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Description of key information

oral

In a subacute and a subchronic study within the framework of the NTP carcinogenicity Study, F344 rats treated with 2,6-xylidine by gavage for 2 or 13 weeks developed haematological alterations (NTP, 1990). Male rats were more sensitive than females. After 2 weeks, anisocytosis, poikilocytosis, and polychromasia of red blood cells, a generalised leukocytosis and a decrease in body weight gain were observed at 310 mg/kg bw/d. After 13 weeks, total leukocyte counts were significantly decreased at 40 mg/kg bw/d, additional haematological changes (decrease of lymphocyte and increase in neutrophil counts, decrease of haemoglobin level, red blood cell count and haematocrit) were observed at higher concentrations. However, the decreases in red blood parameters were not severe enough to be considered as anaemia an no noteworthy clinical signs or histopathological effects were observed. The NOAEL was determined to be 160 mg/kg bw/d based on effects on haematological parameters and organ weights.

 

dermal

No information available.

 

inhalation

No information available.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
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
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.
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.
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
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Dose / conc.:
160 mg/kg bw/day (actual dose received)
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
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.
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.
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
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.
Critical effects observed:
not specified
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
160 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A subchronic repeated dose toxicity study is available to address repeated dose toxicity.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

SCOEL (2009):

Oral

Effects of 2,6-DMA on liver and kidney were studied in Sprague-Dawley rats and Beagle dogs (Magnusson et al., 1971). Both species were treated with 2,6-DMA for 4 weeks. Dogs (1 male + 1 female/group) were given 0, 2, 10 or 50 mg/kg d in capsules.Rats received 0, 20, 100,and 500 mg/kg d by gavage, the highest dose was increased to 700 mg/kg d after 2 weeks. No gross or histological effects were seen on the kidney at any dose in both species. Dogs were much more sensitive than rats. Vomiting occurred in dogs at 10 and 50 mg/kg d. At the highest dose, the body weight was reduced and the dogs had to be sacrificed after 2 weeks. Fatty degeneration of the liver was seen in dogs at all dose levels with a dose-dependent increase in severity (LOAEL dogs: 2 mg/kg d). Further effects at the highest dose were increased liver weight, pale liver with some isolated necrotic liver cells, hypoproteinaemia, hyperbilirubinaemia and icteric tissues. In rats, mortality was increased at the highest dose. Rats also showed lowered haemoglobin levels and haematocrit, and hepatomegaly but only a slight fat accumulation with necrotic foci in the liver at the highest dose (NOAELrat: 100 mg/kgbw/d).

 

Oral administration of 400-500 mg/kg d of 2,6-DMA to rats for 4 weeks caused hepatomegaly which was attributed to the proliferation of the smooth endoplasmic reticulum. Hepatic glycogen content and glucose-6-phosphatase were decreased; the activity of glucuronyltransferase was increased but not of aniline hydroxylase activity and of cytochrome P450 content (Magnusson et al., 1979).

 

Haemosiderosis of the spleen was observed in F344 rats after gavage administration of 157 mg/kg d over a period of 20 days. No other effects could be observed (Short et al., 1983).

 

In a further subacute study, male F344 rats were fed a diet containing 300 ppm 2,6DMA (27 mg/kg d) for one week or 3000 ppm 2,6-DMA (200-226 mg/kg bw/d) for 4 weeks (Yasuhara et al., 2000). Histological changes in nasal tissue, such as atrophy of Bowman"s glands and irregular arrangement of olfactory epithelial cells were observed at 3000 ppm but not at 300 ppm. Other organs were not assessed.

 

Feeding of 2,6-DMA hydrochloride to Osborne-Mendel rats for 26 weeks Ied to an increased relative kidney weight (LOAEL: 2500 mg/kg food; NOAEL 750 mg/kg food). At higher doses, reduced weight gain and renal lesions such as interstitial fibrosis and inflammation, tubular atrophy papillary oedema, cystic tubular dilation, and chronic congestion of the spleen were also observed (Lindström et al., 1963).

 

In a range-finding study including a reproductive toxicity part, the body weight gain of female CD rats was reduced after feeding a diet containing 3,000 ppm 2,6-DMA for 10 weeks (NTP, 1990).

 

In a subacute and a subchronic study within the framework of theNTP carcinogenicity study, F344 rats treated with 2,6-DMA by gavage for 4 or 13 weeks developed haematological alterations (NTP,1990). Male rats were more sensitive than females. After 4 weeks, anisocytosis, poikilocytosis, and polychromasia of red blood cells, a generalised leukocytosis and a decrease in body weight gain were observed at 310 mg/kgbw/d (LOAEL, NOAEL: 160 mg/kgbw/d). After13 weeks, total leukocyte counts were significantly decreased at 40 mg/kgbw/d (LOEL; NOEL: 20 mg/kg bw/d), additional haematological changes (decrease of Iymphocyte and increase in neutrophil counts, decrease of haemoglobin level, red blood cell count and haematocrit) were observed at higher concentrations. However, the decreases in red blood parameters were not severe enough to be considered as anaemia.

 

Non-carcinogenic effects in the 2-year NTP carcinogenicity study with CD rats included a dose-dependent, significant increase of acute inflammations (rhinitis) of the nasal cavity at all dose levels in both sexes. In males, the incidence was 21 % in controls and increased to 38 %, 57 % and 75 % at dose levels of 300 ppm, 1,000 ppm and 3,000 ppm 2,6-DMA in food. In females, the corresponding incidences were 13 %, 25 %, 27 % and 68 %. Additionally, epithelial hyperplasia in the nasal cavity was observed in mid- and high-dose males and females, and squamous metaplasia developed in high-dose males and females. Hyper-and metaplasia were not observed in any animal fed 300 ppm or control diet. Decreases in red blood cellcount, haemoglobin level and haematocrit occurred in males fed diets containing 3000 ppm 2,6-DMA (accordingto HCN (2002): 120mg/kg bw/d, calculated using standard factors) after 18 months and in females at 1000 ppm (50mg/kg bw/d) and 3000 ppm (150 mg/kg bw/d) after 12 months. These haematological changes were small and not considered to be of biological significance (NTP,1990).

 

Dermal

No data available.

 

Inhalation

No data available.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the forteenth time in Regulation (EU) No 2017/776.