m-phenylenebis(methylamine)

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

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): 1,3-bis(aminomethyl)benzene
- Physical state: Colourless transparent liquid easily soluble in water at a vapour pressure of 1.6 x 10E-3 mmHg (25 degrees Centigrade)
- Storage condition of test material: Sealed container in the dark until used

- Molecular weight: 136.22
- Melting point: 14 degrees Centigrade

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 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)

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Remarks:

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

Doses / concentrations 2

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: body weight; food consumption

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.