2,4-xylidine

Endpoint summary

Description of key information

Inhalation:
NOEC (4 weeks, 6h/d, whole body, male/female rat): 0.033 mg/L
Oral:
NOAEL (20 days, gavage, male rat): < 117 mg/kg bw/d
NOAEL (4 weeks, gavage, male/female rat): < 500 mg/kg bw/d
NOAEL (28 days, gavage, male/female rat): 20 mg/kg bw/d
NOAEL (28 days, oral, male/female dog): 10 mg/kg bw/d
NOAEL (6 months, feeding study, male/female rat): 750 ppm
NOEL (10 days, oral, male dog): 25 mg/kg bw/d (study not reliable)
NOAEL (10 days, gavage, male rat): < 117 mg/kg bw/d (study not reliable)
NOAEL (7 days, oral, male/female rat): < 400 mg/kg bw/d
Dermal:
NOAEL (32 days, dog): < 100 mg/kg bw/d 
NOAEL (19 days, cat): < 100 mg/kg bw/d

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

dog

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Dose descriptor:

NOAEC

33 mg/m³

Study duration:

subacute

Species:

rat

Additional information

- A 20 day gavage study was performed in rats. The animals were treated with 117 mg Xylidine/kg bw/d.

The following effects were observed:

2 dead rats after 10d of dosing and 1 dead rat after 20d of dosing

- clinical signs comprised: thinness, rough hair coat

- body weight decreased

- spleen to body weight rations were increased, w/o specific changes in spleen weights

- liver weights and liver to body weight ratio was increased

- kidney weights were unchanged, with a slight increase in kidney to body weight ratio

- histopathological lesions: mild tracheitis, probably due to the administration procedure

- slight congestion of the spleen

- slight hemosiderosis in spleen

- toxic hepatopathy with extensive cloudy swelling, diffuse hepatocellular necrosis, early periacinar connective tissue proliferation, biliary hyperplasia, and periacinar vacuolar degeneration.

Therefore, the NOAEL is considered to be smaller than 117 mg/kg bw/d.

  

- In a further oral toxicity study 20 male and female rats (10 of each sex) were devided into 4 groups (5 male and 5 female). Rats were dosed by gastric intubation for 4 weeks with 400 mg/kg bw/day in the first week and 500 mg/kg bw/day in weeks 2-4. Control rats were treated with saline at the same dosage volume (5mL/kg) as other dose groups. All animals were sacrificed at termination of study and post-mortem examined.

The following observations were made:

- reduction in body weight gain

- livers were enlarged, otherwise no gross abnormalities

- increase in liver weights - apparent enlargement of the hepatocytes, mainly in centrilobular regions

- occasional isolated necrotic hepatocytes

- centrilobular decrease in glycogen, with the decrease being more present in males than in females

- centrilobular decrease in glucose-6-phosphat distribution, i.e. enzyme activity

- electron microscopy revealed proliferation of the smooth endoplasmatic reticulum, as well as isolated degenerative hepatocytes characterised by vaculation and inclusion bodies, dilation of bile caniculi, associated with loss or atrophy of microvilli and occasional pigmented Kupffer cells

- hepatic microsomal protein content was increased in males and females treated with 2,4-xylidine

- CYP was increased after treatment

The NOAEL is smaller than 500 mg/kg bw/d.

 

- Beagle dogs were devided into 10 groups each consisting of one male and one female and treated once daily for 4 weeks with 0 (control), 2, 10 or 50 mg/kg test substance, orally in capsules. Rats of the Sprague Dawley strain were used in ten groups of 5 males and 5 females each and treated by stomach tube once daily for 4 weeks with 0 (control), 20, 100, 500 mg test item. After 2 weeks of treatment the high group dose was increased to 700 mg/kg bw.

