Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment

Data source

Reference Type:
Liver and lung tumors in dogs from 4,4'-methylene-bis(2-methylaniline).
Stula EF, Barnes JR, Sherman H, Reinhardt CF, Zapp Jr JA
Bibliographic source:
J. Environ Pathol Toxicol 1: 339-356.

Materials and methods

Principles of method if other than guideline:
Determination of target organ toxicty including carcinogenicty in dogs following chronc and repeated exposure to 4,4'-methylenedi-o-toluidine.
GLP compliance:
Limit test:

Test material

Details on test material:
- Name of test material (as cited in study report): 4,4'-methylene-bis(2-methylaniline)
The sample was >99 percent active ingredient. It was crystalline, beige in color, and slightly soluble in water. The melting point was 157-159 °C, and the molecular weight was 226.16.

Test animals

Details on test animals and environmental conditions:
The dogs were fed a commercial dog chow ad libitum. They were housed in separate cages in a climate-controlled room and were put into an outside concrete exercise yard daily. Separate exercise yards were used for control and test dogs.

Administration / exposure

Route of administration:
oral: capsule
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
up to 7.0 years
Frequency of treatment:
The dogs were dosed 100 mg per day, 3 times per week for the first 6 weeks, then 5 times per week for 5 weeks, at which time the dose was reduced to 50 mg per day 5 times per week continuously for up to 7.0 years.
Doses / concentrations
Doses / Concentrations:
100 mg/day (11 weeks) then 50 mg/kg until end of the study
actual ingested
No. of animals per sex per dose:
6 female dogs
Control animals:
yes, concurrent no treatment
Details on study design:
Each of six purebred female beagle dogs, approximately one year old, was dosed orally with MeMDA in a gelatin capsule. They were dosed 100 mg per day, 3 times per week for the first 6 weeks, then 5 times per week for 5 weeks, at which time the dose was reduced to 50 mg per day 5 times per week continuously for up to 7.0 years. When a dog exhibited anorexia, MeMDA was not administered until the dog was eating again. Six purebred female beagle dogs were kept as untreated controls and were sacrificed after 8.3 to 9.0 years on test, as they served as controls for several other tests.


Observations and examinations performed and frequency:
Specimens of blood and 24-hour urine samples were collected from each dog, once before the test began, and then after 7, 14, 18, 24, 30, 33, 39, 43, 47, 55, 63, 69, 77, 83, 89, 92, and 102 months on test. The following tests were performed:

HEMATOLOGY: erythrocyte count; hemoglobin concentration; hematocrit; total leucocyte count; differential leucocyte count.

BIOCHEMISTRY (on blood): glucose; urea nitrogen; cholesterol; alkaline phosphatase; glutamic-pyruvic transaminase; total protein; albumin-globulin ratio.

URINALYSIS: volume, pH, appearance, osmolality; protein; sugar; blood; acetone; urobilinogen; bilirubin; microscopic examination of sediment.

URINE SEDIMENT CYTOLOGY: Once each year a 12-hour urine specimen was collected in 250 ml 95 percent ethyl alcohol. Fifty ml of the above mixture was centrifuged at 1500 rpm for 20 minutes. The sediment was fixed and stained and examined microscopically.
Sacrifice and pathology:
NECROPSY: dogs were sacrificed by electrocution and a complete necropsy was performed. Color photographs were taken of various gross lesions. Tissues were fixed in Bouin's fluid and in 10 percent buffered formalin. Tissues were embedded in paraffin and stained with hematoxylin-eosin.
Fisher's Exact Test

Results and discussion

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Mortality: One substance treated dog died after 2.8 years. Four dogs treated with the test substance were sacrificed in extremis after the following time periods: 2.7, 4.0, 5.2, and 6.0 years.

Hematology and clinical chemistry:

The erythrocyte count, hemoglobin concentration, and hematocrit of substance treated dogs were generally lower than those of the untreated controls. Four of six substance treated dogs had elevated blood urea nitrogen during an approximate six-month time period prior to death or being sacrificed in extremis. One dog of the control group had elevated plasma glutamic-pyruvic transaminase and and alkaline phosphatase during the entire test period. All substance treated dogs showed elevated plasma glutamic-pyruvic transaminase and all but one had an elevated plasma alkaline phosphatase.

Microscopic examination of the urine sediment did not reveal neoplastic changes in the genitourinary tract of test or control dogs.


Four of the six substance treated dogs had a thin, pale, irregular renal cortex. Microscopically, there was a severe proximal tubular atrophy with fibrosis. Three of six treated dogs had subintimal fibrosis of inedium sized renal arteries with a reduction in the size of the lumen. These effects on the kidneys were not seen in the control dogs.

Multiple pale focal areas and/or pale nodules in the liver measuring up to 5.0 cm in diameter were found in all substance treated dogs at necropsy. Histologically, these focal areas were classified as focal cell alteration, cholangiofibrosis, nodular hyperplasia, or hepatocellular carcinoma. In focal cell alteration there was no disruption of the liver architecture; the affected

hepatocytes were usually enlarged with a centrally positioned nucleus and a vacuolated cytoplasm. Usually the vacuolation was not spherical to suggest a fatty change, although fatty change was at times a minor component of the lesion designated as focal cell alteration. Cholangiofibrosis was seen in 2/6 dogs of the control group.


The three dogs that survived treatment with the test substance for 5.2 to 7.0 years developed hepatocellular carcinomas. In addition, two them developed lung carcinomas. The tumors contained glandular and epidermoid characteristics and were classified as combined epidermoid and adenocarcinomas, with a possible origin from the bronchus and/or bronchial glands.

No liver or lung tumor were observed in the six dogs of the control group. Thus, the increase in the tumor rate was considered to be statistically relevant (liver tumors: p <0.05; lung tumors: p <0.10).

Four dogs of the control group, treated for 8.3 to 9.0 years, had mammary tumors, whereas no mammary tumors were observed in dogs treated with the test substance for for 2.7 to 7.0 years.

Applicant's summary and conclusion