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Carcinogenicity

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Description of key information

Cf. Scientific opinion on lung toxicity and carcinogenicity in Section 13 for complete weight of evidence and read-across assessments.

 

Several long-term historical occupational inhalation exposures to Sb compounds have been associated with impairments of lung function resulting from chronic inflammation and fibrosis. Two studies of relatively good quality, and other studies of lower quality document such effects in Sb-exposed workers. In general, however, the pneumoconiosis observed in workers tends to be benign with only little evidence of pathological changes (e.g. fibrosis) that would indicate progressive reactivity of the Sb burden in the lungs of workers.

 

Studies confirm the historical incidence of pneumoconiosis in workers employed at Sb processing facilities, with an exposure exceeding the current Occupational Exposure Limit of 0.5 mg/m3 by a factor of 10 or more. This is consistent with medical surveillance data reported to the International Antimony Association by its membership and with the decrease of the incidence of pneumoconiosis at an Sb trioxide production facility after the implementation of OELs.

 

Although epidemiology studies in smelter environments (Jones et al., 1994; Schnorr et al., 1995 and Jones et al., 2007) have reported small increases in lung cancer in workers occupationally exposed to Sb trioxide, attribution to Sb compounds has not been possible due to significant levels of co-exposure to known lung carcinogens such as arsenic and cadmium. An older study of glassblowers suggested an association with colon and stomach cancer (Wingren and Axelson, 1987) but measurements of Sb exposure were not available and the association with the cancer data not presented. An ecological study of breast cancer incidence in the United States (White et al., 2019) failed to find an association with airborne Sb levels (although exposure levels were low). Finally, Deng et al (2019) observed no impacts upon early biomarkers of potential health effects (e.g. the Comet Assay) except for altered expression of microRNAs associated with the metabolism of polycyclic aromatic hydrocarbons. Urinary Sb levels served as the index of exposure and, curiously enough, was higher in the non-exposed controls. No consistent association between cancer incidence and Sb exposure is evident in these studies.

 

Animal studies using exposures of sufficient duration and respirable particle aerosols with a small size facilitating penetration to the deep lung (< 4 µm) confirmed that Sb trioxide can have toxic impacts upon the lung. This affirmation needs to be properly evaluated regarding to the workers exposed in industrial facilities as the occupational aerosols possess a larger particle size distribution (Hughson, 2005) with larger particles (the inhalable fraction) preferentially depositing in the nose, throat and upper airways. The respirable fraction is, on average, only about one third the size of inhalable fraction.

 

Animal inhalation studies are thus designed to maximize the likelihood of damage to tissues of the deep lung. Although three initial experimental inhalation studies deviate from standard protocols (one year of exposure opposed to the two years specified by most cancer bioassay guidelines), it has been demonstrated that Sb trioxide could impair particle lung clearance. More recently, a two-year cancer bioassay (NTP, 2017) reported evidence of a relationship between exposure to respirable Sb trioxide and lung tumors in the mouse and, to a lesser extent, the rat.

 

Evidence is only available for Sb metal, Sb trioxide, and Sb trisulfide. There is no information on the potential to cause lung toxicity or cancer for any other Sb substance. While all studies have reported some degree of lung toxicity, only one reports clear evidence of cancer (in mice). Evidence in rats is either less clear or can be attributed an overload response.

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

Referenceopen allclose all

Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
GLP compliance:
not specified
Specific details on test material used for the study:
Antimony trioxide was obtained from 3N International, Inc. (Akron, OH), in one lot (3N-06159) that was used in the 2-week and 2-year studies. Identity and purity analyses were conducted by the analytical chemistry laboratories at H&M Analytical Services, Inc. [Allentown, NJ; X-ray diffraction (XRD)], and Chemir Analytical Services, Inc. [Maryland Heights, MO; redox titration and ultraviolet (UV) spectrophotometry], and by the study laboratory at Battelle Toxicology Northwest [Richland, WA; inductively coupled plasma/atomic emission spectroscopy (ICP/AES)] (Appendix H). Reports on analyses performed in support of the antimony trioxide studies are on file at the National Institute of Environmental Health Sciences.
Lot 3N-06159 of the chemical, a finely divided white powder, was identified as antimony trioxide by XRD. The purity of lot 3N-06159 was determined using ICP/AES. In addition, redox titration and UV spectrophotometry of bulk chemical samples determined the oxidation state of antimony in the test article.
The ICP/AES analysis indicated a purity of 101.9% based on a theoretical content of 83.5% antimony in antimony trioxide. Of the 18 minor elements measured by ICP/AES, only arsenic (~0.019%) and lead (~0.016%) were detected above 0.01% relative to antimony trioxide. Redox titration and UV spectroscopy confirmed that antimony was present in the +3 oxidation state, consistent with antimony trioxide. The overall purity of lot 3N-06159 was determined to be greater than 99.9%.
Species:
mouse
Strain:
B6C3F1
Details on species / strain selection:
B6C3F1/N mice, obtained from the NTP colony maintained at Taconic Farms, Inc. (Germantown, NY).
Sex:
male/female
Details on test animals and environmental conditions:
Animal care and use are in accordance with the Public Health Service Policy on Humane Care and Use of Animals. All animal studies were conducted in an animal facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. Studies were approved by the Battelle Toxicology Northwest Animal Care and Use Committee and conducted in accordance with all relevant NIH and NTP animal care and use policies and applicable federal, state, and local regulations and guidelines.
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Vehicle:
not specified
Details on exposure:
For the 2-year studies, the generation system used a linear feed device designed and built by Battelle to meter antimony trioxide into a Trost jet mill for aerosolization and particle size reduction. The study laboratory designed the inhalation exposure chambers so that uniform aerosol concentrations could be maintained throughout the chambers with the catch pans in place. The total volume of each chamber was 2.3 m3 with an active mixing volume of 1.7 m3. Tests showed that aerosol concentration could be reliably maintained homogenous within 8% throughout the chambers, provided the aerosol was uniformly mixed before passing through the chamber inlet and provided the test material did not react to a significant extent with animals, animal excrement, or the chamber interior.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of antimony trioxide in the exposure chambers and room air was monitored using three real-time aerosol monitors (RAMs) (MicroDust pro, Casella USA, Amherst, NH).
Duration of treatment / exposure:
6 hours plus T90 (12 minutes) per day
Frequency of treatment:
5 days per week for up to 105 weeks.
Dose / conc.:
0 mg/m³ air
Dose / conc.:
3 mg/m³ air
Dose / conc.:
10 mg/m³ air
Dose / conc.:
30 mg/m³ air
No. of animals per sex per dose:
60 male and 60 female rats
Control animals:
yes, concurrent no treatment
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Exposure-related clinical findings included abnormal breathing and thinness in males and females.
Mortality:
mortality observed, treatment-related
Description (incidence):
Survival of 10 and 30 mg/m3 males and females was significantly less than that of the chamber control groups. Decreases in survival were attributed primarily to alveolar/bronchiolar carcinomas and inflammation of the lung in males and malignant lymphoma and lung inflammation in females.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of 30 mg/m3 males were 10% to 25% less than those of the chamber control group after week 73; mean body weights of 30 mg/m3 females were at least 10% less than those of the chamber control group after week 85.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Lung weights were significantly increased in all exposed groups of males and in 10 and 30 mg/m3 females at the 12-month interim evaluation. Thymus weights of 10 and 30 mg/m3 males and females were significantly increased at the 12-month interim evaluation.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Male mice:
* Lung: infiltration cellular, lymphocyte (13/50, 47/50, 48/50, 45/50); foreign body (0/50, 50/50, 50/50, 50/50); inflammation, chronic active (0/50, 48/50, 50/50, 50/50); alveolus, fibrosis (0/50, 12/50, 30/50, 37/50); pluera, fibrosis (0/50, 36/50, 46/50, 50/50); pleura, inflammation (1/50, 40/50, 47/50, 48/50); alveolar epithelium, hyperplasia (6/50, 39/50, 45/50, 49/50); bronchiole, epithelium, hyperplasia (0/50, 32/50, 44/50, 44/50)
* Bone marrow: hyperplasia (10/49, 19/50, 27/48, 33/50)
* Thymus: depletion cellular (15/41, 14/38, 32/43, 32/39)
* Nose: foreign body (0/50, 48/49, 48/49, 49/50); respiratory epithelium, inflammation, chronic active (3/50, 9/49, 9/49, 6/50)
* Larynx: foreign body (0/50, 15/50, 29/50, 44/50); respiratory epithelium, hyperplasia (1/50, 3/50, 15/50, 30/50); respiratory epithelium, metaplasia, squamous (0/50, 0/50, 8/50, 18/50); squamous epithelium, hyperplasia (2/50, 0/50, 4/50, 13/50)
* Trachea: foreign body (0/49, 3/50, 1/50, 20/50); epithelium, hyperplasia (0/49, 0/50, 2/50, 5/50)
* Lymph node, bronchial: hyperplasia, lymphoid (2/30, 21/43, 26/47, 13/41); foreign body (0/30, 34/43, 47/47, 38/41); infiltration cellular, histiocyte (0/30, 2/43, 4/47, 6/41)
* Lymph node, mediastinal: hyperplasia, lymphoid (2/37, 8/45, 17/48, 34/49); foreign body (0/37, 32/45, 42/48, 48/49); infiltration cellular, histiocyte (0/37, 4/45, 13/48, 34/49)
* Heart: epicardium, inflammation, chronic active (0/50, 2/50, 7/50, 16/50)
* Stomach, forestomach: inflammation, chronic active (2/50, 4/50, 4/49, 7/50)

