2,6-xylidine

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:

other: Well documented study report, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Male and female rats were repeatedly dosed with the test substance for 13 weeks.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,6-xylidine

EC Number:

201-758-7

EC Name:

2,6-xylidine

Cas Number:

87-62-7

Molecular formula:

C8H11N

IUPAC Name:

2,6-dimethylaniline

Test material form:

liquid: viscous

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Ethyl Corporation (Baton Rouge, LA).
- Lot/batch number of test material: E121279.
- Analytical Purity: 98.8%.
- Water content: 0. 105 %.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The bulk 2,6-xylidine was stored at EG&G Mason in a cold box maintained at 0°± 5°C .
- Stability under storage conditions: A stability study indicate that 2,6-xylidine is stable for 2 weeks when stored as the bulk chemical under nitrogen and protected from light at temperatures up to 60°C.
- Stability: bulk chemical remained stable during the study (confirmed by reanalysis, re-analysis result 99.06%).

FORM AS APPLIED IN THE TEST: solution in corn oil.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Male: Harlan Industries (Indianapolis, IN) and Charles River (Kingston, NY), female: Harlan Industries (Indianapolis, IN).
- Age at study initiation: 5-8 weeks.
- Age when killed: 18-21 weeks.
- Housing: five per cage by sex.
- Diet: NIH 07 Rat and Mouse Ration (Zeigler Bros ., Inc ., Gardners, PA); available ad libitum.
- Water: Tap water supplied by automatic watering system (Edstrom Industries, Waterford, WI). Ad libitum.
- Acclimation period: 3 weeks.
- Distribution to cages: Animals with extreme weights were culled; the remaining animals were distributed so that cage weights were approximately equal.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9°-23.3° C.
- Humidity (%): 32.7-77.0 %.
- Air changes (per hr): 12- 15.
- Photoperiod (hrs dark / hrs light): 12/12.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The results of a stability study performed by the National Toxicology Program (NTP) indicated that 2,6-xylidine was not stable when mixed with feed. Therefore, the gavage route of administration was used for the studies conducted at EG&G Mason.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Preperation: 2,6-xylidine solutions in corn oil prepared to allow administration of desired dose when 5 ml/kg body weight given by gavage.
- Storage conditions: sealed in labeled serum vials with a nitrogen headspace and stored at 0°- 5° C.
- Maximum storage time: 2wk.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
- Source: Ethyl Corporation (Baton Rouge, LA)
- Lot/batch no. (if required): E121279

Details on analytical verification of doses or concentrations:

2,6-Xylidine/corn oil mixtures at a concentration of 100 mg/ml were analyzed at MRI and showed no loss of stability after being stored the dark for 14 days at room temperature. Samples stored in the open air and light for 3 hours also showed no loss of stability .

Duration of treatment / exposure:

12 days

Frequency of treatment:

5 d/wk for 13 wks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

80 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

160 mg/kg bw/day (actual dose received)

Doses / concentrations 5

Dose / conc.:

310 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes

Details on study design:

Post-exposure period: 2-3 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: twice daily.

HAEMATOLOGY: Yes.
- Time schedule: Blood collected at day 90.
- leukocytes and erythrocytes were electronically counted, hemoglobin levels were measured.

CLINICAL CHEMISTRY: Yes.
- Time schedule: Blood collected at day 90.
- Analyses for LDH, SGH, SGOT, SGPT, OCT, serum carbon dioxide, cholinesterase, alkaline phosphatase, albumin, globulin,creatinine, blood urea nitrogen, calcium, inorganic phosphate, total bilirubin, and direct bilirubin were performed. Sodium, potassium, and chloride concentrations were measured.

URINALYSIS: Yes.
- Time schedule: Urine collected at day 90.
- The appearance of the urine was described and specific gravity and pH were determined. termined . A microscopic examination was performed to determine the presence of erythrocytes, leukocytes, casts, or other formed elements. Urine reagent strips were used to analyze for protein, glucose, ketone bodies, bilirubin, blood nitrite, and urobilinogen.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
- Necropsy performed on all animals.

HISTOPATHOLOGY: Yes.
- All vehicle control and highest dose animals examined histologically.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No noteworthy clinical signs were observed in any of the animals in these studies.

Mortality:

no mortality observed

Description (incidence):

