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Carcinogenicity

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Description of key information

On the basis of a weight of evidence assessment Alchisor TAL 111 is determined to lack a carcinogenic potential.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to guideline study OECD 453.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
no
GLP compliance:
not specified
Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: F344/N rats: Taconic Laboratory Animals and Services
- Age at study initiation: F344/N rats were approximately 4 weeks old
- Acclimation period: 13-14 days
- Feed and Water: Feed was available ad libitum except during exposure periods; water was available ad libitum.
- Housing: individually
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
not specified
Vehicle:
unchanged (no vehicle)
Details on exposure:
Stoddard solvent IIC was pumped through a preheater and then into the top of a heated glass column filled with glass beads to increase the surface area for evaporation. Heated nitrogen entering the column from below vaporized the chemical as it conveyed it out of the generator.
Generator output was controlled by the delivery rate of the chemical metering pump. Because the vapor leaving the generator was above room temperature, the transport line to the exposure room was heated to prevent condensation. In the exposure room, the vapor was mixed with additional heated air before it entered a short vapor distribution manifold. Concentration in the manifold was determined by the chemical pump rate, nitrogen flow rate, and dilution air flow rate. All three components were monitored by the exposure operator. The pressure in the distribution manifold was kept fixed to ensure constant flows through the manifold and into the chambers. Buildup and decay rates for chamber vapor concentrations were determined with animals present in the chambers. At a chamber airflow rate of 15 air changes per hour, the theoretical value for the time to achieve 90% of the target concentration after the beginning of vapor generation (T90) and the time for the chamber concentration to decay to 10% of the target concentration after vapor generation was terminated (T10) was approximately 12.5 minutes. Based on experimental data, a T90 value of 12 minutes was selected for all studies. The uniformity and persistence of Stoddard solvent IIC vapor concentrations in the inhalation exposure chambers and Stoddard solvent IIC degradation products were evaluated throughout the study using GC. Chamber concentration uniformity was maintained; no evidence of degradation was found, and no extraneous peaks were seen. Stoddard solvent IIC was stable for 273 days in the generator reservoir.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The Stoddard solvent IIC concentrations in the exposure chambers were monitored by an online gas chromatograph. Samples were drawn from each exposure chamber approximately every 24 minutes using a 12-port stream select valve. The online gas chromatograph was checked throughout the day for instrument drift against an on-line standard of an approximately 400 mg/m3 mixture of the n-paraffins decane, undecane, and dodecane in nitrogen supplied by a diffusion tube standard generator. The on-line gas chromatograph was calibrated monthly or when excessive calibration drift was detected by a comparison of chamber concentration data to data from grab samples, which were collected with charcoal sampling tubes. Grab samples were extracted with hexanes containing nonane as an internal standard and analyzed by an off-line gas chromatograph. Known volumes of chamber atmosphere from each chamber were sampled at a constant flow rate ensured by a calibrated critical orifice. The off-line gas chromatograph was calibrated with gravimetrically prepared standards of Stoddard solvent IIC and an internal standard (nonane) in hexanes. The composition of Stoddard solvent IIC in the 2,200 mg/m3 exposure chamber was monitored by a second on-line gas chromatograph in the 3-month and 2-year studies. Samples were drawn from the exposure chamber five or six times during each 6-hour exposure period using a 12-port stream select valve. The on-line gas chromatograph was checked against the on-line standard after exposure termination. The composition monitor provided enhanced chromatographic separation of the components and allowed reporting of the relative amounts of the major n-paraffins of Stoddard solvent IIC. Mean results for decane, undecane, and dodecane of 100.1%, 100.0%, and 99.9%, respectively, during the 2-year studies indicated that the composition of Stoddard solvent IIC did not change significantly during exposure.
Duration of treatment / exposure:
6 hours plus T90 (12 minutes) per day
Frequency of treatment:
5 days per week for 105 weeks
Remarks:
Doses / Concentrations:
0, 138, 550, 1,100, or 2,200 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
50 male and 50 female per dose; Additional groups of 10 male and 10 female rats were exposed to the same concentrations for 13 weeks for renal toxicity analyses.
Control animals:
yes, concurrent no treatment
Details on study design:
Groups of 50 male and 50 female rats were exposed to Stoddard solvent IIC at concentrations of 0, 138, 550, 1,100, or 2,200 mg/m3, 6 hours plus T90 (12 minutes) per day, 5 days per week for 105 weeks. Additional groups of 10 male and 10 female rats were exposed to the same concentrations for 13 weeks for renal toxicity analyses.
Positive control:
none
Observations and examinations performed and frequency:
All animals were observed twice daily. Animals were weighed at the beginning of the studies. Clinical findings and body weights were recorded every 4 weeks from week 5 through 89 and every 2 weeks from week 92 to the end of the studies.
Sacrifice and pathology:
Complete histopathology was performed on all core study animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung with mainstem bronchi, lymph nodes (mandibular, mesenteric, bronchial, and mediastinal), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus. The larynx, lung, nose, and trachea of rats and mice; kidney of rats; and spleen of female mice were also examined in the remaining exposure groups.
Other examinations:
Renal Toxicity Study -Osmotic pumps containing 30 mg/mL bromodeoxyuridine (BrdU) were implanted subcutaneously in the renal toxicity study rats on the Sunday before the 13th week of exposure; the animals were sacrificed on Friday of the 13th week. The duodenum, kidneys, and the portion of the tail with identification were removed from each rat; the remaining carcass was discarded. The kidneys were weighed and alpha 2u-globulin concentration and cell proliferation analyses were performed. For alpha 2u-globulin assessment, the right kidney was frozen in liquid nitrogen and stored at –70° C pending analysis. After thawing, a volume of sodium/potassium phosphate buffer (pH 7.2) equivalent to twice the recorded fresh weight of the sample was added, and the sample was homogenized using a tissue homogenizer. The homogenate was centrifuged at 3,000 g for 15 minutes at 4° C, and the supernatant was drawn off and stored at –70° C. The protein content of each supernatant was measured in a 1:50 dilution in PBS-Tween using a pyrogallol red assay. Homogenates were analyzed for alpha 2u-globulin using a competitive indirect ELISA technique. Ascites fluid containing anti-alpha 2u-globulin monoclonal antibodies was provided by Dr. Susan J. Borghoff. The amount of alpha 2u-globulin was measured by comparing the relative fluorescent signal intensity in the study samples to that observed with known amounts of alpha 2u-globulin present in calibration standards. Calibration standards and ELISA control standards (negative and positive) were plated in predetermined wells on 96-well microtiter plates. Calibration standards and study samples were assayed in triplicate.