Female mice:
* Lung: infiltration cellular, lymphocyte (7/50, 37/50, 37/50, 26/50); foreign body (0/50, 50/50, 50/50, 50/50); inflammation, chronic active (1/50, 50/50, 50/50, 50/50); alveolus, fibrosis (0/50, 13/50, 30/50, 38/50); pluera, fibrosis (1/50, 39/50, 50/50, 50/50); pleura, inflammation (4/50, 27/50, 42/50, 38/50); alveolar epithelium, hyperplasia (1/50, 36/50, 49/50, 48/50); bronchiole, epithelium, hyperplasia (1/50, 34/50, 48/50, 45/50)
* Spleen: hematopoietic cell proliferation (17/50, 19/50, 20/50, 35/50)
* Bone marrow: hyperplasia (3/50, 5/50, 15/50, 28/50)
* Thymus: depletion cellular (9/47, 18/49, 23/49, 29/49)
* Nose: foreign body (1/50, 44/49, 45/50, 48/50); respiratory epithelium, metaplasia, squamous (0/50, 3/49, 2/50, 4/50)
* Larynx: foreign body (0/50, 25/50, 39/50, 48/50); respiratory epithelium, hyperplasia (2/50, 0/50, 14/50, 18/50); respiratory epithelium, metaplasia, squamous (1/50, 0/50, 5/50, 24/50); squamous epithelium, hyperplasia (4/50, 1/50, 1/50, 12/50)
* Trachea: foreign body (0/50, 7/50, 14/50, 20/50)
* Lymph node, bronchial: hyperplasia, lymphoid (2/41, 15/47, 17/48, 11/49); foreign body (0/41, 34/47, 46/48, 43/49); infiltration cellular, histiocyte (0/41, 2/47, 7/48, 7/49)
* Lymph node, mediastinal: hyperplasia, lymphoid (0/46, 3/48, 16/49, 18/50); foreign body (0/46, 28/48, 45/49, 44/50); infiltration cellular, histiocyte (0/46, 6/48, 11/49, 16/50)
* Heart: epicardium, inflammation, chronic active (0/50, 2/50, 7/50, 7/50)
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Significantly increased incidences of alveolar/bronchiolar carcinoma and alveolar/bronchiolar adenoma or carcinoma (combined) occurred in all exposed groups of males in the 2-year study; these incidences occurred with a positive trend and exceeded the historical control ranges for inhalation studies and for all routes of administration. The incidences of multiple alveolar/ bronchiolar carcinoma were also significantly increased in exposed male mice. In female mice, incidences of alveolar/bronchiolar adenoma, alveolar/bronchiolar carcinoma, and alveolar/bronchiolar adenoma or carcinoma (combined) were significantly increased in all exposed groups in the 2-year study and exceeded the historical control ranges for inhalation studies and for all routes of administration. The incidences of multiple alveolar/bronchiolar carcinoma were significantly increased in all exposed groups of females.
Incidences of malignant lymphoma occurred with a positive trend in females, and were significantly increased in all exposed groups in the 2-year study.
In the skin, the incidences of fibrous histiocytoma and fibrous histiocytoma or fibrosarcoma (combined) were significantly increased in 30 mg/m3 males in the 2-year study. In females, the incidence of squamous cell carcinoma was slightly increased at 30 mg/m3.

Male rats:
* Lung: alveolar/bronchiolar carcinoma (4/50, 18/50, 20/50, 27/50)
* Skin: fibrous histiocytoma (0/50, 1/50, 1/50, 4/50); fibrous histiocytoma or fibrosarcoma (0/50, 1/50, 3/50, 4/50)


Female rats:
* Lung: alveolar/bronchiolar adenoma (1/50, 10/50, 19/50, 8/50); alveolar/bronchiolar carcinoma (2/50, 14/50, 11/50, 11/50); alveolar/bronchiolar adenoma or carcinoma (3/50, 22/50, 27/50, 18/50)
* All organs: malignant lymphoma (7/50, 17/50, 20/50, 27/50)
* Skin: squamous cell carcinoma (0/50, 0/50, 0/50, 2/50)
Other effects:
effects observed, treatment-related
Description (incidence and severity):
In the lung, incidences of lymphocytic cellular infiltration, chronic active inflammation, pleura fibrosis, pleura inflammation, alveolar epithelium hyperplasia, and bronchiole epithelium hyperplasia were significantly increased in all exposed groups of males and females at the 12-month interim evaluation and in the 2-year study. Incidences of alveolus fibrosis were significantly increased in all exposed groups of males and in 10 and 30 mg/m3 females at the 12-month interim evaluation; incidences of this lesion were significantly increased in all exposed groups of males and females in the 2-year study. Foreign body, presumed to be the test article, was identified in the lungs of all exposed male and female mice in both the 12-month and 2-year evaluations, and was also seen in the nose, larynx, trachea, and bronchial and mediastinal lymph nodes of many exposed male and female mice at each time point.
In the nose, incidences of chronic active inflammation of the respiratory epithelium were significantly increased in 3 and 10 mg/m3 males in the 2-year study. The incidence of squamous metaplasia of the respiratory epithelium was significantly increased in 30 mg/m3 females in the 2-year study.
In the larynx, incidences of respiratory epithelium hyperplasia were significantly increased in 10 and 30 mg/m3 males and 10 mg/m3 females at the 12-month interim evaluation and in 10 and 30 mg/m3 males and females in the 2-year study. Incidences of respiratory epithelium squamous metaplasia were significantly increased in 30 mg/m3 females at the 12-month interim evaluation and in 10 and 30 mg/m3 males and 30 mg/m3 females in the 2-year study. Incidences of squamous epithelium hyperplasia were significantly increased in 30 mg/m3 males and females in the 2-year study.
In the trachea, the incidence of epithelium hyperplasia was significantly increased in 30 mg/m3 males in the 2-year study.
In the hematopoietic system, incidences of lymphoid hyperplasia in the bronchial lymph nodes were significantly increased in all exposed groups of males and females at the 12-month interim evaluation and in the 2-year study. Incidences of this lesion in the mediastinal lymph node were significantly increased in 30 mg/m3 males at the 12-month interim evaluation and in 10 and 30 mg/m3 males and females in the 2-year study. Incidences of this lesion were also significantly increased in the spleen of all exposed groups of females at the 12-month interim evaluation. The incidences of histiocytic cellular infiltration were significantly increased in the bronchial lymph node of 10 mg/m3 females at the 12-month interim evaluation and 30 mg/m3 males and 10 and 30 mg/m3 females in the 2-year study. Incidences of this lesion were also significantly increased in the mediastinal lymph node of 10 and 30 mg/m3 males and all exposed groups of females in the 2-year study. The incidence of hematopoietic cell prolif- eration was significantly increased in the spleen of 30 mg/m3 females in the 2-year study. Incidences of bone marrow hyperplasia, predominantly due to increased myelopoiesis, were significantly increased in all exposed groups of males and in 10 and 30 mg/m3 females in the 2-year study. Incidences of cellular depletion were significantly increased in the thymus of 10 and 30 mg/m3 males and all exposed groups of females in the 2-year study.
In the heart, incidences of chronic active inflammation of the epicardium were significantly increased in 10 and 30 mg/m3 males and females in the 2-year study.
The incidence of chronic active inflammation of the forestomach was significantly increased in 30 mg/m3 males in the 2-year study.

