Indium phosphide

Carcinogenicity

Description of key information

 Two GLP compliant studies assessing the carcinogenicity of indium phosphide were conducted using a method similar to the OECD TG 453 (with deviations). Groups of 60 B6C3F1 mice and F344/N rats per sex per dose were exposed to particulate aerosols of indium phosphide at concentrations of 0, 0.03, 0.1, or 0.3 mg/ m3, 6 hours per day, 5 days per week, for 21/22 (mice and rats respectively) weeks at doses of 0.1 and 0.3 mg/ m3 or 105 weeks at doses of 0 and 0.03 mg/ m3 groups. No dose-effect correlation could be made due to the exposure groups 0.3 and 0.1mg/m3 being interrupted after 21/22 weeks. Evidence of the carcinogenic activity of indium phosphide was established under the conditions of these studies. Evidence was based on the increased incidences of benign and malignant neoplasms of the lung in male and female rats and female mice. Male mice exhibited increased incidences of malignant neoplasms of the lung and benign and malignant neoplasms of the liver. Female mice also presented increased incidences of neoplasms in the liver which were postulated to be exposure related. In male mice increases in adenomas and carcinomas of the small intestine were thought to have been related to exposure of indium phosphide. In rats, marginal increases in the incidences of mononuclear cell leukaemia in both sexes, skin fibroma in males and mammary gland carcinoma in females were also assumed to be exposure associated. Inhalation exposure to indium phosphide also resulted in nonneoplastic lesions in the lungs of male and female both mice and rats, the adrenal medulla of male and female rats and the liver and heart of male and female mice. Based on the results of these studies a LOAEC of 0.03mg/m3 for local and systemic effects was determined. It was concluded that Indium Phosphide was carcinogenic under the conditions of these studies.

Key value for chemical safety assessment

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 1996 - January 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

Deviations compared to OECD TG 453 regarding organs investigated and lack of some examinations on animals exposed for 105 weeks.

GLP compliance:

yes

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms (Germantown,NY)
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 6 weeks
- Weight at study initiation: male: average =115 (g) female: average= 96.0(g)
- Housing: Individually in stainless steel wire bottom cages.
- Diet: NTP-2000 pelleted diet ad libitum (except during exposure periods)
- Water: Softened tap water ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24° +/- 3
- Humidity (%): 55% +/-15%
- Air changes (per hr): Chamber air changes 15/hr
- Photoperiod (hrs light): 12 hours per day

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Remarks:

Micronized Indium Phosphide

Mass median aerodynamic diameter (MMAD):

ca. 1.2 µm

Geometric standard deviation (GSD):

1.8

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless-steel inhalation exposure chambers (Harford Systems Division of Lab Products, Inc., Aberdeen, MD)
- Source and rate of air: Compressed air
- Method of conditioning air: Aerosol leaving the generator passed through a corona discharge air-ionizing neutralizer into the distribution line. At each chamber location, a pneumatic injector drew aerosol from the distribution line into the chamber inlet, the aerosol was further diluted with HEPA-filtered air to the appropriate concentration.
- System of generating particulates/aerosols:Aerosol generator consisting of a drum, body, and cap. The drum rotated at 60° increments controlled by a compressed-air-driven valve driver. As the drum rotated, indium phosphide filled six metering ports in a disk at the bottom of the drum and was held in each port by a stainless-steel screen. The metering ports aligned with a nitrogen inlet in the body and dispersed indium phosphide when a nitrogen solenoid valve was opened.
- Temperature, humidity, pressure in air chamber: Temperature (°C): 24° +/- 3 Humidity (%):55% +/-15% Pressure: Not Reported
- Air change rate: 15 air changes per hour
- Method of particle size determination: Mercer-style seven-stage impactor.Stages analyzed by inductively coupled plasma/mass spectroscopy (ICP/MS). Relative mass collected on each stage was analyzed by probit analysis. The mass median aerodynamic particle diameter and the geometric standard deviation of each set of samples were estimated.

TEST ATMOSPHERE
- Brief description of analytical method used: Mercer-style seven-stage impactor (In-Tox Products, Albuquerque, NM). The stages (glass coverslips lightly sprayed with silicone) were analyzed by inductively coupled plasma/mass spectroscopy (ICP/MS).
- Samples taken from breathing zone: Uniformity evaluated every 2 to 4 months. Measurements taken from 12 different chamber positions.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber aerosol concentrations were monitored with real-time aerosol monitors using a pulsed-light-emitting diode in combination with a silicon detector to sense light scattered over a forward angular range of 45° to 95° by particles traversing the sensing volume. The particle size distribution in each chamber was determined during the prestudy testing, during the first week of the studies, and monthly thereafter using a Mercer-style seven-stage impactor (In-Tox Products, Albuquerque, NM). The stages (glass coverslips lightly sprayed with silicone) were analyzed by inductively coupled plasma/mass spectroscopy (ICP/MS). The relative mass collected on each stage was analyzed by probit analysis. The mass median aerodynamic particle diameter and the geometric standard deviation of each set of samples were estimated.