For cell proliferation analyses, the left kidney (bisected longitudinally) and a piece of duodenum were removed, fixed in 10% neutral buffered formalin for 24 hours and then transferred to 70% ethanol for 24 hours. The tissues were then processed, embedded in paraffin, and stained immunohistochemically. Cell proliferation assessment was done using a 20× objective and ocular grid; labeled and unlabeled tubular nuclei were counted from each kidney. The duodenum section was examined first for positive BrdU staining in crypts. Then counting started at the second grid in from the outer edge of the cortex of the kidney. After one grid was counted, the slide was moved toward the medulla and every other field encountered by the grid was counted. If 2,000 proximal tubular nuclei were reached before the entire grid had been counted, the remainder of the grid was counted. If 2,000 proximal tubular nuclei were not counted by the time the outer medulla was reached, the slide was moved two grids laterally and the counting process resumed at the second grid in from the edge of the cortex. In addition to the cell proliferation assessment, sections of the left kidney and duodenum were stained with hematoxylin and eosin and with Mallory-Heidenhain stains. These sections were evaluated microscopically for evidence of renal toxicity.
Statistics:
Survival Analyses:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.
Calculation of Incidence:
Survival-adjusted neoplasm rate for each group and each site-specific neoplasm accounts for differential mortality by assigning a reduced risk of neoplasm, proportional to the third power of the fraction of time on study, to animals that do not reach terminal sacrifice.
Analysis of Neoplasm and Nonneoplastic Lesion Incidences:
The Poly-k was used to assess neoplasm and nonneoplastic lesion prevalence. Unless otherwise specified, a value of k=3 was used in the analysis of site-specific lesions. Tests of significance included pairwise comparisons of each exposed group with controls and a test for an overall exposure-related trend. Continuity-corrected Poly-3 tests were used in the analysis of lesion incidence, and reported P values are one sided. The significance of lower incidences or decreasing trends in lesions is represented as 1– P with the letter N added (e.g., P=0.99 is presented as P=0.01N).
Analysis of Continuous Variables:
Organ and body weight data were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Other end points were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test).
Historical Control Data was used.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Survival of 138 and 1,100 mg/m3 males and 2,200 mg/m3 females was significantly less than that of the chamber controls. Mean body weights of exposed groups of males and females were similar to those of the chamber control groups throughout the study. There were no clinical findings related to Stoddard solvent IIC exposure.