No deaths occured.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight gains were over 10% less in male and female rats receiving the highest (0 .31 g/kg) dose and in female rats administered 0.04 and 0.16 g/kg than in controls.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The results of hematologic and clinical chemistry studies indicate that male rats are more sensitive to 2,6-xylidine than are female rats.
Significant decreases in total leukocyte counts occurred in male rats at doses of 0.04 g/kg and above; they were accompanied by decrease the percent of lymphocytes and increases the percent of segmented neutrophils at doses 0.08 g/kg and above. In male rats, significant decreases occurred in hemoglobin levels at 0.16 and 0.31 g/kg and in erythrocyte and hematocrit levels at 0.31 g/kg. A dose-related increase in polychromasia occurred in male rats. In female rats, significant decreases were observed in hemoglobin levels 0.16 and 0.31 g/kg and in hematocrit levels at 0.31 g/kg. The decreases in hemoglobin, erythrocyte, and hematocrit levels appear to be related to administration of 2,6-xylidine, but they are not severe enough to be considered indicators of anemia. The decreases in total leukocytes and lymphocytes may also be compound related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The results of hematologic and clinical chemistry studies indicate that male rats are more sensitive to 2,6-xylidine than are female rats.
Significant decreases in serum glutamic oxaloacetate transaminase (SGOT) and lactic dehydrogenase levels occurred at all doses.
Decreases were also observed for potassium, calcium, and inorganic phosphorus levels in sera of male rats. Cholinesterase levels were increase in male rat sera. Significant decreases in cholinesterase, globulin, and creatinine levels occurred in the sera of female rats. Increases were observed in sorbitol dehydrogenase, serum glutamic pyruvate transaminase, blood urea nitrogen, chloride, and inorganic phosphorus levels in female rat sera.
Decreases in SGOT, lactic dehydrogenase, direct bilirubin, and serum creatinine levels have no known clinical significance. The decreases in potassium, calcium, and inorganic phosphorus levels and in urine specific gravity, and the increase in cholin esterase levels are not biologically significant at the observed levels.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the highest dose (0.31 g/kg), increases in mean liver weights and decreases in body weights resulted in significant increases in the liveribody weight ratios (P = 0.003 for males and females). The liver weight to body weight ratio also was increased for male rats ad ministered 0.16 g/kg. The liver weight to brain weight and kidney weight to brain weight ratios significantly increased in females given 0.31 g/kg.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histologic examinations revealed minimal-to moderate inflammatory changes in the nasal mucosa of each sex, but the vehicle controls had morphologically similar lesions of equal severity. Various inflammatory and degenerative lesions observed in other tissues were considered administration of 2,6-xylidine.

Effect levels

1

Dose descriptor:

LOAEL

Effect level:

310 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

haematology

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Administration of 2,6-xylidine for 13 weeks to male F344/N rats was associated with slight decreases in total leukocyte counts and the percentage lymphocytes. Slight decreases in hemoglobin and hematocrit levels and in erythrocyte counts conccurred in dosed male rats ; hemoglobin and hematocrit levels were decreased in dosed fremale rats.

Executive summary:

Groups of ten male and ten female F344/N rats were administered doses of 0, 20, 40, 80, 160, 310 mg/kg of 2,6 -xylidine in corn oil by gavage 5 days per week for 13 weeks.

All animals were observed twice per day, and clinical observations were recorded. Urine and blood was collected fat day 90. Necropsy was performed on all animals. All vehicle groups and highest dose animals were examined histologically.

No deaths occured.

Mean body weight gains were over 10% less in male and female rats receiving the highest (0.31 g/kg) dose and in female rats administered 0.04 and 0.16 g/kg than in controls.

No noteworthy clinical signs were observed in any of the animals in these studies.

At the highest dose (0.31 g/kg), increases in mean liver weights and decreases in body weights resulted in significant increases in the liveribody weight ratios (P = 0.003 for males and females). The liver weight to body weight ratio also was increased for male rats ad ministered 0.16 g/kg. The liver weight to brain weight and kidney weight to brain weight ratios significantly increased in females given 0.31 g/kg.

Histologic examinations revealed minimal-to moderate inflammatory changes in the nasal mucosa of each sex, but the vehicle controls had morphologically similar lesions of equal severity. Various inflammatory and degenerative lesions observed in other tissues were considered administration of 2,6-xylidine.

The results of hematologic and clinical chemistry studies indicate that male rats are more sensitive to 2,6-xylidine than are female rats.

Significant decreases in total leukocyte counts occurred in male rats at doses of 0.04 g/kg and above; they were accompanied by decrease the percent of lymphocytes and increases the percent of segmented neutrophils at doses 0.08 g/kg and above. In male rats, significant decreases occurred in hemoglobin levels at 0.16 and 0.31 g/kg and in erythrocyte and hematocrit levels at 0.31 g/kg. A dose-related increase in polychromasia occurred in male rats. Significant decreases in serum glutamic oxaloacetate transaminase (SGOT) and lactic dehydrogenase levels occurred at all doses.

Decreases were also observed for potassium, calcium, and inorganic phosphorus levels in sera of male rats. Cholinesterase levels were increase in male rat sera . In female rats, significant decreases were observed in hemoglobin levels 0.16 and 0.31 g/kg and in hematocrit levels at 0.31 g/kg. Significant decreases in cholinesterase, globulin, and creatinine levels occurred in the sera of female rats. Increases were observed in sorbitol dehydrogenase, serum glutamic pyruvate transaminase, blood urea nitrogen, chloride, and inorganic phosphorus levels in female rat sera.

The decreases in hemoglobin, erythrocyte, and hematocrit levels appear to be related to administration of 2,6-xylidine, but they are not severe enough to be considered indicators of anemia. The decreases in total leukocytes and lymphocytes may also be compound related . Decreases in SGOT, lactic dehydrogenase, direct bilirubin, and serum creatinine levels have no known clinical significance. The decreases in potassium, calcium, and inorganic phosphorus levels and in urine specific gravity, and the increase in cholin esterase levels are not biologically significant at the observed levels.