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Administrative data

short-term repeated dose toxicity: oral
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented study report which meets basic scientific principles

Data source

Reference Type:
study report

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): 2,6-Xylidine
- Analytical purity: > 98.7 %
- water content: 0.29 %
- Physical state: clear, dark-brown, slightly viscous liquid
- Lot/batch No.: 37220
- Storage condition of test material: < 4° C
- Stability: bulk chemical remained stable during the study (confirmed by reanalysis
- Supplier: Pfaltz and Bauer (Stanford, CT)

Test animals

other: CD
Details on test animals or test system and environmental conditions:
- Source: Charles River Breeding Laboratories
- Age at study initiation: weanling
- Fasting period before study: over night
- Housing: individual
- Diet: Purina lab chow; available ad libitum
- Water: Tap water supplied by automatic watering system (Edstrom Industries, Waterford, WI)
- Acclimation period: 8 days

- Temperature (°F): 74 ± 3° F
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
corn oil
Details on oral exposure:
2,6-xylidine was suspended in corn oil, mixed with a small amount of Purina Lab Chow ground meal, combined with additional feed and then mixed in a blender until homogeneous. Control and formulated diets contained 50 ml of corn oil per kg of feed. At each concentration, samples were taken at distinct lacations in the blender and analyzed for homogeneity.
Analytical verification of doses or concentrations:
Duration of treatment / exposure:
102 weeks
Frequency of treatment:
Doses / concentrations
Doses / Concentrations:
0, 100, 300, 1000, 3000 ppm
nominal in diet
No. of animals per sex per dose:
56 males and 56 females
Control animals:
yes, concurrent vehicle
Details on study design:
For each dose group, 28 male and 56 female weanling Charles River CRL:COBS CD (SD) BR outbred albino rats were obtained from Charles River Breeding Laboratories. These animals constituted the F0 generation in this multigeneration study. Beginning at 5 weeks of age, the animals were fed diets containing 0, 300, 1,000 or 3,000 ppm 2,6-xylidine. They were mated at 16 weeks of age and pregnant females were allowed to deliver naturally over a 2-week period. The F0 females continued to receive dosed or control diets during pregnancy and lactation. Progeny of this mating, designated the F1, generation, were weaned at 21 days of age, and groups of 56 males and 56 females were fed the same study diets as their parents for 102 weeks.

Diet Formulation and Feed Stability Studies
2,6-Xylidine was suspended in corn oil, mixed with a small amount of Purina Laboratory Chow ground meal, combined with additional feed then mixed in a blender until homogeneous. Control and formulated diets contained 50 ml of corn oil per kg of feed. At each concentration, samples were taken at distinct locations in the blender and analyzed for homogeneity.
Stability studies were performed at Litton Bionetics on formulated diets stored in sealed containers at ambient temperatures. Dosed feed was extracted with benzene. Concentrations of 2,6-xylidine were determined by spectrophotometric analysis after reaction with diazotized sulfanilic acid. Analyses performed on days 1, 7, 14, 21, and 28 indicated respective recoveries of 90.1 %, 81.2 %, 81.0 %, 85.1 %, and 83 % of the 2,6-xylidine from the 3,000-ppm diet. Formulated diets were prepared once per week, stored at -20° C until used, and dispensed in feed cups. The NTP had performed feed stability studies route of administration for its series of toxicity tests at EG&G Mason. The stability of 2,6-xylidine in feed was determined by preparing a 1,000-ppm feed mixture using NIH 07 Rat and Mouse Ration and analyzing it at various intervals after open storage in a rat cage at ambient temperatures and after storage in sealed bottles kept in the dark at –20° C, 5° C or room temperature. Feed samples were extracted with acetonitrile, and the 2,6-xylidine content of the extracts was determined by gas chromatography. Open storage samples were analyzed on days 1, 3, 5, and 7; samples stored in sealed bottles were analyzed on days 7 and 14. The feed stability results indicated that 2,6-xylidine mixed with the basal diet was unstable under all conditions of storage. Formulated feed mixtures stored open for 7 days in a rat cage lost 44 % of the 2,6-xylidine initially present. 2,6-Xylidine concentrations in samples stored for 14 days in the dark in sealed containers at – 20° C, 5° C, or room temperature were reduced 1.5 %, 3.2 %, and 14.6 %. The samples of feed mixtures stored in sealed bottles lost 2,6-xylidine by reacting with feed components. Losses from samples rat cage were caused by reaction with feed and evaporation. An estimated 70 % - 80 % of the loss from the rat cage was due to evaporation. Because of the results of these studies, the NTP decided to use gavage rather than diet for oral administration of 2,6-xylidine in short-term studies. The 2-year studies at Litton Bionetics began under the EPA contract before the NTP feed stability studies were completed.

