2,6-xylidine

Carcinogenicity

Description of key information

In a carcinogenicity study conducted similar to OECD 453, 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.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Well documented study report, which meets basic scientific principles; comparable to guideline

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

no

Specific details on test material used for the study:

- 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)

Species:

rat

Strain:

other: CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories.
- Age at study initiation: weanlings, 4wks.
- Housing: 18 rats per group individual, 38 rats per group were housed 3 per cage.
- Diet: Purina lab chow; available ad libitum.
- Water: available ad libitum.
- Acclimation period: 4 wk.


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

Route of administration:

oral: feed

Vehicle:

corn oil

Details on exposure:

DIET PREPARATION
2,6-xylidine suspended in corn oil and incorporated into Purina Lab Chow meal. Control diets were prepared by mixing corn oil with Chow.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The feed stability results indicated that 2,6-xylidine mixed with the basal diet was unstable under all conditions of storage.
Formulated feed mixtured 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 stored open in a 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.

Duration of treatment / exposure:

102 weeks

Frequency of treatment:

continuously

Post exposure period:

none

Dose / conc.:

100 ppm (nominal)

Dose / conc.:

300 ppm (nominal)

Dose / conc.:

1 000 ppm (nominal)

Dose / conc.:

3 000 ppm (nominal)

No. of animals per sex per dose:

56 males and 56 females

Control animals:

yes, concurrent vehicle

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 represents 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.

Statistics:

see below section "Any other information on materials and methods".

Clinical signs:

no effects observed

Description (incidence and severity):

No compound-related clinical signs were observed.

Mortality:

mortality observed, treatment-related

Description (incidence):

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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Decreases were detected in erythrocyte counts and haemoglobin levels at 18 month in the 3000 ppm group of male rats and in erythrocyte counts, hemoglobin 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.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No biologically significant changes in clinical chemistry parameters were attributable to administration of 2,6-xylidine.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

Description (incidence and severity):

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.

Dose descriptor:

LOAEL

Remarks:

carcinogenicity

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: carcinogenic lesions not significantly increased at this dose

Critical effects observed:

yes

Lowest effective dose / conc.:

3 000 ppm

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

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. Evaporation of the test substance from the feed was verified by stability anaylsis. Therefore, additional exposure via the inhalation path throughout the study cannot be ruled out. 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.

Executive summary:

In an NTP study, CD-rats were fed diets containing 0, 300, 1,000, or 3,000 ppm 2,6-DMA before breeding, during pregnancy, and through the lactation period. The carcinogenicity of 2,6-DMA was studied in 56 male an 56 female offspring from each dose group which were treated with the same dose (corresponding to 0, 12, 40 or 120 mg/kg d in male and 0, 15, 50 or 150 mg/kg d in female rats, according to HCN (2002)) after weaning for 102 weeks. Body weight gain was reduced in both male and female offspring at the high dose during the study and survival was reduced in males at mid and high dose level. Lesions of the nasal epithelium were the main neoplastic and non-neoplastic effects. The incidences of both papillomas and carcinomas of the nasal cavity were significantly increased in both sexes at the high dose. The combined number of carcinomas and adenocarcinomas was 28/56 in high dose males, 24/56 in high dose females, and 1/56 in mid dose females. Papillary adenomas were observed in 10/56 high dose males, 2/56 mid dose males, and 6/56 high dose females. None occurred in the other groups. The carcinomas were very invasive and metastasis to the brain was present in 5/56 male and 7/56 females at the high dose. In two male and two female rats of the high dose group rhabdomyosarcomas of the nasal cavity were observed, a malignant tumour extremely rare in rats. Non-neoplastic lesions observed in the nasal cavity included acute inflammation (all doses), epithelial hyperplasia (≥ 300 ppm), and squamous metaplasia (1,000 ppm, see above). Furthermore, dose-related increases in the incidence of subcutaneous tissue fibromas and fibrosarcomas were observed in both sexes and there was a significant positive trend for neoplastic nodules in the liver of female rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

chronic

Species:

rat

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

SCOEL (2009):