Relevance of carcinogenic effects / potential:
No equivocal findings for male rats, but for female rats, the following skin-related findings are reported:
Skin: squamous cell carcinoma (0/50, 0/50, 0/50, 2/50)

The NTP-study concluded that there was clear evidence of carcinogenic activity to mice (male & female).
Conclusions:
Reduced body weight gain and survival at all treatment levels indicating MTD approached or exceeded. Dose-dependent increase in benign and malignant lung tumors in both sexes. Dose-dependent lymphoma increase (predominantly B cell) especially in female mice. Benign and malignant skin neoplasms were also observed. Significant lung inflammation, fibrosis and abnormal breathing.
Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
GLP compliance:
not specified
Specific details on test material used for the study:
Antimony trioxide was obtained from 3N International, Inc. (Akron, OH), in one lot (3N-06159) that was used in the 2-week and 2-year studies. Identity and purity analyses were conducted by the analytical chemistry laboratories at H&M Analytical Services, Inc. [Allentown, NJ; X-ray diffraction (XRD)], and Chemir Analytical Services, Inc. [Maryland Heights, MO; redox titration and ultraviolet (UV) spectrophotometry], and by the study laboratory at Battelle Toxicology Northwest [Richland, WA; inductively coupled plasma/atomic emission spectroscopy (ICP/AES)] (Appendix H). Reports on analyses performed in support of the antimony trioxide studies are on file at the National Institute of Environmental Health Sciences.
Lot 3N-06159 of the chemical, a finely divided white powder, was identified as antimony trioxide by XRD. The purity of lot 3N-06159 was determined using ICP/AES. In addition, redox titration and UV spectrophotometry of bulk chemical samples determined the oxidation state of antimony in the test article.
The ICP/AES analysis indicated a purity of 101.9% based on a theoretical content of 83.5% antimony in antimony trioxide. Of the 18 minor elements measured by ICP/AES, only arsenic (~0.019%) and lead (~0.016%) were detected above 0.01% relative to antimony trioxide. Redox titration and UV spectroscopy confirmed that antimony was present in the +3 oxidation state, consistent with antimony trioxide. The overall purity of lot 3N-06159 was determined to be greater than 99.9%.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Han [Crl:WI (Han)] rats.
The Wistar Han rat, an outbred rat stock, was selected because it was projected to have a long lifespan, resistance to disease, large litter size, and low neonatal mortality.
Sex:
male/female
Details on test animals and environmental conditions:
Animal care and use are in accordance with the Public Health Service Policy on Humane Care and Use of Animals. All animal studies were conducted in an animal facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. Studies were approved by the Battelle Toxicology Northwest Animal Care and Use Committee and conducted in accordance with all relevant NIH and NTP animal care and use policies and applicable federal, state, and local regulations and guidelines.
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Vehicle:
not specified
Details on exposure:
For the 2-year studies, the generation system used a linear feed device designed and built by Battelle to meter antimony trioxide into a Trost jet mill for aerosolization and particle size reduction. The study laboratory designed the inhalation exposure chambers so that uniform aerosol concentrations could be maintained throughout the chambers with the catch pans in place. The total volume of each chamber was 2.3 m3 with an active mixing volume of 1.7 m3. Tests showed that aerosol concentration could be reliably maintained homogenous within 8% throughout the chambers, provided the aerosol was uniformly mixed before passing through the chamber inlet and provided the test material did not react to a significant extent with animals, animal excrement, or the chamber interior.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of antimony trioxide in the exposure chambers and room air was monitored using three real-time aerosol monitors (RAMs) (MicroDust pro, Casella USA, Amherst, NH).
Duration of treatment / exposure:
6 hours plus T90 (12 minutes) per day
Frequency of treatment:
5 days per week for up to 105 weeks.
Dose / conc.:
0 mg/m³ air
Dose / conc.:
3 mg/m³ air
Dose / conc.:
10 mg/m³ air
Dose / conc.:
30 mg/m³ air
No. of animals per sex per dose:
60 male and 60 female rats
Control animals:
yes, concurrent no treatment
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Exposure-related clinical findings included abnormal breathing, cyanosis, and thinness in males and females. Lung weights were significantly increased in all exposed groups of males and females at the 12-month interim evaluation.
Mortality:
mortality observed, treatment-related
Description (incidence):
Survival of 10 and 30 mg/m3 females was significantly less than that of the chamber control group. The decreased survival in females was attributed primarily to lung proteinosis. In males, the trend towards reduced survival was attributed primarily to lung inflammation and proteinosis.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of 30 mg/m3 males were at least 10% less than those of the chamber control group after week 69 and decreased to 80% of that of the chamber controls by the end of the study. Mean body weights of 3, 10, and 30 mg/m3 females were at least 10% less than those of the chamber control group after weeks 99, 81, and 65, respectively, and those of 10 and 30 mg/m3 females were 20% and 28% less, respectively, than that of the chamber control group by the end of the study.
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
Incidences of retinal atrophy were significantly increased in all exposed groups of females in the 2-year study. Incidences of acute inflammation of the ciliary body of the eye were significantly increased in 30 mg/m3 males and females in the 2-year study.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Male rats:
* Lung: foreign body (1/50, 50/50, 50/50, 50/50); inflammation, chronic active (18/50, 50/50, 50/50, 50/50); alveolus, inflammation, suppurative (0/50, 12/50, 24/50, 28/50); perivascular, infiltration cellular, lymphocyte (3/50, 25/50, 19/50, 9/50); proteinosis (0/50, 47/50, 50/50, 50/50); alveolar epithelium, hyperplasia (4/50, 50/50, 48/50, 49/50); bronchiole, epithelium, hyperplasia (3/50, 34/50, 36/50, 33/50); fibrosis (2/50, 50/50, 49/50, 49/50)
* Adrenal medulla: hyperplasia (1/49, 2/50, 4/49, 8/50)
* Nose: foreign body (0/50, 0/49, 17/50, 40/50); respiratory epithelium, hyperplasia (6/50, 15/49, 13/50, 25/50); respiratory epithelium, metaplasia, squamous (0/50, 0/49, 2/50, 6/50)
* Larynx: foreign body (0/50, 50/50, 50/50, 50/50)
* Trachea: foreign body (0/50, 28/50, 43/50, 48/50)
* Bone marrow: hyperplasia (0/50, 3/50, 4/50, 8/50)
* Lymph node, bronchial: foreign body (0/41, 35/40, 45/48, 42/47); hyperplasia, lymphoid (0/41, 21/40, 29/48, 26/47); pigmentation (1/41, 4/40, 5/48, 10/47)
* Lymph node, mediastinal: foreign body (0/42, 41/45, 41/49, 43/49); hyperplasia, lymphoid (1/42, 24/45, 30/49, 26/49)
* Mediastinum: artery, inflammation, chronic active (0/0, 1/1, 2/2, 10/10)
* Pancreas: artery, inflammation, chronic active (1/50, 0/50, 2/50, 8/50)
* Mesentery: artery, inflammation, chronic active (0/50, 0/50, 0/50, 6/50)
* Lung: artery, inflammation, chronic active (0/50, 0/50, 1/50, 1/50)
* Kidney: renal tubule, accumulation, hyaline droplet (0/50, 1/50, 3/50, 14/50); artery, inflammation, chronic active (0/50, 0/50, 1/50, 4/50)
* Artery (all tissues combined): inflammation, chronic active (1/50, 1/50, 5/50, 16/50)
* Eye: ciliary body, inflammation, acute (0/49, 0/49, 1/50, 6/49)