Duration of treatment / exposure:

22 weeks or 105 weeks

Frequency of treatment:

6 hours plus T90 per day, 5 days per week for 22 weeks (0.1 and 0.3 mg/m3 groups) or 105 weeks (0 and 0.03 mg/m3 groups)

Post exposure period:

approx. 82 weeks for 0.1 and 0.3 mg/m3 groups

Dose / conc.:

0.03 mg/m³ air

Dose / conc.:

0.1 mg/m³ air

Dose / conc.:

0.3 mg/m³ air

Dose / conc.:

0 mg/m³ air

No. of animals per sex per dose:

60 animals/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on the results of a 14-week inhalation repeated dose toxicity study performed before hand
- Rationale for animal assignment : Random
- Rationale for selecting satellite groups: Tissue burden analysis were performed on 20 male rats-no justification provided
- Post-exposure recovery period in satellite groups: 16 weeks

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Every 4 weeks from week 4 to week 92 (except between weeks 44 and 52), and every 2 weeks thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: every 4 weeks from week 4 through 92 (except between weeks 44 and 52), and every 2 weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

FOOD EFFICIENCY:No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3 Month interim evaluation
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: Not specified
- How many animals: 17 males/ 18 Females
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 3 Month interim evaluation
- Animals fasted: Not specified
- How many animals:17 males/ 18 Females
- Parameters checked in table [No.2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: See Table No. 2
HISTOPATHOLOGY: Yes: See Table No. 2

Statistics:

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958).The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence.Two approaches were employed to assess the significanceof pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971,1972).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings were generally observed after 18 months and included lethargy in 0.03 mg/m3 males and females and 0.3 mg/m3 males, and abnormal breathing in all exposed groups of males.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

Survival rates of males and females were similar to those of the controls.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights of exposed males and females were similar to those of the chamber controls throughout the study.

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:

effects observed, treatment-related

Description (incidence and severity):

At the 3-month interim evaluation, all groups of exposed male and female rats demonstrated an exposure concentration-related increased erythron evidenced by increased hematocrit values, hemoglobin concentrations, and erythrocyte counts.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Slight increase in serum iron concentrations in 0.1 and 0.3 mg/m3 males. No changes in iron-binding capacity or alterations in iron concentration occurred in exposed females.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 3 months, lung weights of males exposed to 0.1 or 0.3 mg/m3 and of all groups of exposed females were significantly greater (1.2-to 2.1-fold) than those of the chamber controls and increased with increasing exposure concentration and duration of exposure. Following exposure termination, lung weights in the 0.1 and 0.3 mg/m3 groups remained significantly greater than those of the chamber controls.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A broad spectrum of inflammatory and proliferative (neoplastic and nonneoplastic) lesions occurred in the lungs of exposed rats. Proliferative lesions included alveolar/bronchiolar neoplasms and squamous cell carcinomas as well as alveolar epithelial hyperplasia and atypical hyperplasia. The majority of exposed males and females exhibited increased occurrences of chronic inflammation, foreign body (indium phosphide particles) within the alveoli and phagocytic inflammatory cells, and alveolar proteinosis. An increase in the quantities of foreign body in the bronchial and mediastinal lymph nodes of exposed males and females was also recorded and found to be due to the increase of the exposure concentration. The severity of lesions was proportionate to the increase in exposure concentration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the 0.03 and 0.1 mg/m3 animals at 3 months, alveolar proteinosis was typically in areas of inflammation. The lesions were comparably severe in both male and female rats in the 0.03 and 0.3 mg/m3 groups and less severe in the 0.1 mg/m3 groups. Analysis performed on lungs of animals designated for tissue burden studies showed that lesions progressed in the 0.03 mg/m3 (continuously exposed) group while severities of lung lesions in the 0.1 and 0.3 mg/m3 groups (stopped exposure) groups was comparable.
At 2 years, non-neoplastic lesions of the lung included atypical hyperplasia, chronic active inflammation, alveolar epithelial metaplasia, foreign body, alveolar proteinosis, and interstitial fibrosis, and the incidences were significantly increased in all exposed groups.
Bronchial lymph node hyperplasia was increased in all exposed groups foreign body and were observed in 38% to 82% of rats.
The incidences and severities of alveolar epithelial hyperplasia were increased in 0.1 and 0.3 mg/m3 males and females. Furthermore, the incidence of squamous cysts was significantly increased in 0.3 mg/m3 females.
At the end of the study, the incidence of pars distalis hyperplasia was significantly increased in males exposed to 0.3 mg/m3. The increased incidence of hyperplasia was not supported by an increased incidence of pituitary gland neoplasms. Therefore, the increased incidence of hyperplasia was not considered to be related to exposure to indium phosphide.
At 2 years, hyperplasia in the adrenal medulla were significantly increased in females exposed to 0.03 or 0.3 mg/m3 but were not significantly increased in exposed males.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Lung: At 2 years alveolar/bronchiolar adenoma in all groups of the exposed female rats and in male rats exposed to 0.1 or 0.3 mg/m3 were significantly greater than those in chamber controls. Alveolar/bronchiolar carcinoma were also significantly increased in all groups of exposed males and in females exposed to 0.3 mg/m3. The incidences of alveolar/bronchiolar adenoma or carcinoma (combined) were augmented in all groups of males and in females exposed to 0.03 or 0.3 mg/m3. Squamous cell carcinoma of the lung occurred in four male rats exposed to 0.3 mg/m3 at the end of the study. The incidence exceeded the historical control range for controls given and was considered to be exposure related. Squamous cell carcinomas were observed in four males exposed to 0.3 mg/m3.

