m-phenylenediamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

m-phenylenediamine

EC Number:

203-584-7

EC Name:

m-phenylenediamine

Cas Number:

108-45-2

Molecular formula:

C6H8N2

IUPAC Name:

m-phenylenediamine

Details on test material:

- Purity: 99%.
- Source: Fluka No. 78420 (Fluka AG, Buchs, Switzerland.)
- Physical state: Pale-gray, finely crystalline and odorless.
- Stability under test conditions: Test substance solution did not seem to change significantly in the period from dissolution to application on the test animals.
- Other: Protected from light and kept under nitrogen in a refrigerator.

Test animals

Species:

rat

Strain:

other: OFA(SD)SPD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Research Institute for the Breeding of Test Animals (A-2325 Himberg, Austria.)
- Age at study initiation: No details
- Average weight at study initiation: 157 g for males and 136 g for females
- Housing: Kept in pairs in Type III Makrolon cages with sterilized softwood chips for bedding
- Diet: ad libitum - Altromin Maintaining Diet No. 1324, pelletized
- Water: ad libitum -Tap in bottles acidified with sulfuric acid to a pH of 5
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 60
- Photoperiod (hrs dark / hrs light): 10/14

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: deionized water

Details on oral exposure:

The test substance was administered once daily in the mornings Monday- Saturday (inclusive) using a metal esophageal probe. The test substance was freshly dissolved in deionized water.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks (90 days)

Frequency of treatment:

Once daily in the mornings Monday-Saturday (inclusive) given a volume of 10 ml/kg at various doses.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 other: mg/kg/day

Doses / concentrations 2

Dose / conc.:

6 other: mg/kg/day

Doses / concentrations 3

Dose / conc.:

18 other: mg/kg/day

No. of animals per sex per dose:

20 (and control group)
160 animals total (80 male, 80 female)

Control animals:

yes

Details on study design:

Male and female animals were separated and subdivided into groups based on increasing body weight using a random number series. The animals were put pairwise into cages according to consecutive numbers. The arrangement of the cages in the room was not randomized. The animals were individually marked in the right ear with brass ear markers. The animals with odd numbers received an additional notch in the left ear muscle.

- Dose selection rationale: The doses were selected based on previously obtained results from a dose identification study (4-week oral gavage).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: Daily Mon-Sat by caretakers and once per week by the veterinarian.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily Mon-Sat by caretakers and once/week by the veterinarian

BODY WEIGHT: Yes
- Time schedule for examinations: Once/week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Once/week

WATER CONSUMPTION: Yes
- Time schedule for examinations: Once/week

OPHTHALMOSCOPIC EXAMINATION: Yes
-Performed during last test week

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before substance was administered 6 days prior to study start and at test days 30 and 86
- Anaesthetic used for blood collection: Yes (light ether narcosis)
- Animals fasted: No
- How many animals: 10 males and 10 females from all groups

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before substance was administered 6 days prior to study start and at test days 30 and 86
- Animals fasted: No
- How many animals: 5 males and 5 females from all groups (at first two collection time points); 10 males and 10 females from all groups (at last collection time point)

URINALYSIS: Yes
- Time schedule for collection of urine: 6th, 7th, 28th, 29th, 84th, 85th days.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- How many animals: 10 males and 10 females from all groups

Sacrifice and pathology:

All animals that were still living at the end of the test and any that died prematurely during the study were sacrificed, dissected and examined on the 91st-93rd test days. Organs were weighed, fixed, and stained with hematoxillin and eosin. Eyes were fixed according to Susa method. The histopathology evaluation took place on a Zeiss photo microscope III.
Gross and relative organ weight and histopathology was performed on selected organs.

Organs weighed and prepared for histopathology: kidneys, suprarenal bodies, spleen, testicles, heart, liver, brain, and hypophysis (pituitary gland). Organs in pairs were weighed together if obvious size differences were not present. All organs except for the liver were analyzed histopathologically from the control and high dose groups. The liver was analyzed histopathologically from the control and all test groups.

Statistics:

The mean, standard deviation and sample size of the median together with the smallest and largest values are presented in this study. Frequencies above approximately 10 (e.g., number of neutrophils) and measured values were examined with simple analysis of variance followed by the Scheffe test for uniformity of the group means. Frequencies below approximately 10 (e.g., number of monocytes) and ordinal data (e.g., urine evaluation) were compared using the H-test. The Chi-square test and/or Fisher's Exact Test was used for events that were counted (numerated events). A value of p <=0.05 was established as the significance limit.

All testing methods were taken from the book "Applied Statistics" by Sachs L (Springer Publishing House, 4th edition, 1974)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

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:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

In addition to spontaneous alterations and probably random differences between the groups, the following substance-related effects were observed or measured:
-An abundance of incriminating, degenerative lesions of the liver in the group receiving the highest dose (statistically significant only in one case: pycnosis).
-A definite and dose-related increase in the absolute and relative liver weights in the highest dose group.
-An increase in kidney weight, although statistically significant only with females in the group receiving the highest dose.

All toxic effects mentioned above were only observed in the highest dose group (18 mg/kg of body weight).

Effect levels

Dose descriptor:

NOAEL

Effect level:

6 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: 1) an increase in the amount of serious degenerative liver damage (nuclear pyknosis), 2) a clear and dose-dependent increase in the absolute and relative liver weight, and 3) an increase in the kidney weight (females only).

Target system / organ toxicity

1

Critical effects observed:

yes

Lowest effective dose / conc.:

18 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

liver

Treatment related:

yes

2

Critical effects observed:

yes

Lowest effective dose / conc.:

18 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Applicant's summary and conclusion

Conclusions:

The rat oral 90 day gavage NOAEL identified in this study for the test substance was 6 mg/kg bw.

Executive summary:

Male and female rats were orally gavaged with the test substance for 90 days at levels of 2, 6, and 18 mg/kg bw (6 days per week). The following statistically significant test-substance related effects were observed in the 18 mg/kg bw: 1) an increase in the amount of serious degenerative liver damage (nuclear pyknosis), 2) a clear and dose-dependent increase in the absolute and relative liver weight, and 3) an increase in the kidney weight (females only). Based on this information, the rat oral 90 day gavage NOAEL identified in this study for the test substance was 6 mg/kg bw.