Female rats:
* Lung: foreign body (0/50, 50/50, 50/50, 50/50); inflammation, chronic active (21/50, 50/50, 50/50, 50/50); alveolus, inflammation, suppurative (0/50, 5/50, 6/50, 5/50); perivascular, infiltration cellular, lymphocyte (0/50, 18/50, 11/50, 8/50); proteinosis (0/50, 50/50, 50/50, 50/50); alveolar epithelium, hyperplasia (5/50, 50/50, 49/50, 50/50); bronchiole, epithelium, hyperplasia (6/50, 26/50, 25/50, 27/50); alveolar epithelium, metaplasia, squamous (0/50, 5/50, 3/50, 1/50); fibrosis (1/50, 50/50, 50/50, 49/50)
* Adrenal medulla: hyperplasia (0/49, 0/49, 3/49, 5/50)
* Nose: foreign body (0/50, 5/50, 26/50, 45/50); respiratory epithelium, hyperplasia (4/50, 6/50, 7/50, 16/50); respiratory epithelium, metaplasia, squamous (0/50, 2/50, 3/50, 5/50)
* Larynx: foreign body (0/50, 50/50, 50/50, 50/50); inflammation, chronic active (0/50, 8/50, 0/50, 3/50)
* Trachea: foreign body (0/50, 39/50, 47/50, 49/50)
* Bone marrow: hyperplasia (8/50, 5/50, 11/50, 20/50)
* Lymph node, bronchial: foreign body (0/35, 35/36, 23/28, 36/41); hyperplasia, lymphoid (0/35, 21/36, 9/28, 11/41)
* Lymph node, mediastinal: foreign body (0/46, 27/46, 32/46, 33/46); hyperplasia, lymphoid (0/46, 14/46, 10/46, 15/46)
* Mediastinum: artery, inflammation, chronic active (0/0, 0/0, 2/2, 9/9)
* Pancreas: artery, inflammation, chronic active (0/50, 0/50, 3/50, 8/50); artery, necrosis (0/50, 0/50, 0/50, 4/50)
* Mesentery: artery, inflammation, chronic active (0/50, 0/50, 0/50, 6/50)
* Lung: artery, inflammation, chronic active (0/50, 0/50, 1/50, 2/50)
* Kidney: renal tubule, accumulation, hyaline droplet (0/50, 0/50, 5/50, 11/50); nephropathy (16/50, 15/50, 20/50, 24/50); artery, inflammation, chronic active (0/50, 0/50, 0/50, 2/50)
* Artery (all tissues combined): inflammation, chronic active (0/50, 0/50, 5/50, 15/50)
* Eye: retina, atrophy (6/49, 21/50, 18/49, 19/49); ciliary body, inflammation, acute (0/49, 0/50, 1/49, 6/49)
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At the 12-month interim evaluation, a single incidence of alveolar/bronchiolar adenoma of the lung occurred in a 30 mg/m3 female. There was a positive trend in the incidences of alveolar/bronchiolar adenoma in females in the 2-year study, and the incidences were significantly increased in females exposed to 10 or 30 mg/m3; 30 mg/m3 females also had one squamous cell carcinoma and two cystic keratinizing epitheliomas. In all exposed groups of males, the incidences of alveolar/bronchiolar adenoma and of alveolar/bronchiolar adenoma or carcinoma (combined) exceeded the historical control ranges for inhalation studies and for all routes of administration.

Male rats:
* Lung: alveolar/bronchiolar adenoma (3/50, 4/50, 6/50, 8/50); alveolar/bronchiolar adenoma or carcinoma (3/50, 4/50, 8/50, 8/50)
* Adrenal medulla: benign pheochromocytoma (1/49, 0/50, 2/49, 7/50)

Female rats:
* Lung: alveolar/bronchiolar adenoma (0/50, 2/50, 6/50, 5/50)
* Adrenal medulla: benign pheochromocytoma (0/49, 2/49, 2/49, 6/50); benign or malignant pheochromocytoma (0/49, 2/49, 2/49, 7/50)
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Incidences of chronic active inflammation, alveolar epithelium hyperplasia, proteinosis, and fibrosis in the lung were significantly increased in all exposed groups of males and females at the 12-month interim evaluation and in the 2-year study. Foreign body, presumed to be the test article, was identified in the larynges and lungs of all exposed male and female rats in both the 12 month and 2 year evaluations, and was also seen in the trachea and bronchial and mediastinal lymph nodes of most exposed male and female rats at each time point. Incidences of lymphocytic perivascular cellular infiltration were significantly increased in 3 and 10 mg/m3 males and females at the 12-month interim evaluation and in 3 and 10 mg/m3 males and all exposed groups of females in the 2-year study. Incidences of bronchiole epithelium hyperplasia were significantly increased in all exposed groups of males at the 12-month interim evaluation and in all exposed groups of males and females in the 2-year study. The incidences of suppurative alveolar inflammation were significantly increased in all exposed groups of males and females in the 2-year study. The incidence of squamous metaplasia of the alveolar epithelium was significantly increased in 3 mg/m3 females in the 2-year study.
In the adrenal medulla, the incidences of benign pheochromocytoma were significantly increased in 30 mg/m3 males and females and the incidence of benign or malignant pheochromocytoma (combined) was significantly increased in 30 mg/m3 females in the 2-year study. Incidences of adrenal medullary hyperplasia occurred with a positive trend in both males and females in the 2-year study, and the incidences were significantly increased in 30 mg/m3 males and females.
In the 2-year study, incidences of respiratory epithelium hyperplasia in the nose were significantly increased in 3 and 30 mg/m3 males and 30 mg/m3 females. The incidences of respiratory epithelium squamous metaplasia in 30 mg/m3 males and females were significantly increased in the 2-year study.
In the 2-year study, the incidence of chronic active inflammation was significantly increased in the larynx of 3 mg/m3 females.
In the bone marrow, incidences of hyperplasia, predominantly due to increased erythroid precursors, were significantly increased in 30 mg/m3 males and females in the 2-year study.
Incidences of lymphoid hyperplasia in the bronchial and mediastinal lymph nodes were significantly increased in 10 mg/m3 males and 3 mg/m3 females at the 12-month interim evaluation and in all exposed groups of males and females in the 2-year study. The incidence of pigmentation was significantly increased in the bronchial lymph nodes of 30 mg/m3 males in the 2-year study.
Chronic active arterial inflammation was observed in multiple tissues in males and females in the 2-year study,including the mediastinum, pancreas, mesentery, lung, and kidney. The combined incidences of chronic active arterial inflammation in all tissues were increased in 10 and 30 mg/m3 males and females. These increases were significant in 30 mg/m3 males and 10 and 30 mg/m3 females.
In the kidney, the incidences of renal tubule hyaline droplet accumulation were significantly increased in 30 mg/m3 males and 10 and 30 mg/m3 females in the 2-year study. The incidence of nephropathy was significantly increased in 30 mg/m3 females in the 2-year study.
Relevance of carcinogenic effects / potential:
No equivocal findings for male rats, but for female rats, the following lung-related findings are reported:
Lung: cystic keratinizing epithelioma or squamous cell carcinoma (0/50, 0/50, 0/50, 3/50)