Adrenal Medulla: At 2 years, increased incidences of benign pheochromocytoma and benign or malignant pheochromocytoma (combined) in males exposed to 0.03 mg/m3 and in males and females exposed to 0.3 mg/m3 were observed. There were increased incidences of bilateral pheochromocytomas in all exposed groups of males, while only two were observed in 0.3 mg/m3 females and none were observed in the chamber control group. The occurrences of malignant pheochromocytomas in males exposed to 0.03 or 0.1 mg/m3 exceeded the historical control range for 2-year studies. The incidence of benign or malignant pheochromocytoma (combined) in 0.03 mg/m3 males and the incidences of benign pheochromocytoma and benign or malignant pheochromocytoma (combined) in 0.3 mg/m3 females exceeded the historical control ranges for 2-year studies. The increased incidences of neoplasms and nonneoplastic lesions in the adrenal medulla were considered to be exposure related.

Mammary Gland: mammary gland carcinoma in females exposed to 0.03 mg/m3 at 2 years were observed. There was no increase in mammary gland carcinoma in females exposed to 0.1 or 0.3 mg/m3. A single carcinoma occurred in each of the 0.1 and 0.3 mg/m3 groups of males. The carcinomas were typical of those observed spontaneously.

Skin: Incidences of fibroma and fibroma or fibrosarcoma (combined) were slightly increased in male rats exposed to 0.1 mg/m3 after 2 years. Increases were not observed in the 0.03 or 0.3 mg/m3 groups. It is uncertain if the occurrence of skin neoplasms was exposure related.

Mononuclear Cell Leukemia: Increased in 0.1 mg/m3 males and 0.3 mg/m3 females at 2 years. Because increased incidences occurred in both males and females, the increased incidences were thought to be exposure related by the authors of the study.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Tissue burden analyses were performed on male rats during exposure and post exposure periods and on female rats following 22 weeks of exposure. Lung burdens increased with time and exposure concentration in all exposed groups.
Lung burden data for the 0.03 mg/m3 group increased linearly over time, demonstrating a very low clearance rate. The lung deposition and clearance model were used to estimate the total amount of indium deposited in the lung after exposure to 0.03 mg/m3 for 2 years or to 0.1 or 0.3 mg/m3 for 22 weeks. The 0.1 mg/m3 group received less total exposure than the continuously exposed 0.03 mg/m3 group or the 0.3 mg/m3 group exposed for 22 weeks. The concentrations of indium in serum were low relative to those measured in the lung. Serum indium concentrations increased in proportion to concentration and duration of exposure. Although there was some decline in serum concentrations after termination of exposure for the 0.1 and 0.3 mg/m3 groups, there was no consistent evidence of elimination of indium from serum, which is consistent with the continuing slow elimination of indium from the lungs.

Relevance of carcinogenic effects / potential:

Clear evidence of carcinogenic activity of indium phosphide has been shown in this study. F344/N rats exhibited increased incidences of benign and malignant neoplasms of the lung, pheochromocytoma of the adrenal medulla and increases in mononuclear cell leukemia in males and females, fibroma of the skin in males, and carcinoma of the mammary gland in females.

Key result

Dose descriptor:

LOAEC

Effect level:

0.03 mg/m³ air

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEC

Effect level:

0.03 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

clinical signs

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.03 other: mg/m3

System:

respiratory system: lower respiratory tract

Organ:

alveoli

bronchi

bronchioles

lungs

lymph node

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.1 other: mg/m3 air

System:

integumentary

Organ:

skin

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Key result

Lowest effective dose / conc.:

0.03 other: mg/m3 air

System:

respiratory system: upper respiratory tract

Organ:

larynx

nasal cavity

trachea

Conclusions:

A chronic 2-year repeated dose toxicity study on 60 male and 60 female F344/N rats was conducted via the inhalation route. Under the conditions of this study, there was clear evidence of carcinogenic activity of indium phosphide based on increased frequency of benign and malignant neoplasms in the lung. Increased incidences of pheochromocytoma of the adrenal medulla in males and females were also considered to be exposure related. Marginal increases in incidences of mononuclear cell leukemia in males and females, fibroma of the skin in males, and carcinoma of the mammary gland in females have also been attributed to indium phosphide exposure. Based on the findings of the study a LOAEC of 0.03 mg/m3 was determined and it was concluded that the substance was carcinogenic under the conditions of the study.