Upon assuming responsibility for the 2-year studies, the NTP conducted further studies on the loss of 2,6-xylidine from feed. In these studies, 2,6-xylidine was blended with a series of feed ingredients and stored in sealed containers for 12 days at room temperature or at – 20° C. Little or no reactivity was observed when 2,6-xylidine was mixed with starch, fat, or protein. The compound reacted moderately with unbleached whole wheat flour and significantly with nonfat dry milk. The two reactive ingredients contained reducing compounds. Dried skim milk typically contains 50 %- 52 % of the reducing carbohydrate, lactose. Flour contains enediol compounds that react like typical aldehydes. Significant reactivity was observed in 2,6-xylidine blends with both ß-D-lactose and D-glucose, whereas no reactivity was observed with sucrose. Mixing 2,6-xylidine and D-glucose resulted in a color change from white to brown. This "browning" phenomenon considered to be due to irreversible reaction with components containing aldehydes groups.

Animal maintenance
Eighteen rats per group were housed individually, and 38 rats per group were housed 3 per cage in polycarbonate shoebox-type hanging cages covered with nonwoven polyester filter sheets. Animal rooms were controlled for temperature, humidity, and duration of light. No other chemicals were on study in the animal room. Control animals were housed in a separate room.

Positive control:
not done


Observations and examinations performed and frequency:
Clinical Examinations
Rats were observed once per day, and clinical signs were observed and recorded once per week. Body weight data for all animals and feed consumption data for animals individually housed were recorded once per week for 26 weeks and once per month thereafter, except for the last 5 months when two measurements for feed and three for body weight were recorded. At 12 months, 18 months, and the end of the studies, 10 males and 10 females were selected randomly and bled via the orbital sinus route. Analyses were performed for total erythrocyte count, hemoglobin, hematocrit, total leukocyte count, differential leukocyte count, blood urea glucose, SGOT, and alkaline phosphatase.

A Coulter Counter (Model FN) was used to determine total white blood cell counts and total red blood cell counts. A Coulter Hemoglobinometer was used to measure hemoglobin levels. Packed cell volume was determined after samples were centrifuged at 10,000 rpm in a microhematocrit centrifuge.
Peripheral blood smears were prepared and examined. The Baker Centrifichem Model 4000 analyzer was used for blood urea nitrogen, SGOT, glucose, and alkaline phosphatase determinations. Specific standards for each test were used for calibration. Two reference material samples (normal and abnormal levels) supplied by the manufacturer were assayed in duplicate along with each run of 20 test samples. Necropsies were performed at Litton Bionetics on all animals that died early or were killed while in a moribund state. Animals found moribund and those surviving to the end of the studies were humanely killed. A necropsy was performed by Experimental Pathology Laboratories, Inc., at the Litton Bionetics facility on all animals including those found dead, except for tissues that were excessively autolyzed or missing. Thus, the number of animals from which particular organs or tissues were examined microscopically varies and is not necessarily equal to the number of animals that were placed on study.
Sacrifice and pathology:
During necropsy all organs and tissues were examined for grossly visible lesions. Tissues were preserved in 10 % neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