The NTP-study concluded that here was some evidence of carcinogenic activity in rats (male & female).
Conclusions:
Reduced body weight gain and survival at all treatment levels indicating MTD approached or exceeded. Dose-dependent increase in benign and malignant lung tumors and adrenal pheochromocytomas in both sexes. Significant lung inflammation and fibrosis accompanied by abnormal breathing and cyanosis indicative of hypoxia.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
0.51 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
Test was conducted with diantimony trioxide. A weight of evidence approach is applied to assess the toxicological endpoint. Cf. scientific opinion in Section 13.

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Cf. Scientific opinion on lung toxicity and carcinogenicity in Section 13 for complete weight of evidence and read-across assessments.

 

In order to assess the potential lung toxicity and carcinogenicity of Sb substances, it is important to nature of the effects which have been reported and try to understand the mechanism(s) by which Sb compounds may damage the lung, and the chemical species involved in such a response.

 

Lung toxicity

 

The historical medical surveillance literature, documenting the impacts of inhalation exposure to Sb trioxide in occupational setting, is concordant with the animal studies in that impairments of pulmonary function (e.g. spirometry deficits and radiographic indications of mild pulmonary fibrosis) were associated with occupational exposures experienced prior to the adoption of modern OELs (McCallum, 1967; Potkonjak and Pavlovich, 1983). Although the pulmonary changes associated with occupational exposures were much less severe than those evident in rats and mice, they confirm that the human lung can be adversely impacted by inhalation exposure to Sb trioxide and ore materials containing Sb trisulfide (stibnite). The combined animal and human exposure data support a STOT RE classification for impacts upon the lung after repeated inhalation exposure to Sb trioxide and Sb trisulfide.

 

STOT RE classifications are further assigned either category 1 or category 2 (ECHA, 2017). Category 1 classifications are indicative of high potency for the product of significant to severe health effects whereas a category 2 classification indicates moderate potency to induce significant health effects. Although the ECHA Classification and Labelling guidance indicates that Category 1 designations are often indicated when there is “good quality evidence from human case or epidemiology studies”, it is further noted that “In exceptional cases human evidence can also be used to place a substance in Category 2” (ECHA, 2017). Category assignment of a STOT RE substance thus entails a weight of evidence analysis that results in a classification which accurately conveys both the potency of the substance and the severity of the health effects observed.

 

STOT RE Category 1 designations are triggered by the observation of significant or severe impacts in rats at aerosol concentrations less than 0.02 mg/liter/6h/day (20 mg/m3) in a 90-day exposure study, whereas category 2 is indicated for effects induced between 0.02 and 0.2 mg/liter/6r/day (20 – 200 mg/m3). These values are not intended as strict demarcation points but as general guidance to be used in conjunction with expert judgement. Adjustment of these values in accordance with Haber’s rule is also suggested – thus the demarcation value of 20 mg/m3would be reduced by a factor of at least 4 (to < 5 mg/m3) in comparing results of a 90-day study and to those from a 1 – 2-year inhalation study.

 

The comprehensive two-year inhalation studies of NTP (2017) observed a LOAEL for pulmonary impacts of 3 mg/m3, just below the Haber’s law adjusted Category 1 demarcation value. However, the NTP (2017) studies utilized experimentally generated respirable Sb trioxide aerosols capable of deep lung penetration and deposition. Studies of real-world occupational aerosols indicate that their particle size distribution has a relatively low content of respirable particles. On average, for exposed humans, inhalable aerosols capable of yielding pulmonary deposition fractions comparable to those produced by experimentally generated Sb trioxide aerosols used in rodent inhalation studies would require a 5-fold higher concentration of Sb trioxide in air (Hughson, 2005; Vetter, 2018).

 

Using the Multiple-Path Particle Dosimetry Model (v. 3.01) described by Ashgarian and Price (2009), one can further compare the pulmonary deposition of the experimental aerosols used by NTP (2017), with those of the real-world occupational Sb trioxide aerosols measured by Hughson (2005). Whereas the NTP aerosols (MMAD 1.2 µm +/- 1.9 GSD) would yield a pulmonary deposition rate in rats of 7.6%, the average particle size distribution of the aerosols sampled by Hughson (2005), as calculated by Vetter (2018) had an MMAD of 17.2 µm with a GSD of 2.7. This would yield a pulmonary deposition rate in the rat of 0.3%. In terms of potency for the rat, a real-world Sb trioxide occupational aerosol of 75 mg/m3would be required to produce the pulmonary impacts observed by NTP (2017) at their airborne LOAEL of 3 mg/m3. This total aerosol Sb trioxide concentration is significantly above the 5 mg/m3demarcation point for chronic exposure potency in establishing category 1 vs. category 2 in STOT RE inhalation classifications.

 

Granulometry studies are described in the CSRs for Sb metal powder and Sb trisulfide, and predict the characteristics of the aerosols each would produce. Sb metal powder would be expected to generate an occupational aerosol with an MMAD of 19.05 µm +/- 2.75 GSD. A bimodal distribution is predicted for Sb trisulfide aerosols with 11% of the particle mass having an MMAD of 2.69 µm +/- 2.38 GSD, and 88.7% of the aerosol mass with a MMAD of 28.48 µm +/- 1.56 GSD). MPPD modelling predicts that pulmonary deposition rates of 0.05% and 0.22% would result from aerosols of Sb metal powder and Sb trisulfide, respectively. Pulmonary deposition rates equivalent to those for rats exposed to 3 mg/m3of Sb trioxide in the NTP studies would thus require Sb metal powder aerosols of approximately 100 mg/m3and Sb trisulfide aerosols of 450 mg/m3. Real-world aerosols of Sb trioxide, Sb trisulfide and Sb metal powder would be judged to have a moderate to low potency as pulmonary toxicants when viewed from the perspective of deposition rates in the lung regions that are the targets for pulmonary toxicity.

 

Historical exposures capable of producing human pulmonary impacts after years of chronic exposure, although not precisely defined, were most likely in significant excess of 10 mg/m3(ECHA, 2008). The historical exposure levels associated with changes to lung pathology and function confirm that Sb trioxide has only moderate potency for inducing pulmonary impacts in humans. The nature of the pulmonary alterations associated with exposure of humans to Sb trioxide provides further indications that Sb trioxide has only moderate potency as a pulmonary toxicant.

 

Inhalation of Sb trioxide by rats and mice produced severe impairment of both pulmonary structure and function (NTP, 2017). The severity of the impacts in rodents, contrasts with the observed impacts in humans. Although impacts upon human lung function are judged as clinically significant, the pulmonary function impacts observed are generally mild. The underlying alterations to human lung tissue that mediate these modest functional changes are in turn associated with comparatively modest inflammatory responses and rather benign and generally non-progressive fibrotic changes.

