No Observed Effect Level (NOEL) is considered to be 150 mg/kg/day.

Endpoint conclusion

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

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:

from 2013-02-14 to 2013-

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conforming GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

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
- 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

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

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

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.

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

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'

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

30 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Only one GLP study according to OECD TG 413 is available.

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:

from 2013-02-14 to 2013-

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conforming GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

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
- 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

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

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

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.

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

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'

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

5 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Only one GLP study according to OECD TG 413 is available.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Critical effects observed:

not specified

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Critical effects observed:

not specified

Additional information

- Repeated dose toxicity, oral

During a 28-day repeated dose oral toxicity study (Yamamoto et al, 1996), the substance was administered to rats by gavage at doses of 0, 10, 40, 150 and 600 mg/kg bw/day. No treatment-related changes were observed in any observation or investigation parameters of the 10, 40 and 150 mg/kg/day groups. However, among the 12 males and 12 females of the 600 mg/kg dose group, ten males and eight females showed increased salivation, three males and seven females decreased spontaneous locomotion, and one male and four females died (on day 15 to 19). No abnormalities were detected during the treatment free period although gastric changes were found in all animals, the changes being more severe in animals killed after the completion of the treatment period. Thus the leading toxic change was considered to be gastric disorder and the NOEL was reported as 150 mg/kg bw/day.

Further data on the effects of repeated oral doses of the test substance are reported as part of a reproduction/developmental toxicity screening study.

- Repeated dose toxicity, inhalation

MXDA is deemed to show a classifiable specific target organ toxicity since at the highest administered concentration at 30 mg/m^3 in the reliable inhalation study conducted according to OECD TG 413, local adverse effects were identified (Randazzo, 201X). During 90 -day repeated dose inhalation study, the substance was administered to rats by aerosol inhalation at doses of 0, 0.6, 5.0 and 30 mg/m^3. Groups 1 and 4 each consisted of 20 animals/sex and groups 2 and 3 each consisted of 10 animals/sex. 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 and macroscopic or organ weight findings at the scheduled necropsies.

At 30 mg/m^3 concentrations the local effect findings like minimal to mild bronchial epithelial degeneration and minimal bronchial squamous metaplasia were considered to be adverse. Therefore, the local NOAEC is determined at 5 mg/m^3. The systemic NOAEC is assumed to be at 30 mg/m^3.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
The repeated dose toxicity of the substance is characterized from a valid subchronic inhalation toxicity study conducted with MXDA. Since the observed adverse effects were expressed localy, the systemic NOAEC is assumed to be the highest administered concentration 30 mg/m^3.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
The repeated dose toxicity of the substance is characterized from a valid subchronic inhalation toxicity study conducted with MXDA. The local NOAEC is determined at 5 mg/^3. The local LOAEC is at 30 mg/m^3.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: stomach

Justification for classification or non-classification

- Repeated dose toxicity, oral:

No abnormalities were detected during the treatment free period although gastric changes were found in all animals, the changes being more severe in animals killed after the completion of the treatment period. Thus the leading toxic change was considered to be gastric disorder and the NOEL was reported as 150 mg/kg bw/day. The systemic LOAEL is 600 mg/kg bw/day.

Based on the above stated assessment of the specific target organ toxicity potential after repeated oral exposure, MXDA does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) or according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.

- Repeated dose toxicity, inhalation:

The present 13-week inhalation toxicity study with MXDA does not show test substance related systemic effects up to and including 30 mg/m3, resulting in a systemic NOAEC of ≥30 mg/m3. Local effects in the lungs were however observed. Test substance-related, minimal bronchial epithelial degeneration was observed at 5.0 mg/m3. In the 30 mg/m3 group, minimal to mild bronchial epithelial degeneration, minimal bronchial squamous metaplasia, and minimal to mild subacute inflammation in the lungs were observed. These histologic changes in the lung are reversible as these effects were not observed after a 4 week recovery period.

As systemic effects were not observed in this repeated dose toxicity study, only the local effects induced by the test substance need to be considered for STOT-RE classification. It should be noted that theclassification criteria for STOT-RE are based on significant changes, e.g. organ dysfunction, functional changes, in which these systemic effects show a time dependent increase of severity (Guidance on the application of the CLP criteria, November 2012). As MXDA induces only local effects which are mild without observed functional impairment, the substance does not have to be classified based on this study, according tothe Globally Harmonised System of Classification and Labeling of Chemicals (GHS UN) with regard to repeated dose inhalation toxicity.