Portions of nasal tumors were retrieved from the posterior nasal cavities of three rats held in formalin fixation; these tumors were submitted for electron microscopy in an attempt to confirm diagnoses made by light microscopy. Recognizable nasal tumor tissue was minced into 1-mm cubes, washed in phosphate buffer, and fixed in 2.5% phosphate buffered glutaraldehyde. After being washed in buffer three times, the specimens were fixed in 1 % osmium tetroxide, dehydrated in ascending concentrations of ethanol, and embedded in Epon 812. One-micron sections of multiple blocks from each specimen were stained with toluidine blue and mounted on glass slides for examination by light microscopy. The optimal block for electron microscopy was selected from these slides, and each block was finely trimmed and thin-sectioned at 600 - 800 Å. These sections were collected on bare copper grids, doubly stained with uranyl acetate and lead citrate, and examined in a Hitachi 12A electron microscope operated at 12 kV.

When the pathology examination was completed, the slides, individual animal data records, and summary tables were sent to an independent quality assurance laboratory. The individual animal records and tables were compared for accuracy, slides and tissue counts were verified, and histotechnique was evaluated. All tumor diagnoses, all target tissues, and all tissues from a randomly selected 10 % of the animals were evaluated by a quality assessment pathologist. The quality assessment report and slides were submitted to the Pathology Working Group (PWG) Chairperson, who reviewed all target tissues and those about which there was a disagreement between the laboratory and quality assessment pathologists.

Representative slides selected by the Chairperson were reviewed by the PWG without knowledge of previously rendered diagnoses. When the consensus diagnoses of the PWG differed from that of the laboratory pathologist, the laboratory pathologist was asked to reconsider the original diagnosis. The final diagnosis represent a consensus of contractor pathologists and the NTP Pathology Working Group.

Slides/tissues are generally not evaluated in a blind fashion (i.e., without knowledge of dose group) unless the lesions in question are subtle or unless there is an inconsistent diagnosis of lesions by the laboratory pathologist. Non-neoplastic lesions are not examined routinely by the quality assessment pathologist or PWG unless they are considered part of the toxic effect of the chemical.
see below section "Any other information on materials and methods".

Results and discussion

Results of examinations

Details on results:
Body Weights, Feed Consumption, and Clinical signs
Mean body weight gains relative to those of controls were markedly reduced (greater than 10 %) for high dose male and female rats throughout most of the studies. Decreased mean body weight gain also was seen in mid dose female rats. Mid dose male rats and low dose male and female rats showed some evidence reduced weight gains, but these decreases were generally in the 5 % - 9 % range. Feed consumption by dosed and control groups was comparable. Feed consumption by low, mid, and high dose males was 102 %, 100 %, and 97 % that of the controls; feed consumption by low, mid, and high dose female rats was 105 %, 99 %, and 98 % that by controls.

No compound-related clinical signs were observed.

Survival in high dose male rats was significantly reduced relative to that of controls. Survival in mid dose males also was reduced. In groups of female rats, differences in survival were not significant. The increased mortality in high dose male rats does not appear to have been caused primarily by nasal cavity tumors, since a comparable increase in nasal cavity neoplasms in female rats was not associated with increased mortality.

Pathology and Statistical Analyses of Results
This section describes statistically significant or biologically noteworthy increases in the incidences of rats with neoplastic or non-neoplastic lesions in the nasal cavity, subcutaneous tissue, liver, pituitary gland, and adrenal gland. Summaries of the incidences of neoplasms and non-neoplastic lesions, individual animal tumor diagnoses, statistical analyses of primary tumors that occurred with an incidence of at least 5 % in at least one animal group, and historical control incidences for the neoplasms mentioned in this section are presented for male and female rats, respectively.