Renal Toxicity Study
In the satellite groups, cell proliferation analyses were performed on the left kidney of males and females after 3 months of exposure. The mean numbers of labeled cells and the labeling indices in 550 and 1,100 mg/m3 males were significantly increased. The labeling index in 1,100 mg/m3 males was significantly greater than that in the 550 mg/m3 group, suggesting an exposure concentration-related increase in renal cell proliferation in exposed males. No significant differences in labeling indices were noted in females. Concentrations of soluble protein and alpha 2u-globulin were measured in the right kidney of males and females. The amount of alpha 2u-globulin was normalized to either soluble protein concentration or kidney weight. In males, the amount of alpha 2u-globulin increased with increasing exposure concentration; the amounts of alpha 2u-globulin in 550 and 1,100 mg/m3 males were significantly greater than that in the chamber controls.

Microscopic lesions occurred in male left kidneys at 3 months. Hyaline droplets occurred in all chamber control and all exposed males; the severity increased with increasing exposure concentration. The incidences of granular casts in 550 and 1,100 mg/m3 males, cortical tubule degeneration in 1,100 mg/m3 males, and cortical tubule regeneration in 550 and 1,100 mg/m3 males were significantly increased. Granular casts were characterized by distended tubular lumens filled with cellular debris and proteinaceous material and are indicative of cell death and believed to result from epithelial cells damaged by excessive accumulations of alpha 2u-globulin. The hyaline droplets and granular casts are consistent with alpha 2u-globulin nephropathy. Degeneration was a minimal lesion of the cortical tubular epithelium characterized by pyknotic nuclei, shrunken eosinophilic cytoplasm, and loss of cell membranes and may have been a component of the alpha 2u-globulin nephropathy. Regeneration was characterized by tubules lined by the epithelial cells with basophilic cytoplasm and enlarged nuclei. Regeneration of renal tubule epithelium is an early component of chronic progressive nephropathy, a common spontaneous syndrome in F344/N male rats.

Kidney: In the standard evaluation of a single hematoxylin and eosin stained section of the left and right kidney, the incidences of mild to moderate renal tubule and transitional epithelial hyperplasia in 550 and 1,100 mg/m3 males were significantly increased. The incidences of renal tubule adenoma and renal tubule carcinoma in exposed groups of male rats were similar to those in the chamber controls at the standard evaluation. Renal tubule hyperplasia, adenoma, and carcinoma are thought to represent a continuum in the progression of proliferative lesions of the renal tubule epithelium. Because there were increased incidences of renal tubule hyperplasia (a preneoplastic lesion) in male rats, additional kidney sections were evaluated, and additional renal tubule hyperplasias and adenomas were identified. In the extended evaluation, the significantly increased incidences of renal tubule hyperplasia in 550 and 1,100 mg/m3 males were confirmed. In the extended evaluation, the incidence of renal tubule adenoma was greater in 1,100 mg/m3 males than in the chamber controls however, the increase was not significant; the incidences of renal tubule carcinoma in exposed groups of males were similar to that in the chamber control group.