Blood and liver examinations were conducted at pathology and the following observations/effects were obtained:

DOGS

- clinical signs: low dose no effects, medium and high doses showed vomiting

- highest dose group: poor general condition, body weight decreased

- laboratory investigation: increased BSP retention at the highest dose level

- gross pathology: livers were slightly enlarged at the highest dose level, liver appeared to be pale, kidneys were normal in all dogs

- microscopic pathology: high dose level induced a slight fatty degeneration

RATS

- clinical signs: decreased weight gain in high dose group

- 6 mortalities in the high dose level

- laboratory investigation: hemoglobin and hematocrit (less pronounced) was decreased (high dose)

- 7 rats with increased OGT-values

- gross pathology: livers of all treated rats were enlarged, high dose: occasional reddish and greyish foci of a size of 0.5-2mm, these foci were most evident in the rats which died

- microscopic pathology:

high dose:

-necrosis and vacuolization of the hepatocytes; necrosis appeared as scattered foci, chiefly in the midzone of some of the hepatic lobule, necrotic foci were generally small and well defined, while the larger foci were more irregular in shape

- fresh hemorrhage within some necrotic areas

- cellular infiltration of mainly histiocytes and to a small extent neutrophilic granulocytes

- no fatty change was found, while in the centrolobular areas various vacuoles were common in the hepatic cells

- vacuoles appeared empty with a somewhat filamentous content which stained palely with eosin, was negativ for both PAS and Sudan III, Weigert, and Mallory

- slight proliferation of bile ducts

all dose levels:

- kidneys were normal

The NOAEL(dog) is derived at 10 mg/kg bw/d, and the NOAEL(rat) is set at 20 mg/kg bw/d.

- In a 6 months feeding study 2,4 -Xylidine was incorporated into batches of ground commercial feed by means of a food mixer. Groups of 20 weanling Osborn-Mendel rats (50 % male / 50 % female) were fed the test material in dietary levels of 10.000, 5.000, 2.500, 750 and 375 ppm. After 3 months 4 rats of each dose level, and after 6 months the remaining animals were sacrificed.

The following observations were made:

-3 mortalities in each of the treatment groups in the course of 6 months (vs 1 mortality in control group)

- growth inhibition was observed

- all levels of 2,4-xylidine showed definite type of target cell anemia, the effect being roughly proportional to the doses administered

- after 6 months liver and kidney showed difference in weight and grossly visible changes: pale foci (0.5-2mm), slightly present surface vessels, below 2500ppm no foci were observed; kidney: moderate irregular pitting or depressed scarring (all at 10000ppm); right and left kidney were often unequally affected

- all dose levels had enlarged livers

LIVER - MICROSCOPIC EXAMINATION (10000ppm)

- most evident (hence not most frequent) foci from 0.5 - 3mm diameter of cholangiofibrosis

- a moderate amount of scattered new small bile duct formation, often shown as well-formed darkly staining ducts

- foci of hepatic cell hyperplasia, generally rounded and about 0.5mm in diameter (up to 4mm); several of these in each liver section

- active necrosis of hepatic cells was almost absent; at 13 weeks necrosis was somewhat more pronounced

- no sex-related difference in effects

- with a reduction from 10000ppm to 5000ppm there was a definite decrease in microscopic liver damage (no cholangiofibrotic foci, formation of new small bile ducts was considerably reduced, especially in males; the hyperplastic foci were present but less well defined

- 2000ppm-group: liver appeared to be relatively normal; particularily in females there was a slight formation of new bile ducts and a few poorly-defined hyperplastic hepatic cell foci

- the low-dose-groups were without any visible damage

KIDNEY

- 10000ppm: cortical foci of tubular atrophy and interstitial fibrosis and chronic inflammation, going to depressed scar formation, represented by the pits seen grossly; various deleterious papillary changes: edema, cast formation in the small looped tubules and progression to necrosis in the lower end of the papilla, with more or less repair

- at 13-weeks, changes were similar but in an earlier stage

- 5000ppm and below: negative

OTHER ORGANS

- forestomach: slight hyperkeratosis

Based on these observations the NOAEL is set at 750 ppm in diet.

- Male Beagles were orally dosed for one or 10 days with 2,4-Dimethylaniline at 25 mg/kg bw. Male Fischer 344 rats were gavaged with 2,4-Dimethylaniline at 25 % of their respective LD50 (117 mg/kg) for the same time period.