Nasal Cavity: High dose male and female rats significantly increased incidences of carcinomas. Two adenocarcinomas were observed in high dose males. High dose rats of each sex had increased incidences of papillary neoadenomas. A few unusual neoplasms of the nasal cavity were observed in high dose male and female rats: One undifferentiated sarcoma was present in a high dose female; rhabdomyosarcomas occurred in two high dose male and two high dose females; and neoplasms with features associated with both adenocarcinomas and rhabdomyosarcomas (malignant mixed tumors) were observed in one high dose male and one high dose female rat.

The benign neoplasms (adenoma and papillary adenoma) were located in the anterior part of the nasal cavity in the region of the respiratory turbinates. The malignant neoplasms (carcinoma, adenocarcinoma, sarcoma, rhabdomyosarcoma, and malignant mixed tumors) were usually located in the posterior nasal cavity in the region of the ethmoturbinates and the posterior part of the nasal septum. The adenocarcinomas and carcinomas varied structure from well-differentiated growths composed of acini lined by cuboidal cells (adenocarcinomas) to poorly differentiated neoplasms in which a glandular pattern was not prominent (carcinomas). Most of these malignant neoplasms appeared to arise from the submucosal gland in the dorsal posterior portion of the nasal turbinates. As the neoplasms became more differentiated, it was difficult to determine whether they were arising from the surface epithelium or from the submucosal glands. The carcinomas were composed of infiltrating sheets of pleomorphic hyperchromic epithelial cells which varied from small pleomorphic cells with indistinct basophilic cytoplasm and round basophilic nuclei to large anaplastic cells with abundant eosinophilic cytoplasm and large pleomorphic nuclei containing distinct nucleoli. Mitotic figures were numerous in malignant neoplasms; focal squamous metaplasia was sometimes present. The carcinomas were highly invasive, frequently destroying the nasal turbinates and nasal septum. Invasion into the adjacent maxillary bone and overlaying frontal bone occurred in several rats. Metastasis to the brain was detected in 5/56 male and 7/56 female rats fed 2,6-xylidine at 3,000 ppm. The undifferentiated sarcoma was present in the olfactory region of the nasal cavity. The neoplasm was composed of interlacing bundles of spindle-shaped cells having little cytoplasm and elongated nuclei. Mitotic figures were numerous throughout the neoplasm. The center of the neoplasm was necrotic. Invasion into the nasal septum and frontal bone occurred.
The rhabdomyosarcomas were composed of anaplastic pleomorphic cells with abundant eosinophilic cytoplasm. The cells had round-to-oval vesicular nuclei with distinct nucleoli; multinuclear giant cells were common. The neoplastic cells varied from round to spindle-shaped, and elongated "strap-like" cells with centrally located multiple nuclei were present. In some areas, the neoplasms were less differentiated; in these areas, cells had hyperchromatic nuclei, numerous mitotic figures, and less cytoplasm. Cross striations were readily apparent in 1 µm sections stained with toluidine blue, whereas striations were barely visible in sections stained with hematoxylin and eosin.

Electron microscopy revealed Z bands; M bands were sometimes apparent. Some slightly elongated cells having filaments or myofibrils and no Z bands may be analogous to the "strap-like" cells observed by light microscopy. Multinucleated giant cells contained a few myofibrils or filaments and a suggestion of Z bands. These findings confirm that these mesenchymal nasal tumors are rhabdomyosarcomas.