Executive summary:

A GLP compliant study assessing the carcinogenicity of indium phosphide was conducted using a method similar to the OECD TG 453 (with deviations). Groups of 60 rats per sex per dose were exposed to particulate aerosols of indium phosphide at concentrations of 0, 0.03, 0.1, or 0.3 mg/m3, 6 hours per day, 5 days per week, for 22 weeks (0.1 and 0.3 mg/m3 groups) or 105 weeks (0 and 0.03 mg/m3 groups). Owing to the extent of the lung weight surges and the severity of the lung lesions in rats exposed to 0.1 or 0.3 mg/m3, it was established that these effects were adequately widespread to stop exposure of these groups. Exposure was stopped at 22 weeks ensuing pathology assessment and animals in the 0.1 and 0.3 mg/m3 groups were maintained on filtered air from exposure termination until the end of the studies. Male and female rats (10 per group) were randomly selected and evaluated at 3 months.

At the 3-month interim evaluation all groups of exposed male and female rats showed microcytic erythrocytosis and enlarged lungs and lesions in the respiratory tract and lung associated lymph nodes. The severity of the lesions was proportionate to the increase in exposure concentration. Clinical findings were observed after 18 months and finding included lethargy in the 0.03 mg/m3 males and females as well as abnormal breathing in all exposed males. The survival rates and mean body weights of both exposed male and female rats were similar to those of the chamber controls. When compared to chamber controls (at two years), exposure to indium phosphide showed increased incidences of alveolar/bronchiolar adenomas in male rats exposed to 0.1 or 0.3 mg/m3 and in all groups of exposed female rats. Alveolar/bronchiolar carcinomas were also found in both sexes exposed to 0.3 mg/m3. Further to this squamous cell carcinoma of the lung occurred in four male rats exposed to 0.3 mg/m3 and was considered to be exposure related.

In all exposed groups, a spectrum of inflammatory and proliferative lesions was observed in both male and female rats. In general, the lesions progressed in the continuously exposed 0.03 mg/m3 group, while the severities of lung lesions remained comparable in animals exposed to 0.1 or 0.3 mg/m3 ensuing exposure cessation. Lesions included atypical hyperplasia, chronic inflammation, alveolar epithelial hyperplasia and metaplasia, alveolar proteinosis, and interstitial fibrosis. Additionally, two rare spontaneous lesions, squamous metaplasia and squamous cysts, occurred in exposed groups, and the incidence of squamous cysts was significantly increased in 0.3 mg/m3females.

Exposure to indium phosphide also caused increased incidences of benign and malignant pheochromocytomas of the adrenal gland in the male group exposed to 0.03 mg/m3 and the female group exposed to 0.3 mg/m3. Additionally, in the female groups exposed to 0.3 mg/m3 or 0.03 mg/mg incidences of hyperplasia were also increased. The increased incidences of neoplasms and non-neoplastic lesions in the adrenal medulla were considered to be exposure related. Marginal increases of mononuclear cell leukaemia in the 0.1 mg/m3 group for males and 0.03 mg/m3 groups of females were noted and were thought to be exposure related. Similarly, incidences of fibroma or fibrosarcoma in the male rats exposed to 0.1 mg/m3 were noted, however as there was no increase of incidences in the 0.03 or 0.3 mg/m3 there was uncertainty if this was exposure related. Finally, carcinoma of the mammary gland in females exposed to 0.03 mg/m3 at 2 years may also have been related to indium exposure.

Under the conditions of this 2-year inhalation study clear evidence of carcinogenic activity of indium phosphide was established based on increased frequency of benign and malignant neoplasms of the lung and pheochromocytoma of the adrenal medulla. Increased incidences of mononuclear cell leukaemia in males and females, fibroma of the skin in males, and carcinoma of the mammary gland in females were also attributed to indium phosphide exposure. Based on the results of the study a LOAEC of 0.03 mg/m3 for systemic effects was determined. It was concluded that Indium Phosphide was carcinogenic under the conditions of this study.

Reference 2

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 January 1996-23 january 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

Deviations compared to OECD TG 453 regarding organs investigated and lack of some examinations on animals exposed for 105 weeks. Opthalmological examination, urinalysis not included.

GLP compliance:

yes

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Taconic Farms (Germantown,NY)
- Females nulliparous and non-pregnant: yes
- Age at study initiation:6 weeks
- Weight at study initiation:male: average =22.5(g) female: average= 19.0(g)
- Housing: Individually in stainless steel wire bottom cages (Lab Products, Inc., Harford System Division, Aberdeen, MD).
- Diet: NTP-2000 pelleted diet ad libitum (except during exposure periods)
- Water: Softened tap water ad libitum (except during urine collection periods)
- Acclimation period:13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C):24° +/- 3
- Humidity (%):55% +/-15%
- Air changes (per hr):15/hr
- Photoperiod (hrs light): 12h/12h

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

ca. 1.2 µm

Geometric standard deviation (GSD):