In males, the severity of chronic nephropathy of the kidney and the incidences and severity of mineralization increased with increasing exposure concentration. Nephropathy is an age-related disease process characterized by a spectrum of lesions, including varying degrees of tubular dilation; proteinaceous tubular casts; atrophy, degeneration, regeneration, and hypertrophy of the tubular epithelium; thickening of tubular and glomerular basement membranes; glomerulosclerosis; interstitial fibrosis; and varying numbers and aggregates of mononuclear inflammatory cells within the interstitium. Minimal nephropathy was characterized by a few scattered foci of tubular regeneration. These regenerative tubules had increased numbers of more intensely stained basophilic cells. Basement membranes, both in glomeruli and around tubules, were slightly thickened. As nephropathy became more severe, tubular dilatation, proteinaceous casts, and interstitial fibrosis were evident. Mineralization was characterized by the presence of lamellated intraluminal or intracellular concretions within the collecting tubules of the renal papilla usually forming linear deposits. Mineralization of the renal medulla oriented in a linear fashion is characteristic of alpha 2u-globulin inducers in 2-year studies, as is exacerbated nephropathy.

Pathology and Statistical Analyses

Adrenal Medulla: The incidences of benign and benign or malignant pheochromocytoma (combined) occurred with positive trends in males, and the incidences in the 550 and 1,100 mg/m3 groups were significantly increased. The incidences of benign pheochromocytoma in 550 and 1,100 mg/m3 males and benign or malignant pheochromocytoma in 1,100 mg/m3 males exceeded the historical ranges in chamber controls. Benign pheochromocytomas were characterized by a proliferating mass of adrenal medullary cells that compressed adjacent tissue. Malignant pheochromocytomas were generally larger with invasion of or beyond the adrenal capsule. The incidence of hyperplasia of the adrenal medulla in 550 mg/m3 males was significantly increased. Medullary hyperplasia was characterized by an increase in basophilia of medullary cells that sometimes accompanied increased size and minimal compression of the adjacent tissue. These results may be the result of the male rat specific alpha 2u-globulin nephropathy noted above.

Clitoral and Preputial Glands: The incidences of adenoma and adenoma or carcinoma (combined) of the clitoral gland occurred with positive trends in females. The incidences of adenoma in the 1,100 and 2,200 mg/m3 groups and adenoma or carcinoma (combined) in the 2,200 mg/m3 group were significantly increased. However, the incidences were within the historical ranges in chamber controls for adenoma [20/295 (7% ± 6%), range 0%-17%] and adenoma or carcinoma (combined) [24/295 (8% ± 6%), range 2%-19%]. The incidence of adenoma or carcinoma (combined) of the preputial gland in 550 mg/m3 males was significantly increased; however, the incidence was within the historical range [11/298 (4% ± 5%), range 0%-13%]. The incidences of clitoral and preputial gland hyperplasia were not significantly increased. Proliferative lesions of the clitoral and preputial glands comprise a morphologic continuum from hyperplasia to adenoma and carcinoma. Clitoral and preputial gland hyperplasia was characterized by an increased number of sebaceous cells with normal cell orientation and morphology forming crowded large acini, often associated with cyst formation. In hyperplasia, the cell cytoplasm is more basophilic and less vacuolated. Because the incidence of clitoral gland adenoma in chamber control females was low (0%) compared to historical chamber controls (6.8%), the incidences in exposed females were within the historical control range, and because of the absence of exposure-related hyperplasia and carcinoma, the increased incidences of clitoral gland adenoma were not considered chemical related. The increased incidence of preputial gland adenoma or carcinoma (combined) was also not exposure related.
Relevance of carcinogenic effects / potential:
Pheochromocytomas that occur in animal experiments currently appear to have little relevance for conditions at the work place. When sufficiently documented and evaluated, such secondary pheochromocytomas are not relevant for classification and human risk assessment
Dose descriptor:
NOAEC
Effect level:
>= 2 200 mg/m³ air (nominal)
Sex:
female
Basis for effect level:
other: Highest concentration tested.
Dose descriptor:
NOAEC
Effect level:
138 mg/m³ air (nominal)
Sex:
male
Basis for effect level:
other: NOAEC is due to male rat specific alpha 2u-globulin nephropathy. Not relevant to humans as they do not have this protein.
Conclusions:
The NOAEC for female rats was determined to be 2,200 mg/m3 which was the highest concentration tested. The NOAEC for male rats was determined to be 138 mg/m3. The male NOAEC was based on the male rat specific alpha 2u-globulin nephropathy, which is not relevant to humans as humans lack this protein. The NTP concluded that under the conditions of these 2-year inhalation studies, there was some evidence of carcinogenic activity of Stoddard solvent IIC in male F344/N rats based on increased incidences of adrenal medulla neoplasms; the slightly increased incidences of renal tubule adenoma may have been related to Stoddard solvent IIC exposure. There was no evidence of carcinogenic activity of Stoddard solvent IIC in female F344/N rats exposed to 550, 1,100, or 2,200 mg/m3.
Executive summary:

Groups of 50 male and 50 female rats were exposed to Stoddard solvent IIC at concentrations of 0, 138, 550, 1,100, or 2,200 mg/m3, 6 hours plus T90 (12 minutes) per day, 5 days per week for 105 weeks. Clinical findings were recorded twice daily. The animals were weighed initially, weekly, and at the end of the studies. Necropsies were performed on all rats and mice. The heart, right kidney, liver, lung, right testis, and thymus were weighed. Histopathologic examinations were performed on all rats and mice. The NOAEC for female rats was determined to be 2,200 mg/m3, which was the highest concentration tested. The NOAEC for male rats was determined to be 138 mg/m3. The male NOAEC was based on the male rat specific alpha 2u-globulin nephropathy, which is not relevant to humans as humans lack this protein. The NTP concluded that under the conditions of these 2-year inhalation studies, there was some evidence of carcinogenic activity of Stoddard solvent IIC in male F344/N rats based on increased incidences of adrenal medulla neoplasms; the slightly increased incidences of renal tubule adenoma may have been related to Stoddard solvent IIC exposure. There was no evidence of carcinogenic activity of Stoddard solvent IIC in female F344/N rats exposed to 550, 1,100, or 2,200 mg/m3.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 200 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
In excess of REACH requirements.

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available read-across data, the substance does not meet the criteria for classification.

Additional information

The carcinogenicity assessment of Alchisor TAL 111 is based on read-across data from C9-C14 aliphatics (2-25% aromatics).

 

Two inhalation carcinogenicity studies gave no evidence of carcinogenic activity in female F344/N rats or in B6C3F1 male mice exposed to up to 2200 mg/m3.

 

The incidences of benign pheochromocytoma in 550 and 1100 mg/m3 male rats and benign or malignant pheochromocytoma exceeded the historical control ranges. However, the adrenal pheochromocytoma are not considered relevant to humans.The increased incidences of adrenal pheochromocytoma that occurred in male rats are rarely observed in humans and other animals (Nyska et al., 1999).

 

The NTP concluded there was equivocal evidence of carcinogenic activity in female B6C3F1 mice based on increased incidences of hepatocellular adenoma. However liver tumors in B6C3F1 mice are known to be sensitive to body weight changes, and statistical analysis of the results confirm that it is the case in this study. For these reason, these tumors should no be used for cancer classification.

 

In support of the carcinogenicity assessment, the available mutagenicity and sub-chronic studies have also been evaluated. The mutagenicity studies gave negative results and there was no evidence of hyperplastic responses or pre-neoplastic lesions in sub-chronic studies.

 


Justification for selection of carcinogenicity via inhalation route endpoint:
There are two high quality inhalation studies available. The rat study has been selected on the basis that there is a larger data base of studies on the rat.

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