Neoplasms diagnosed as malignant mixed tumors of the nasal cavity had two distinct cellular components. Some portions of the neoplasm were composed of well-differentiated cuboidal epithelium arranged in a glandular pattern. Sarcomatous proliferations of elongated "strap-like" cells (containing multiple elongated nuclei and abundant eosinophilic cytoplasm) were mixed with neoplastic glandular epithelium. Marked pleomorphism and numerous mitotic figures were present in all areas of this neoplasm. When this neoplasm was stained by Mallory's phospho acid hematoxylin, rhabdomyosarcomas and cross striations in the cytoplasm of the "strap-like" cells were observed. Papillary adenomas of the nasal cavity varied in size and originated from the epithelium of the nasal cavity and maxillary turbinates or from the nasal septum. These neoplasms formed papillary projections into the lumen of the nasal cavity and were usually lined by single layers of respiratory epithelium. The neoplasms were well-differentiated and characterized by an absence of invasion into the underlying tissue. Multiple papillary adenomas were occasionally present; five high dose male and one high dose female rat with papillary adenomas also had malignant epithelial neoplasms in other regions of the nasal cavity. Acute inflammation (rhinitis), epithelial hyperplasia, and squamous metaplasia occurred at increased incidences in high dose male and female rats. Inflammation of the epithelium lining the nasal cavity occurred in both control and dosed rats. The increase in acute rhinitis was dose related. Animals with rhinitis had an accumulation of suppurative exudate and cellular debris in the lumen of the nasal cavity. Many of the rats with acute inflammatory changes in the nasal mucosa had erosion and ulceration of the epithelial lining of the nasal cavity and hyperplasia of the submucosal glands. In a few animals with subacute rhinitis, the cellular infiltrate was a mixture of neutrophilic and mononuclear cells; in some animals with acute rhinitis, the infiltrate was primarily mononuclear cells.

Subcutaneous Tissue: The incidences of high dose male and female rats with fibromas were greater than those in controls. Subcutaneous fibrosarcomas were observed in three high dose females, one high dose male, one mid dose female, one low dose male, and one control female. The incidences of high dose males and females with fibromas were significant by the life table test.

Liver: Neoplastic nodules occurred in female rats with a significant positive trend. Hepatocellular carcinomas were observed in one control, one mid dose, and one high dose female rat. Incidences of dosed male rats with liver tumors were not increased; a high dose male had neoplastic nodule, and a control and a mid dose male had an hepatocellular carcinoma.

Pituitary Gland: Adenomas occurred in female rats with 4 significant positive trend, but pairwise comparisons did not yield significant results (control 29/56; mid dose 30/56, high dose 20/56; p<0.05). The incidence of male rats with pituitary gland adenomas was significantly increased only in the low dose group (9/55; 18/54; 13/54; 9/54;. p<0.05).

Adrenal Gland: The increased incidence of high dose females with cortical adenomas was significant by the life table test (control, 7/56; low dose 9/56; mid dose, 6/55; high dose, 12/55; P<0.05). Since this lesion is not usually life threatening, life table analysis may not be the most appropriate statistical method. No dose-related adrenal tumors were seen in male rats.

Other Sites: Several microscopic non-neoplastic changes were detected in dosed and control animals; e.g., nephropathy, alveolar macrophages, hemosiderosis and hematopoiesis in the spleen, foci of cellular alteration in the liver, cortical vacuolization and focal hyperplasia in the adrenal cortex and medulla, and cystic changes in the and ovaries. These lesions are commonly observed in aging CD rats.

Hematologic and Clinical Chemistry Analyses
Decreases were detected in erythrocyte counts and haemoglobin levels at 18 months in the 3000 ppm group of male rats and in erythrocyte counts, haemoglobin levels, and hematocrit at 12 months in the 1,000- and 3,000-ppm groups of female rats. These changes were not severe enough to be considered indicative of anemia. The biologic significance of changes in leukocyte counts during the last 6 months of the study is not known. No biologically significant changes in clinical chemistry parameters were attributable to administration of 2,6-xylidine.

Effect levels

Dose descriptor:
Effect level:
3 000 ppm
Basis for effect level:
other: body weight gain

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Conclusion by the authors:

Under the conditions of these studies, 2,6-xylidine was clearly carcinogenic* for male and female Charles River CD rats, causing significant increases in the incidences of adenomas and carcinomas of the nasal cavity. Rhabdomyosarcomas, rare tumors of the nasal cavity, were observed in dosed rats of each sex. In addition, the increased incidences of subcutaneous fibromas and fibrosarcomas in male and female rats and the increased incidence of neoplastic nodules of liver in female rats may have been related to the administration of 2,6-xylidine.

Applicant's summary and conclusion