 

The concentrations of Sb trioxide associated with pulmonary toxicity in both humans and rodents indicate moderate potency that is consistent with a STOT RE category 2 classification. The relatively benign and non-progressive nature of the structural alterations documented in workers with high-level historical occupational exposures similarly indicates relatively mild potency consistent with STOT RE category 2 classification for lung toxicity from inhalation exposure. Modelling of the alveolar deposition fractions predicted for rats exposed to aerosols of Sb metal powder and Sb trisulfide further indicates that the potency of these substances could be lower than Sb trioxide and thus also consistent with a category 2 STOT RE classification.

 

(Lung) carcinogenicity

 

As just noted, chronic inhalation of Sb trioxide by rats and mice can produce damage to the lungs characterized by the progressive development of pulmonary inflammation, tissue damage and fibrotic changes. These dose-dependent changes, at sufficiently high exposures, can produce significant impairment of pulmonary function and severe systemic hypoxia that induces adaptive physiological changes (e.g. erythroid hyperplasia).

 

Activation and alteration of oncogenes

 

NTPs (2017) analysis of mouse and rat lung tumors extended to an evaluation of oncogene alterations associated with tumor formation. The presence of activated oncogenes in tumors is informative but can be the result of a myriad of direct and indirect processes. Focusing on the mouse lung tumors, which were observed with far higher frequency, permits more robust analysis of the “molecular pathology” responsible for activated oncogenes in spontaneous and induced neoplasms. Spontaneous lung tumors were found to contain altered Kras genes with the activating mutations generally mapping to established “hot spots” (i.e. G to A transitions in codon 12). Altered Kras oncogenes were detected in 43% of the tumors observed in Sb trioxide treated animals. NTP notes that tumors in Sb trioxide treated animals possessed base sequence changes in hot spots similar to those observed in spontaneous tumors, and suggests that the Kras altered genes observed in the tumors of Sb trioxide treated animals were the result of spontaneous lesions, permitted to undergo clonal expansion by the pulmonary toxicity of Sb trioxide. This suggestion is consistent with the observation that spontaneous activated oncogenes are now known to be present in the normal tissues of animals used in cancer bioassays (Parsonset al., 2009), exhibiting both tissue and animal strain specificity with respect to the prevalence of different activated oncogenes.

 

In addition to Kras alterations, 46% of lung tumors in Sb trioxide treated mice were observed to contain altered Egfr oncogenes. The high prevalence of tumors with Egfr alterations in exposed animals could be interpreted as evidence of mutagenic oncogene alterations induced by Sb trioxide. However, the origin of Egfr alterations is potentially more complex than is described. In humans, lung cancer tumors are increased in subjects with disease syndromes (e.g. chronic obstructive pulmonary disease) that impair lung function and lead to hypoxic conditions. Signaling pathways involving EGFR appear to play a role in the growth of such tumors under hypoxic condition (Karooret al., 2012). Egfr alterations are further linked to the ability of cancer cells to survive in hypoxic microenvironments (Murakami et al., 2014). The prevalence of Egfr alterations in Sb trioxide treated animals may thus be a result of selection for tumors capable of undergoing rapid clonal expansion, under the hypoxic conditions associated with the pulmonary toxicity produced by Sb trioxide. The activated Egfr oncogenes may thus be spontaneous in origin or produced by a variety of indirect processes during tumor progression (e.g. ROS generation, error prone DNA repair) with an increased prevalence in tumors that is more indicative of the conditions that permitted clonal expansion of neoplastic lesions. The mere observation of an activated oncogene in a tumor, in and of itself, confers little information that permits determination of the mechanism(s) that may have produced it.

 

These oncogene structural alterations do not represent the only potential means by which Sb trioxide might influence the expression of oncogenes or other cellular constituents. The electrophilic nature of Sb is such that binding to a variety of cellular macromolecules occurs (Verdugo et al., 2017) and could facilitate neoplastic development. For example, although Sb has not been linked to prostate cancer, Sb 3+ ions will activate signaling pathways that stabilizes the c-myc oncogene that stimulates cell proliferation (Zhang et al., 2018).

 

Whereas NTP suggests that many of the lung tumors in mice originate from cells with spontaneous Kras oncogene activation that are permitted to undergo clonal expansion in response to the pulmonary toxicity induced by Sb trioxide, mouse lung tumors with Egfr lesions may similarly reflect selection for, and clonal expansion of, cells with enhanced proliferative capacity under hypoxic conditions. It is not possible to ascertain whether Egfr alterations are spontaneous or induced. The etiology of Kras and Egfr oncogene alterations observed in lung tumors merits investigation to determine if they are pre-existing spontaneous lesions, lesions induced by Sb via indirect mechanisms of genotoxicity and/or lesions selected for clonal expansion as a consequence of pulmonary toxicity and hypoxia.

 

Body weight suppression

 

The impacts of Sb trioxide exposure upon the overall health status of rats and mice should not be neglected, and may explain other adverse effects observed in the NTP studies. Exposure of rats to 3, 10 or 30 mg/m³ Sb trioxide was associated with end of study body weight suppression of 7, 8 and 20% in male rats; and 10, 20 and 28% in female rats, respectively. Corresponding body weight suppression in male mice was 8, 11 and 25%; and 3, 8 and 21% in female mice. Much of the data generated by the NTP bioassays reflects effects near, or in excess of, the maximum tolerated dose for Sb trioxide. This conclusion is bolstered by the observations of labored breathing, hypoxia and premature mortality due to pulmonary inflammation in exposed animals. These observations do not negate the induction of pulmonary lesions, but indicate that care must be exercised in the interpretation of systemic effects that might be associated with inhalation exposure to Sb trioxide.

 

Pheochromocytomas

 

Adrenal gland neoplasms (pheochromocytomas) are cited by the US NTP Monograph (p. 65 – 66) as supporting classification of Sb trioxide as a carcinogen. However, adrenal gland neoplasms (pheochromocytomas) lesions are expected to develop under conditions of pulmonary inflammation and hypoxia. As reviewed by Greimet al.(2009), the association of this adrenal lesion with pulmonary impairment is sufficiently robust that, within the context of the EU REACH process, pheochromocytomas secondary to pulmonary impairment are not considered as relevant for cancer classification or risk assessment. The adrenal lesions are a response to pulmonary damage induced by Sb trioxide and not a direct substance-specific effect of Sb trioxide. Indeed, they can be interpreted as confirmation that maximum tolerated doses have been exceeded in the rat.

 

Lymphomas

 

Sb trioxide exposures in mice were also associated with an increase in lymphomas. Interpretation of increased lymphoma incidence in female mice poses diagnostic challenges that were not addressed by NTP’s histopathological analysis. Whereas lymphomas induced by chemicals are usually T cell in origin (Ward, 2005), those associated with Sb trioxide exposure were predominantly B-cell or mixed B- and T-cell in origin, and many appeared to be reactive lesions responding to Sb trioxide lung toxicity. Mouse B-cell lymphomas are further difficult to interpret due to their high spontaneous incidence and complex etiology that likely includes endogenous retrovirus activity. In NTP inhalation studies, the average historical control incidence of lymphomas in B6C3F1 female mice is 25.2% (range 14 – 36%). Thus, lymphoma incidence at 10 and 30 mg/m3Sb trioxide, but not 3 mg/m3,was significantly elevated over historical controls. The complex and diverse mechanisms for B-cell lymphoma induction have prompted the development of histopathological diagnosis and classification strategies to distinguish between spontaneous and induced lesions (Ward, 2005). Unfortunately, none of these diagnostic criteria were applied in the NTP study. Based upon the limited data provided, the excess lymphomas associated with Sb trioxide exposure appear to be similar to the naturally occurring lesions in the B6C3F1 mouse; it can be plausibly postulated that the chronic inflammation and hypoxic conditions in the Sb trioxide exposed lung produced adaptive responses in the lung and spleen that promoted the development of what is already a high incidence spontaneous neoplasm in the female mouse. As such, the increased incidence of lymphomas would not provide clear evidence of carcinogenicity.