1.8

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless-steel inhalation exposure chambers (Harford Systems Division of Lab Products, Inc., Aberdeen, MD)
- Source and rate of air: Compressed air
- Method of conditioning air: The aerosol leaving the generator passed through a corona discharge air-ionizing neutralizer into the distribution line. At each chamber location, a pneumatic injector drew aerosol from the distribution line into the chamber inlet, where the aerosol was further diluted with HEPA-filtered air to the appropriate concentration.
- System of generating particulates/aerosols:Aerosol generator consisting of a drum, body, and cap. The drum rotated at 60° increments controlled by a compressed-air-driven valve driver. As the drum rotated, indium phosphide filled six metering ports in a disk at the bottom of the drum and was held in each port by a stainless-steel screen. The metering ports aligned with a nitrogen inlet in the body and dispersed indium phosphide when a nitrogen solenoid valve was opened.
- Temperature, humidity, pressure in air chamber: Temperature (°C): 24° +/- 3 Humidity (%):55% +/-15% Pressure: Not Reported
- Air change rate: 15 air changes per hour
- Method of particle size determination: Mercer-style seven-stage impactor.Stages analyzed by inductively coupled plasma/mass spectroscopy (ICP/MS). Relative mass collected on each stage was analyzed by probit analysis. The mass median aerodynamic particle diameter and the geometric standard deviation of each set of samples were estimated.

TEST ATMOSPHERE
- Brief description of analytical method used: Mercer-style seven-stage impactor (In-Tox Products, Albuquerque, NM). The stages (glass coverslips lightly sprayed with silicone) were analyzed by inductively coupled plasma/mass spectroscopy (ICP/MS).
- Samples taken from breathing zone: Uniformity evaluated every 2 to 4 months. Measurements taken from 12 different chamber positions.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber aerosol concentrations were monitored with real-time aerosol monitors using a pulsed-light-emitting diode in combination with a silicon detector to sense light scattered over a forward angular range of 45° to 95° by particles traversing the sensing volume. The particle size distribution in each chamber was determined during the prestudy testing, during the first week of the studies, and monthly thereafter using a Mercer-style seven-stage impactor (In-Tox Products, Albuquerque, NM). The stages (glass coverslips lightly sprayed with silicone) were analyzed by inductively coupled plasma/mass spectroscopy (ICP/MS). The relative mass collected on each stage was analyzed by probit analysis. The mass median aerodynamic particle diameter and the geometric standard deviation of each set of samples were estimated.

Duration of treatment / exposure:

21 weeks or 105 weeks.

Frequency of treatment:

6 hours per day, 5 days per week for 21 weeks or 105 weeks.

Post exposure period:

approx. 82 weeks for 0.1 and 0.3 mg/m3 groups

Dose / conc.:

0 mg/m³ air

Dose / conc.:

0.01 mg/m³ air

Dose / conc.:

0.03 mg/m³ air

Dose / conc.:

0.1 mg/m³ air

No. of animals per sex per dose:

60 animals/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on the results of a 14-week inhalation repeated dose toxicity study performed beforehand
- Rationale for animal assignment : Random
- Rationale for selecting satellite groups: Tissue burden analysis were performed on 20 male mice-no justification provided
- Post-exposure recovery period in satellite groups: 16 weeks

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: week 5 through 93 and approximately every 2 weeks thereafter

DERMAL IRRITATION (if dermal study): No

BODY WEIGHT: Yes
- Time schedule for examinations:every 4 weeks from week 5 through 93, and every 2 weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3 month interim evaluation
- Anaesthetic used for blood collection: Yes (CO2/air-mixture)
- Animals fasted: No
- How many animals: 25 males or 26 females
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 3 month interim evaluation
- Animals fasted: No
- How many animals: 25 males or 26 females
- Parameters checked in table [No.2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Tissue Burden Studies
-Time schedule: 3, 5, 9, or 12 months or 2, 4, 6, 8, or 12 months after exposure termination
- Parameters checked: Lung and serum . Complete necropsy, histopathologic examination of the lung, or lung burden and serum indium concentration evaluation were performed.
- How many animals: 3/4
- Parameters checked: Histopathologic examination of lung, and lung burden and serum indium concentration evaluation

Sacrifice and pathology:

GROSS PATHOLOGY: Yes See Table No. 2

HISTOPATHOLOGY: Yes: See Table No. 2

Statistics:

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958).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.The Poly-k test (Bailer andPortier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence.Two approaches were employed to assess the significanceof pairwise comparisons between exposed and control groups in the analysis of continuous variables. For neoplasms and non-neoplastic lesions detected at the interim evaluation, the Fisher exact test (Gart et al., 1979) was used. Organ and body weight data were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971,1972).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings observed were most common in the 0.03 mg/m3 groups, followed by the 0.3 and 0.1 mg/m3 groups and included abnormal breathing, thinness, and ruffled fur. Thinness and ruffled fur were noted at 5 months, and abnormal breathing at 14 months.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Survival rates of all exposed groups were lower than those of the chamber controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of 0.03 and 0.3 mg/m3 males and all groups of exposed females were less than those of the chamber controls for most of the study. Decreases were marginally more severe in females. Mean body weights of the 0.03 and 0.3 mg/m3 groups were mostly comparable.