In all treated dogs the test item caused no detectable lesions. In rats 2,4-Dimethylaniline induced hepatomegaly and a subtle but distinct change charaterized by swollen cells with volumnious homogenous cytoplasm and segregation or clumping of cytoplasmic substructures. The major urinary metabolite of the test item in the dog was 6-Hydroxy-2,4-dimethylaniline. Minor metabolites included 4-Amino-3-methyl-benzoic acid and N,2,4-Trimethylaniline. Rats produced N-Acetyl-4-Amino-3-Methyl-Benzoic acid as the major metabolite and N,2,6-Trimethylaniline in small amounts.

The NOEL(dog) ist derived at 25 mg/kg bw/d, the NOAEL(rat) is smaller than 117 mg/kg bw/d.

- Rats of the treatment group were given once daily oral dose of 2,4-Xylidine in saline for 7 consecutive days. Control rats were given equal volume of saline per os.

The significant hepatic lesions revealed in rats treated with 2,4-Xylidine for a week induced liver cell enlargement, occasional liver cell necrosis and/or degeneration and minimal bile duct hyperplasia. Histochemically, there was an overall reduction in the canalicular ATPase in the treated rats. Salient features of the electron microscopical examination of Xylidine treated rats included proliferation of smooth endoplasmic reticulum and dilation of bile canaliculi with loss of microvili. Significant elevations were seen in serum levels of GPT and GDH. The levels were highest among the treated males. Alkaline phosphatase levels showed no significant change. Serum bile acids were markedly raised with highest levels among the male rats. The NOAEL is smaller than 400 mg/kg bw/d.

- 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. There was little evidence of an injurious effect on the kidneys.

Another study shows that continued dermal exposure to 100 mg/kg bw/d Xylidine for up to 19 days causes in cats progressive depression of the central nervous system, incorordination, loss of appetite, vomiting, and jaundice. With continued exposure the number of red blood cells and the Hemoglobin are decreased and there may be moderate lymphopenia, but the white blood cell picture is affected very little otherwise. Continued exposure to Xylidine causes a moderate increase of the Methemoglobin, which, however, becomes significant if the Hemoglobin is reduced on account of the anemizing action of Xylidine. In Xylidine poisoning the Prothrombin time, the icterus index, and the Van den Bergh reaction are increased, indicating injury of the liver, as is also indicated by the presence of Urobilin and bile pigments in the urine. There may be some injurious effect on the kidneys, as indicated by albuminuria. The percent inorganic sulfate ratio is decreased, indicating an increased excretion of phenolic compounds, whereas the excretion of substituted hippuric acids is decreased, indicating presumably injury of liver or kidney. The gross pathology of cats poisoned with Xylidine is mainly characterised by jaundice, mottling, and dimpling of the liver, and less marked injury of the kidneys. The experiments show further that Xylidine is readily absorbed through the skin and that it or some of its diazotizable metabolites may be present in the blood several days after discontinuation of the exposure. The same compounds are also excreted in bile and urine to a large extent. In both studies the NOAEL is smaller than 100 mg/kg bw/d.

- The available inhalation study indicates that inhalation of 2,4-Xylidine by rats at concentrations of 0.1 or 0.3 mg/L over a 4-week period causes changes indicative predominantly of effects on the liver. At the higher dose level there were also changes in the stomach and an elevation of water consumption. At the lowest dose level used in this study (0.03 mg/L) there were no microscopic pathological changes. However there were slight effects on weight gain and food consumption, signs of eye irritation and statistically significant changes in plasma GPT and calcium. It is concluded that the NOEL for inhalation of 2,4-Xylidine by rats over a 4-week period is 0.03 mg/L.

In summary, the major target organ for toxicity of m-Xylidine was the liver in all tested species. These effects were also observed via the oral, dermal as well as the inhalatory route.

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

Repeated dose toxicity: inhalation - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

Bsed on the effects observed in all repeated dose studies m-Xylidine has to be classified as

- Xn, R48/20/22 - danger of serious damage to health by prolonged oral/inhalatory exposure

- STOT(RE) cat.1, H372 - Causes damage to liver trough prolonged or repeated exposure if inhaled

- STOT(RE) cat. 2, H373 - May cause damage to liver trough prolonged or repeated exposure if swallowed

regarding systemic and target organ toxicity after repeated exposure according to the criteria laid down in the EU Dangerous Substances Directive (67/548/EEC) and in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).