 

Skin lesions

 

Neoplastic skin lesions were also observed in mice exposed to Sb trioxide and different types of skin lesions were pooled to yield statistical significance. Given the high-level whole-body inhalation exposures employed by NTP, the appearance of histiocytomas (a benign skin lesion) is mostly likely an immunological response, as opposed to neoplastic response, and not a precursor lesion to fibrosarcoma (malignant tumors of fibrous tissues). Histiocytomas are not generally known to be precursor lesions to fibrosarcoma and there appears to be no legitimate scientific rationale to support data pooling. The observation of two squamous cell carcinomas in Sb trioxide treated female mice is unusual but is similarly difficult to interpret in the absence of preneoplastic precursor lesions. Moreover, no other study has suggested skin as a target organ for Sb trioxide carcinogenesis. There is no legitimate scientific rationale to support that skin tumors are induced by Sb trioxide.

 

Lung cancer

 

According to the experimental studies, Sb compounds might pose a carcinogenic risk to the lungs of rats through particle overload (Newton et al., 1994; Schroeder, 2003). Rat’s lungs do not have the capacity to remove excessive quantity of respirable particle and this triggers a cascade of inflammatory responses leading to a tumor formation, by accumulation of inert particles. This response to inflammation from particle overload is not observed in mice or humans. There is no statistically significant increase in rat lung tumors at Sb trioxide concentrations (3 mg/m3) that do not produce pulmonary overload. Rat lung tumor incidence at higher exposure levels is low, lacking in dose-response and most likely the result of pulmonary overload. As such, the rat pulmonary lesions are not reflective of human risk. Therefore, rat lung tumors, if induced by particle overload, would be of questionable significance for hazard classification or risk assessment.

 

NTP (2017) concluded that overload did not occur in rats at an Sb trioxide exposure of 3 mg/m³ and therefore that pulmonary overload is not required for the induction of lung neoplasms in the rat lung. The rationale for this conclusion is tenuous in that 3 mg/m³ is indeed associated with impaired clearance in the rat in the NTP studies – the departure from modeled clearance rates is just not sufficient to attain the lung burden levels that meet an arbitrary criterion for overload. Moreover, significant impairment of clearance has been reported at levels much lower than those used in the NTP studies (e.g. Newtonet al., 1994). Finally, the incidence of lung neoplasms in both male and female rats is not statistically elevated over that in controls at 3 mg/m³ Sb trioxide exposures. The lack of both overload and a carcinogenic response in the rat at 3 mg/m³ Sb trioxide cannot be taken as an indication that tumors produced in the rat lung at higher levels of exposure were not the result of the pulmonary overload. Particle overload and the subsequent cascade of inflammatory responses leading to a tumor formation can be retained as a possible mode of action for lung cancer in rats, but would be of questionable relevance for hazard classification or risk assessment.

 

As discussed in the scientific opinion on genotoxicity, any Sb genotoxicity that might facilitate neoplastic development is likely mediated by indirect mechanisms, such as induction of oxidative stress or interference with DNA repair processes. The available data do not permit discrimination between alternative mechanisms, nor do the mechanisms need to be mutually exclusive, butthere is relatively high confidence that the lung carcinogenicity is not a result of direct genotoxicity of Sb. Excess tumors observed may reflect the clonal expansion of pre-existing preneoplastic cells with activated oncogenes in the absence of genotoxicity (direct or indirect). If lesions are induced, it is most likely via a local indirect genotoxic mode of action. The most probable indirect modes of action (e.g. overload in the rat, inflammation and ROS generation in the mouse) would be expected to exhibit effect thresholds that produce neoplastic response only above a given level of inhalation exposure.

 

Sb trioxide appears to induce cancer at tissue sites (adrenal, lymphoma and skin) that are likely side-effects of pulmonary toxicity or the irritant properties of Sb trioxide. These lesions are not relevant to an evaluation of the carcinogenic properties of Sb trioxide. The primary target organ of inhaled Sb substances appears to be the lung, and mode of action considerations should look at local effects in the lung rather than systemic effects. The inhalation exposure route is the only route of exposure relevant for the assessment of carcinogenicity properties. Exposure route specificity (the lung by inhalation exposure) is further evidenced by lack of pulmonary changes after sub-chronic oral exposures to high doses of Sb trioxide (Hext et al., 1999) and high sub-chronic i.p. dosing with the Sb (III) potassium tartrate (Dieter, 1992).

 

Data from experimental animal studies do not yield compelling evidence of cancer risk at exposure levels, or via mechanisms, that are likely to be relevant to present occupational or consumer exposure scenarios. Epidemiological studies have failed to demonstrate elevated cancer risk that can be attributed to Sb trioxide exposure. According to the ECHA Guidance on the Application of CLP criteria, the present evidence satisfies, and likely exceeds, that required for a Category 2 cancer via inhalation classification. Indeed, according to the ECHA Guidance on the Application of the CLP Criteria (July 2017), suspected human carcinogens are those for which the evidence obtained from human and/or animal studies is not sufficiently convincing to place the substance in Category 1A or 1B, in particular, when e.g. the data suggest a carcinogenic effect but are limited for making a definitive evaluation because there are unresolved questions regarding the adequacy of the interpretation of the results of the studies. In light of the discussion presented above, there are clear interpretation issues which do not permit to conclude on a category 1 carcinogenicity, and rather suggest maintaining the current Category 2 cancer classification:

·       Tumour background incidence - comparison of the tumour incidence with historical control tumour data. This can be particularly relevant for animal strains which have a propensity to develop a particular type of tumour spontaneously with variable and potentially high incidence. In such a case, the tumour incidence may not be providing reliable evidence of treatment related carcinogenicity;

·       The possibility of a confounding effect of excessive toxicity at test doses. In lifetime bioassays, compounds are routinely tested using at least three dose levels, of which the highest dose needs to induce minimal toxicity, such as characterized by an approximately 10% reduction in body weight gain (maximal tolerated dose, MTD dose). The MTD is the highest dose of the test agent during the bioassay that can be predicted not to alter the animal’s normal longevity from effects other than carcinogenicity. If a test compound is only found to be carcinogenic at the highest dose(s) used in a lifetime bioassay, and the characteristics associated with doses exceeding the MTD are present, this could be an indication of a confounding effect of excessive toxicity/excessive loading. This may support a classification of the test compound in Category 2 or no classification; and

Mode of action and its relevance for humans, such as cytotoxicity with growth stimulation, mitogenesis, immunosuppression, mutagenicity. The various international documents on carcinogen assessment all note that mode of action in and of itself, or consideration of comparative metabolism, should be evaluated on a case-by-case basis and are part of an analytic evaluative approach. One must look closely at any mode of action in animal experiments taking into consideration comparative toxicokinetics/toxicodynamics between the animal test species and humans to determine the relevance of the results to humans. The criteria for a Carcinogenicity Category 1B classification are not met due to the uncertain relevance of both rat and mouse lung tumors for humans.

Additional information

Upon dissolution in aqueous media at physiologically relevant concentrations and pH conditions, the only aqueous antimony species emerging from all considered trivalent antimony substances is the trivalent antimony cation. In vitro bioaccessibility testing in various artificial body fluids (Hedberg et al., 2010) has shown that diantimony tris(ethylene glycolate) compared to diantimony trioxide has a lower release rate of antimony ions, thus read-across warrants an intrinsic conservatism.

With respect to systemic toxicity, read-across from diantimony trioxide toward diantimony tris(ethylene glycolate) is justified. 