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:

effects observed, treatment-related

Description (incidence and severity):

At 3 months 0.3mg/m3 males and females and 0.1 mg/m3 females showed an increased erythron evidenced by increased hematocrit values, hemoglobin concentrations, or erythrocyte counts. Diminutions in mean cell volume and mean cell hemoglobin and an surges in platelet counts accompanied the increased erythron. There was evidence of an exposure-related increase in leukocyte and neutrophil counts in all exposed groups of female mice, consistent with the pulmonary inflammation observed microscopically.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 3 months, there was an increase in unbound iron-binding capacity in the 0.3 mg/m3 males and in all exposed groups of females and total iron-binding capacity (0.1 and 0.3 mg/m3 females) there was no change in serum iron concentration.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Lung weights of all exposed groups of males and females were significantly greater (1.4-to 2.4-fold) than those of the chamber controls at 3 months. Lung weights of exposed male mice were significantly increased comparative to chamber control lung weights and increased with increasing exposure concentration and duration of exposure. Ensuing cessation of exposure, lung weights in the 0.1 and 0.3 mg/m3 groups remained elevated, showing very little recovery.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 3 months, the most prominent lesions induced by exposure to indium phosphide occurred in the lungs and consisted of chronic active inflammation, foreign body (indium phosphide particles), and alveolar proteinosis. At 2 years, there were increased incidences of chronic active inflammation, alveolar proteinosis and foreign body (indium phosphide particles) in the lungs of exposed mice.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 3 months, the most prominent lesions induced by exposure to indium phosphide occurred in the lungs and consisted of chronic active inflammation, foreign body (indium phosphide particles), and alveolar proteinosis. The severity of these lesions was similar between animals exposed to 0.03 and 0.1 mg/m3 and increased in animals exposed to 0.3 mg/m3 indium phosphide. Subsequent to discontinuation of exposure to the 0.1 and 0.3 mg/m3 groups (at 2 months), chronic inflammation, alveolar epithelial hyperplasia and alveolar proteinosis were less severe than at 145 days. At 4 and 6 months after cessation of exposure, proteinosis and hyperplasia appeared less severe, however the chronic inflammation appeared similar to that observed at 145 days. The only mice evaluated at 12 months on test were still being exposed to 0.03 mg/m3. The lesions were clearly more severe than those observed in mice exposed to 0.03 mg/m3 at 145 days on test and were similar to those observed in the 0.3 mg/m3 group at 145 days. At 2 years, there were increased incidences of chronic active inflammation, alveolar proteinosis and foreign body (indium phosphide particles) in the lungs of exposed mice. This inflammation was more severe in mice exposed to 0.03 mg/m3 and was least severe in the 0.1 mg/m3 group. Alveolar proteinosis was observed only in mice exposed to 0.03 and 0.3 mg/m3 and was characterized by the presence of amorphous, granular to homogeneous eosinophilic material (surfactant) in alveoli. Foreign body (indium phosphide) was minimal in all groups and was not always present in animals exposed to 0.1 mg/m3. At 2 years, the incidences of hematopoietic cell proliferation in the spleen were increased in all exposed groups of males and in females exposed to 0.03 and 0.1 mg/m3 and minimal hematopoietic cell proliferation was considered to be common in the spleen of mice.

Bronchial lymph nodes were markedly enlarged at 3 months. Hyperplasia of the mediastinal lymph nodes was more subtle. Minimal foreign body (indium phosphide particles) were observed in the bronchial lymph nodes. Incidences of pleural mesothelial hyperplasia of the lung were increased in both male and female mice exposed to 0.03 and 0.3 mg/m3. The pleural mesothelium from numerous animals was hypertrophic/hyperplastic which is associated with chronic inflammation and fibrosis. At two years hyperplasia and foreign body presence in the bronchial and mediastinal lymph nodes increased in all exposed groups the severities and incidences were greater in the 0.03 mg/m3 groups of both males and females that were continually exposed for the two-year period. In the cardiovascular system increased inflammation of the arteries of the heart (mainly coronary arteries) and proximal aorta occurred. Additional chronic inflammatory lesions were observed in the pericardium of both males and females exposed to 0.03 mg/m3 and 0.3 mg/m3 these microscopic arears of inflammation correlated to the adhesions noted grossly. Reduced incidences of qualitatively similar (to the coronary arteries and proximal aorta) vasculitis occurred in the lung, mediastinal and bronchial lymph nodes, mesentery and the kidney.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Lung: At 2 years marked increases of alveolar/bronchiolar carcinoma arose in both male and female groups of mice exposed to 0.03 or 0.3 mg/m3. The occurrence of adenomas was increased in the 0.1 mg/m3 female group while the incidences of alveolar/bronchiolar carcinoma combined were increased in all exposed groups of females. Incidences of alveolar/bronchiolar adenoma or alveolar/bronchiolar carcinoma and combined alveolar/bronchiolar adenoma or carcinoma exceeded the historical ranges for the 2- year NTP studies with the exception of the 0.03 mg/m3 and 0.1 mg/m3 female groups which did not exceed.

