2,4-xylidine

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1947

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Some aspects (treatment, exminations etc.) are in line with the current guideline; restrictions in reporting.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1947

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

yes

Remarks:

3 test animals, one dose level, restricted reporting

Principles of method if other than guideline:

In these experiments the effect of repeated application of Xylidine to the skin of dogs was studied over a period of 7 weeks. 3 dogs treated with 100 mg/kg, 3 served as controls but were treated the same way except for the admission of Xylidine.
During the first 3 weeeks the substance was left in contact with the skin for periods of 4 - 4 1/2 h daily. Thereafter, the substance was left in contact with the skin for 24 h/d so that th animals were continuously exposed.

GLP compliance:

no

Remarks:

study performed before GLP guidelines

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Xylidine

EC Number:

215-091-4

EC Name:

Xylidine

Cas Number:

1300-73-8

IUPAC Name:

2,3-dimethylaniline

Details on test material:

no data

Test animals

Species:

dog

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

no data

Administration / exposure

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

In these experiments the effect of repeated application of Xylidine to the skin of dogs was studied over a period of 7 weeks. For 2 weeks all animals were watched prior to study. 3 dogs treated with 100 mg/kg, 3 served as controls but were treated the same way except for the admission of Xylidine.
During the first 3 weeeks the substance was left in contact with the skin for periods of 4 - 4 1/2 h daily. The skin was swabbed with diluted acetic acid to remove the product remaining on the surface of the skin. Because no toxic effects became apparent the substance was then left in contact with the skin for 24 h so that th animals were continuously exposed.

All treated dogs had to be killed for human reasons (last on the eleventh day of the 24 h/d exposure period).

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

2 weeks acclimatisation
3 weeks 4-4.5 h/d exposure
11 days 24 h/d exposure

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
100 mg/kg bw/d
Basis:
nominal per unit body weight

No. of animals per sex per dose:

3

Control animals:

yes, sham-exposed

Details on study design:

Please refer to "Details on exposure"

Positive control:

none

Examinations

Observations and examinations performed and frequency:

Clinical signs/mortality: daily
Hematology: daily
Urinalysis: daily

Sacrifice and pathology:

gross pathology

Other examinations:

no data

Statistics:

none

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Dermal irritation:

not specified

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Clinical signs/mortality:
The cutaneous application of Xylidine for 4 to 4.5 hours resulted in vomiting in some of the dogs on several occasions, but animals had a good appetite for 12 to 16 days, after which they began tp take less food.
Soon after beginning of the 24 hour exposures they refused food, became inactive, salivated, and one dog (No. 2)developed neurologic symptoms charaterised by spasticity of the fore and hind legs, stiffness of the neck, and convulsions. It discharged blood from mouth and nose and its sclerae were slightly icteric. Shortly before death, which occurred on the fourth day of the prolonged exposure, the animal developed Cheyne-Stokes respiration, indicating that death was due to respiratory paralysis.
The remaining two dogs showed signs of jaundice on the fourth day of the prolonged exposure. They became very weak and markedly depressed. One (No. 3) began to bleed spontaneously after 1 week, and puncture of the veins for collection of the blood samples resulted in hemorrhages. At this time the spinal fluid pressure in the cisterna magna was so low that spinal fluid could be obtained only by compression of the jugular vein. This animal was killed on the ninth day of the intensified exposure by intercardiac injection of Magnesium sulfate.
The last dog, (No. 2) which was extremely jaundiced, dehydrated, and markedly depressed and weak, was killed in the same way on the eleventh day of the intensified exposure. Neither of these two animlas showed the neurologic symptoms of the first dead dog.

Absorption:
After 4 h 35 % of Xylidine and after 24 h 65 % were absorbed in the skin.