Please also refer to the study results presented in section 4.8 and 7.1.1 of the technical dossier (IUCLID) and in section 1.3 and 5.1.1 of the CSR.

The following conclusions can be drawn for diantimony trioxide and by read across, also for diantimony tris(ethylene glycolate):

Considering the available genotoxicity data, antimony trioxide does not induce gene mutations in vitro, but has been shown to induce structural chromosome aberrations in cultured mammalian cells in vitro. However, negative in vivo results on induction of chromosome aberrations and/or micronuclei were obtained in two different species – mouse (Elliot et al., 1998) and rat (Covance Laboratories Ltd, 2005), (Kirkland et al., 2007). An in vivo UDS assay in rats was also negative (Elliot et al., 1998). These tests were performed according to GLP and using OECD test guideline protocols and oral administration. According to a recent guideline-conform toxicokinetic study conducted under GLP at a dose of 1000 mg/kg as used in these mutagenicity studies, tissue distribution data confirmed that the bone marrow was exposed. It can therefore be concluded that diantimony trioxide does not cause systemic mutagenicity in vivo after oral administration.

Three chronic inhalation studies in rats are available for carcinogenicity assessment of diantimony trioxide (Watt, 1983; Groth et al., 1986a, Newton et al., 1994). Two animal studies indicate neoplastic properties of diantimony trioxide, whereas one animal study showed negative results. There is also one human study available (Jones, 1994). However, due to lack of exposure data the human study is regarded inconclusive. The exposure duration in all three animal studies is 12 months and thus all studies deviates from the OECD guideline on chronic toxicity/carcinogenicity, which prescribes an exposure period of 24 months for rats. In the first animal study (Watt, 1983) inhalation of 5.0 mg Sb2O3/m³ for 12 months produced lung neoplasms in 44% of the animals tested (only females were exposed). In the second study, (Groth et al.,1986a) a 9 times higher dose (45 mg Sb2O3/m³) produced pulmonary neoplasms in 32% of the female rats exposed under similar conditions, but none in male rats. It is noted that the female survival rate was significantly higher than the male counterparts in the study by Groth et al., (1986a). The differences in incidence between the studies might be explained by a longer observation period (12 months vs 20 weeks) and by the use of older animals (8 months vs 14 weeks) in the study by Watt (1983). The study by Newton et al., (1994) showed no diantimony trioxide-related lung tumours, neither in males nor females, at any dose level up to 4.5 mg/m³. This is in contrast with the data reported by Watt and Groth and the cause to the difference is not entirely clear. However, the histopathology slides from the negative study were re-evaluated by the pathologist who evaluated the slides from the Groth and of the Watt studies. The re-examination confirmed a lack of antimony trioxide-related neoplastic changes in the study. In addition, the comparison of the Watt and the Newton studies, which were conducted at similar exposure levels, showed that the exposed rats had more lung damage and appeared to have considerably more antimony deposited in the lungs in the Watt study than in the Newton et al.study. This may suggest that the exposure levels in the Watt study may have been above those reported. Given that the dose level in the study by Groth is 10 times higher and also the dose levels in the study by Watt were likely higher than 1.9 and 5.0 mg/m³ the dose levels in the study most probably fit in the dose range where no tumours were observed. However, the difference could also be due to different particle generation techniques or different strains of rats. The particle size, which will affect lung deposition, clearance and retention and hence target organ dose, was similar among the studies although they were all measured using different techniques. In the study by Newton and co-workers it was shown that diantimony trioxide reduced the pulmonary clearance rate in a dose dependent manner, interpreted by the authors as a toxic effect of diantimony trioxide rather than a general effect due to pulmonary overload. However, it is well known that reduced lung clearance rate at chronic exposure of rats to poorly soluble particles (PSPs) can result in pulmonary overload, subsequently followed by an inflammatory response, epithelial cell hypertrophy and/or hyperplasia and squamous metaplasia. The persistence of these tissue responses over chronic time periods can lead to secondary development of lung tumours (Hext, 1994). Thus, it could be speculated that the neoplastic effects seen in the Watt and Groth studies is a result of pulmonary overload and an inflammatory response to particulate diantimony trioxide. The tumour development as a consequence of pulmonary overload is an inflammatory-driven process which usually takes over a year (15-18 months) of PSP exposure via inhalation (Driscollet al., 1997). In the present studies on antimony trioxide, development of lung tumours occurred earlier – already at 12 months of antimony inhalation.

Due to deviations from the OECD guidelines and the critical shortcoming in all three studies, US NTP (National Toxicology Program) has embarked on a testing programme leading to a new, full 2 -year bioassay. A 14d range-finder on rats and mice was already conducted at the end of 2007 and preliminary reporting was already conducted and will be further evaluated for inclusion into the REACH dataset for ATO. The chronic toxicity studies in both rats and mice have already started; Pathology Quality Assessment is in progress and reporting is expected 2014 -2015. 

The overall expert judgement by TC NES is that the most likely mechanism for carcinogenicity appears to be impaired lung clearance and particle overload followed by an inflammatory response, fibrosis and tumours. Consequently, diantimony trioxide can be regarded as a threshold carcinogen and as a starting point for a quantitative risk characterisation the NOAEC of 0.51mg/m³ derived for local repeated dose toxicity is also used for carcinogenicity. However, in this context, it is questionable whether effects caused by pulmonary overload in the rat are also relevant for humans. Positive (Hext, 1994), (Oberdorster, 1995) and negative (Tran and Buchanan, 2000; Kuempel et al., 2001) findings of particle overload in human lungs are reported. Macrophage transport of particles into the alveolar interstitium is the major clearance mechanism in humans but of minor importance to the rat. These species differences are related to morphological features of the lung, i. e. to the relative short pathway length from the alveoli to the ciliated terminal bronchioles in rats (Bailey et al., 1989; Kreyling, 1990; Kreyling et al., 1991). In the absence of mechanistic data to the contrary, it must be assumed that the rat model of tumorigenicity can identify potential carcinogenic hazards to humans and the rat presently remains the appropriate model for both neoplastic and non-neoplastic responses to PSP exposure (ILSI Risk Science Institute Workshop Participants., 2000).

Justification for selection of carcinogenicity via inhalation route endpoint:

Key study

Carcinogenicity: via inhalation route (target organ): respiratory: lung

Justification for classification or non-classification

Cf. Scientific opinion on lung toxicity and carcinogenicity in Section 13 for complete weight of evidence and read-across assessments.

 

The combined animal and human exposure data support a Carcinogenicity category 2 via inhalation classification for Sb trioxide. Based on physical form/particle size, water solubility, and Sb speciation/valency, the same classification can be applied to Sb metal and Sb trisulfide. Sb trichloride and Sb tris (ethylene glycolate) do not satisfy the criteria to be grouped with Sb metal, Sb trioxude and Sb trisulfide for purpose of lung carcinogenicity classification, and are not classified for carcinogenicity.

 

The main uncertainty underpinning this pertains to the mode of action related to the carcinogenicity, but the conclusion can be established (based on human exposure evidence), with a relatively high level of confidence, but the following research options could reinforce the classification justification:

·       Determine whether the Kras and Egfr oncogene alterations observed in lung tumors are pre-existing spontaneous lesions, lesions induced by Sb via indirect mechanisms of genotoxicity, and/or lesions selected for clonal expansion as a consequence of pulmonary toxicity and hypoxia;

·       Clarify the (local) genotoxicity of Sb substances (cf. scientific opinion on genotoxicity);

·       Run one or more in vivo inhalation study to confirm the observations gathered so far.

 

The research strategy developed by the International Antimony Association, which supports REACH registrants with their Registration and Evaluation obligations, already foresees the above research options.