 

2,6-DMA is a carcinogen in rats causing invasive tumours of the nasal cavity. These tumours develop in tissues which show inflammatory processes at the same time and prior to the occurrence of tumours. It remains open whether inflammatory and toxic effects play a rote in tumour development. However, 2,6-DMA is N-oxidised to genotoxic products, binds to DNA at the target tissue, and forms a type of DNA-adduct analogue to that observed with genotoxic carcinogenic polycyclic arylamines.Hence, the mode of action of nasal tumour development in experimental animals remains open. Under these conditions, no health-based OEL can be derived. Moreover, no inhalation study exists to derive quantitative risk estimates.

Justification for classification or non-classification

Classification, Labeling, 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 classified for carcinogenicity Cat. 2 under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2018/1480.

Furthermore, 2,6 -xylidine is included in Annex VI of Regulation (EC) No. 1272/2008 with the following legal classification:

• Carcinogenicity Cat. 2 (H351: Suspected of causing cancer)

Additional information

SCOEL (2009):

 

In an NTP study, CD-rats were fed diets containing 0, 300, 1,000, or 3,000 ppm 2,6-DMA before breeding, during pregnancy, and through the lactation period. The carcinogenicity of 2,6-DMA was studied in 56 male an 56 female offspring from each dose group which were treated with the same dose (corresponding to 0, 12, 40 or 120 mg/kg d in male and 0, 15, 50 or 150 mg/kg d in female rats, according to HCN (2002)) after weaning for 102 weeks. Body weight gain was reduced in both male and female offspring at the high dose during the study and survival was reduced in males at mid and high dose level. Lesions of the nasal epithelium were the main neoplastic and non- neoplastic effects. The incidences of both papillomas and carcinomas of the nasal cavity were significantly increased in both sexes at the high dose. The combined number of carcinomas and adenocarcinomas was 28/56 in high dose males, 24/56 in high dose females, and 1/56 in mid dose females. Papillary adenomas were observed in 10/56 high dose males, 2/56 mid dose males, and 6/56 high dose females. None occurred in the other groups. The carcinomas were very invasive and metastasis to the brain was present in 5/56 male and 7/56 females at the high dose. In two male and two female rats of the high dose group rhabdomyosarcomas of the nasal cavity were observed, a malignant tumour extremely rare in rats. Non-neoplastic lesions observed in the nasal cavity included acute inflammation (all doses), epithelial hyperplasia (≥ 300 ppm), and squamous metaplasia (1,000 ppm, see above). Furthermore, dose- related increases in the incidence of subcutaneous tissue fibromas and fibrosarcomas were observed in both sexes and there was a significant positive trend for neoplastic nodules in the liver of female rats (NTP, 1990).

 

The occurrence and development of 2,6-DMA-related nasal tumours was studied in an initiation-promotion carcinogenesis mode) with male F344 rats. Initiation was carried out by a single subcutaneous injection of N-bis(2-hydroxypropyl)nitrosamine (DHPN) followed by administration of 2,6-DMA with the diet (3000 mg/kg) for 52 weeks. While no untreated animals developed nasal neoplasias, animals treated with DHPN developed nasal adenomas and carcinomas and the number of tumourbearing animals was further increased in the group treated with DHPN and 2,6-DMA. The incidences or multiplicity of epithelial hyperplasia and dysplastic foci was also increased in this group. Detailed histopathological and ultrastructural investigations revealed that the lesions typically arose in the Bowman's glands of the olfactory mucosa and that dysplastic foci developing in this gland progress to carcinomas while proliferation of the glands lead to hyperplasia and adenomas. Atrophy of Bowman's glands was apparent after 4 weeks, proliferation of undifferentiated cells after 13 weeks. Hyperplasia, dysplastic foci and adenoma could be observed from 26 weeks on and carcinomas at 52 weeks (Koujitani et al., 1999, 2000, 2001).