Liver: At 2 years increased occurrences of hepatocellular adenoma occurred in the 0.03 mg/m3 and 0.3 mg/m3 groups of male mice and incidences of hepatocellular carcinoma in the 0.1 mg/m3 group of males and 0.03 mg/m3 group of both males and females occurred. Combined hepatocellular adenoma or carcinoma were increased in all groups of exposed males and in the 0.03 mg/m3 group of females. The incidences of adenoma, carcinoma, and adenoma or carcinoma (combined) in exposed males and females exceeded the ranges in NTP-2000 studies and the incidence of adenoma in the 0.3 mg/m3 males exceeded the historical control range in the larger NIH-07 database. Hepatocellular adenoma or carcinoma (combined) in the 0.03 mg/m3 female mice exceeded the historical control range in the NIH-07 database. Incidences of eosinophilic foci were increased in all groups of exposed males and in the 0.3 mg/m3 females. Foci of hepatocellular alteration, hepatocellular adenoma, and hepatocellular carcinoma are believed to characterise a spectrum that establishes the progression of proliferative liver lesions. The increased incidences of liver lesions observed were considered to be related to indium phosphide exposure.

Small Intestine: Carcinoma of the small intestine in 0.1 mg/m3 males was slightly increased and was equal to the highest incidence in the NTP-2000 historical control database. Although the increased incidence in the 0.1 mg/m3 males was within the historical control range for the NTP-2000 studies, it fell well outside the range for the larger NIH-07 database and was considered an uncertain finding.

Hematopoietic System: At 3 months, hematopoietic cell proliferation was observed in the liver of six exposed female mice but not in any chamber controls. Incidences of hematopoietic cell proliferation in the spleen were increased in exposed mice at 3 months. A slight positive trend in the incidences of malignant lymphoma in female mice and the incidences in the 0.1 and 0.3 mg/m3 groups exceeded the historical control range for NTP-2000 studies. The slight positive trend was not considered related to exposure to indium phosphide.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Lung burdens, like lung weights, increased with time and with increasing exposure concentration in all groups. For the 0.1 and 0.3 mg/m3 groups, lung burdens were reduced by 2 months after exposure and continued to decline until 12 months after exposure when they had decreased to 16% and 28% of the values observed at the end of exposure, respectively. Thus lung burdens decreased to a greater extent during the clearance phase of the study than did lung weights, which remained elevated due to the pathological changes present in the lung. Lung burdens, when normalized to exposure concentrations, remained constant across exposure concentrations, indicating linear toxicokinetics. Lung deposition rates increased proportionately to exposure concentration in male mice. There were no differences in the calculated lung deposition fraction, clearance rate constant, or clearance half-times among exposed groups. Although estimated clearance half-times for indium in the lung were 230, 144, and 163 days respectively for the three exposed groups, there was considerable overlap in their uncertainties. The overall mean clearance half-time for the exposed groups was 179 days.

Relevance of carcinogenic effects / potential:

There was clear evidence of carcinogenic activity of indium phosphide in male B6C3F1 mice based on increased incidences of malignant neoplasms of the lung and benign and malignant neoplasms of the liver. Marginal increases in occurrences of adenoma and carcinoma of the small intestine may have been related to exposure to indium phosphide. Evidence of carcinogenic activity of indium phosphide in female B6C3F1 mice was based on increased incidences of benign and malignant neoplasms of the lung. Increased incidences of liver neoplasms in females were also considered to be exposure related.

Key result

Dose descriptor:

LOAEC

Effect level:

0.03 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.03 other: mg/m3

System:

respiratory system: upper respiratory tract

Organ:

bronchi

bronchioles

lungs

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.03 other: mg/m3 air

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

no

Critical effects observed:

no

Lowest effective dose / conc.:

0.1 other: mg/m3 air

System:

gastrointestinal tract

Organ:

intestine

Critical effects observed:

no

Lowest effective dose / conc.:

0.1 other: mg/m3 air

System:

haematopoietic

Organ:

spleen

Critical effects observed:

no

Lowest effective dose / conc.:

0.03 other: mg/m3 air

System:

cardiovascular

Organ:

aorta

artery

heart

vascular system

Conclusions:

A chronic 2-year repeated dose toxicity study on 60 male and 60 female B6C3F1 mice was conducted via the inhalation route. Under the conditions of this study, there was clear evidence of carcinogenic activity of indium phosphide in male B6C3F1 mice based on increased incidences of malignant neoplasms of the lung and benign and malignant neoplasms of the liver. Marginal increases in occurrences of adenoma and carcinoma of the small intestine may have been related to exposure to indium phosphide. Evidence of carcinogenic activity of indium phosphide in female B6C3F1 mice was based on increased incidences of benign and malignant neoplasms of the lung. Increased incidences of liver neoplasms in females were also considered to be exposure related. Based on the findings of the study a LOAEC of 0.03 mg/m3 was determined and it was concluded that the substance was carcinogenic under the conditions of this study.