Hematology:
One dog developed anemia to a mild degree as indicated by the reduction of the number of red blood cells, the hemoglobin values, and the hematoctrit. None of the dogs showed significant changes of the white cell and differential counts and a definite increase of the Methemoglobin level in the blodd during either the 4-4.5 h/d or the 24 h/d exposure periods. This lack of methemoglobinemic action was also characterised by a conspicuous absence of increased turbidity ration and Heinz bodies which are closely related and are both due to the presence of denatured Globin in the blood cells. The Prothrombin time ration shows a definite increase above normal during the last 2 weeks of the study. The last Prothrombin time ratio could not be determined in dog No. 3 because of failure of the blood sample to form a clot, and it was found that in this sample Prothrombin was entirely absent whereas sufficient fibrinogen was present to form a clot. At the time the Prothrombin time ratio started to increase the icterus rose until it reached + 100 and similarly the Van den Bergh test showed a very considerable increase up to 8.0 and 10.0 mg/100 cc in dogs No.1 and 3, respectively.
It appears, therefore, that the hepatotoxic action was very definite and marked, in pite of the conspicuous absence of distinct anamia or methemoglobinemina. The blood concentration of Xylidine of the dogs was definitely lower than that found in cats under comparable conditions (please refer to 7.5.2, Sievers (3)), and only during the last 2 weeks, when Xylidinewas applied for 24 h/d, did the blood level approach values similar to those observed in cats, indicating that Xylidine passes less readily through the skin of dogs than though that of cats. This indication is supported by the observation that a higher percentage of Xylidine reamined on the patches applied to dogs than on those applied to cats.

Urinalysis:
the urinary findings and the pH of the urine showed no particular trend during the entire exposure period whereas the specific gravity of the urine had a slight tendency to decline during the last week of the exposure. During the same period the color of the urine changed from yellow to brown. the amounts of Urobilin and Bile pigment were definitely increased during the last weeks, simultaneously with the increase of the icterus index and the Van den Bergh test. No sugar or blood and no definite increase of Albumine were found in the urine of these animals. the total and inorganic Sulfates decreased slightly at the beginning of the expousre, decreased further during the third and fourth weeks, and rose slightly during the fifth week without reaching the preexpousre level. The ethereal Sulfates increased notably during the fourth week of the exposure in all three dogs but showed a considerable drop during the fifth week, especially in dog No. 1 which survived the continued exposure for the longest period of time. accordingly the inorganic Sulfate ration dropped definitely in all three dogs during the fourth week and rose again in dogs No. 2 and 3 during the fifth week. This behaviour of the inorganic Sulfate ratio may be interpreted as indicating that Xylidine is metabolised with the formation of a phenolic compound which is conjugated with sulfuric acid, and that during the last week either the oxidative or the conjugative functions of the liver, or both, were sufficiently impaired with the formation of ethereal Sulfates. The presence of an increase of Urobilin and Bile pigments in the urine is indicative of liver injury.

Pathology:
The gross pathological findings were essentially the same in all three dogs treated with Xylidine. The subcutaneous tissue, sclerae, dura mater, and viscera showed slight to intense yellow discoloration. Blood was present in the nostrils and oral cavities of two dogs without any evidence of trauma. the bile was dark to greenish black and of high viscosity. In all dogs the liver was moderately enlarged, firm, of light color, mottled, and dimpled, and it was judged to contain large amounts of fat. The spleens were at least twice their normal size and were soft and friable. The kidneys, heart, lungs, brain, spinal cord, adrenals, and lymph nodes were essentially normal. One dog (No. 2) had about 40 cc of yellow, blood-tinged ascitic fluid in the peritoneal cavity. It was shown that Xylidine or other Diazotizable derivatives are excreted, to a large extent, with the bile.

Effect levels

Dose descriptor:

NOAEL

Effect level:

< 100 mg/kg bw/day (nominal)

Sex:

not specified

Basis for effect level:

other: mortality

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the effects of this dermal toxicity study with Xylidine in dogs (5 weeks) the NOAEL is considered to by < 100 mg/kg bw/d.

Executive summary:

The cutaneous application of 100 mg/kg bw/d Xylidine for 4 -4.5 h/d for 3 weeks to dogs caused vomiting, probably of the central origin, and after 3 weeks salivation, loss of appetite, and weakness. With application for 24 h/d the animals developed shivering and, finally, jaundice, marked depression of the central nervous system and muscular weakness; one dog developed convulsions, spasticity, and Cheyne-Stokes respiration during the final stage. The methemoglobinemic and anemising actions and the injurious effect on the blood cells, as indicated by increased turbidity ratio, were slight or negligible in contrast to similar experiments performed with cats. Injury of the liver was severe, as shown by increased icterus index, the Prothrombin time ration, the Van den Bergh test, and the appearance of Urobilin and Bile pigments in the urine. The behaviour of the inorganic Sulfate ratio in the urine was also indicative of liver injury. This hepatotoxic action appears to be of the same order in dogs and cats (please refer to 7.5.2., Sievers (3)). There was little evidence of an injurious effect on the kidneys. Comparison of the Xylidine levels in the blood of dogs and cats shows that the former absorb Xylidine less readily through the skin than the latter.