Executive summary:

A GLP compliant study assessing the carcinogenicity of indium phosphide was conducted using a method similar to the OECD TG 453 (with deviations). Groups of 60 B6C3F1 mice per sex, per dose, were exposed to particulate aerosols of indium phosphide at concentrations of 0, 0.03, 0.1, or 0.3 mg/m3, 6 hours per day, 5 days per week, for 21 weeks (0.1 and 0.3 mg/m3 groups) or 105 weeks (0 and 0.03 mg/m3 groups). Exposure to indium phosphide for 3 months affected the circulating erythroid mass and caused enlarged lungs and lesions in the respiratory tract and lung associated lymph nodes. Owing to the extent of the lung weight surges and the severity of the lung lesions in rats exposed to 0.1 or 0.3 mg/m3, it was established that these effects were adequately widespread to stop exposure of these groups. Exposure was stopped at 21 weeks ensuing pathology assessment and animals in the 0.1 and 0.3 mg/m3 groups were maintained on filtered air from exposure termination until the end of the studies. Male and female mice (10 per group) were randomly selected and evaluated at 3 months. Additional groups of 20 male mice were designated for tissue burden analysis.

The majority of clinical findings observed were most common in the 0.03 mg/m3 groups, followed by the 0.3 and 0.1 mg/m3 groups and involved abnormal breathing (14 months), thinness, and ruffled fur (5 months). Exposure to indium phosphide for 2 years reduced survival and body weight gain in exposed males and females. Survival rates of the 0.1 mg/m3 groups were the highest followed by the 0.3 and 0.03 mg/m3 groups, respectively. Mean body weights of the 0.03 and 0.3 mg/m3 males and all exposed groups of females were less than those of the chamber controls throughout the study.

At two years, exposure to indium phosphide gave rise to incidences of alveolar/bronchiolar carcinomas in male 0.03 or 0.3 mg/m3 groups and alveolar/bronchiolar adenomas and carcinomas in all female groups. Additionally, alveolar proteinosis, chronic inflammation, serosa fibrosis and pleural mesothelial hyperplasia were also seen. Hepatocellular neoplasms and eosinophilic foci of the liver were also increased in both male and female mice exposed. Hepatocellular adenoma occurred in the 0.03 and 0.3 mg/m3 groups of male mice where hepatocellular carcinoma increased in incidence in the 0.03 mg/m3 of both males and females. Incidences of combined hepatocellular adenoma or carcinoma were increased in all exposed groups of males and in the 0.03 mg/m3 group of females. Minor increases in the occurrences of neoplasms of the small intestines in 0.1 mg/m3 group of males were thought to be exposure related. exposure also caused inflammation of the arteries of the heart, chiefly in the coronary arteries and proximal aorta, and to a smaller degree in the lung associated lymph node in both male and female mice.

Lung burden during the study was relative to the exposure concentration and duration and no variances in elimination rates of indium from the lungs were observed as a result of exposure concentration. Mice exhibited a clearance half-time of 144 days for the 0.1 mg/m3 group and 163 days for the 0.3 m, g/m3 groups. Estimates at the end of the 2 years indicated that the lung burdens in the continually exposed 0.03 mg/m3 groups were greater than those groups where the exposure was stopped at 3 months i.e. the 0.1 and 0.3 mg/m3 groups. Lung burdens were the lowest in the 0.1 mg/m3 groups followed by the 0.3 mg/m3 groups and therefore 0.1 mg/m3 may be considered as the low dose group.

Under the conditions of this 2-year inhalation study clear evidence of carcinogenic activity of indium phosphide in male B6C3F1 mice was established based on increased frequencies of malignant neoplasms of the lung and benign and malignant neoplasms of the liver. Marginal increases in the incidences of adenoma and carcinoma of the small intestine were affiliated with indium exposure. evidence of carcinogenic activity of indium phosphide in female B6C3F1 mice was based on increased incidences of benign and malignant neoplasms of the lung, increased incidences of neoplasms in the females were also affiliated with indium exposure. Overall, exposure to indium phosphide by inhalation resulted in non-neoplastic lesions in the lungs, liver and heart of both male and female mice. Based on the results of the study a LOAEC of 0.03 mg/m3 for systemic effects was determined. It was concluded that Indium Phosphide was carcinogenic under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

0.03 mg/m³

Study duration:

chronic

Species:

other: F344/N rats and B6C3F1 Mice

Quality of whole database:

The Carcinogenicity of Indium Phosphide was evaluated using a method similar to OECD guideline 453 (GLP) without any significant deviations.

System:

respiratory system: lower respiratory tract

Organ:

lungs

Justification for classification or non-classification

The studies performed and reported by the NTP (2001) was used as basis by the ECHA Committee for Risk Assessment to set the Harmonised Classification of Indium Phosphide as Carc. 1B; H350: May cause cancer following exposure via the inhalation route.

This classification was based mainly on clear evidence of increased incidence of lung tumours observed in male and female rats and mice exposed to 0.03, 0.1 and 0.03 mg/m3 of Indium Phosphide.