Gallium arsenide

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP toxicity study conducted under the US National Toxicology Program (NTP)

Reason / purpose for cross-reference:

reference to same study

Objective of study:

other: lung and tissue burden study

Guideline:

other: The 2-year study was conducted in compliance with FDA Good Laboratory Practice Regulations (21 CFR, Part 58). In addition, as records from the 2-year study were submitted to the NTP Archives, this study was auditied retrospectively.

Principles of method if other than guideline:

Tissue burden study: lung, testes, blood and serum from male rats were evaluated. At 1, 2, 4, 6, 12, and 18 months, 5 male rats from the 0, 0.1, and 1.0 mg/m³ groups were evaluated. 4 or 5 male rats from the 0.01 mg/m³ group were evaluated at 2-, 12-, and 18-month time points.
The 2-year study was conducted in compliance with FDA Good Laboratory Practice Regulations (21 CFR, Part 58). In addition, as records from the 2-year study were submitted to the NTP Archives, this study was auditied retrospectively by an independent quality assurance contractor. Seperate audits covered completeness and accuracy of the pathology data, pathology speciems, final pathology tables, and a draft of this NTP Technical Report. Audit procedures and findings are presented in the reports and are on file at NIEHS. The audit findings were reviewed and assessed by NTP staff, and all comments were resolved or otherwise addressed during the preparation of this Technical Report.

GLP compliance:

yes

Radiolabelling:

no

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms (Germantown, NY)
- Age at study initiation: approx. 6 weeks old (after being quarantined for 14 days)
- Housing: individually; stainless steel wire bottom (Hazzelton System, Inc., Aberdeen, MD), changed weekly
- Diet: ad libitum, except during exposure and urine collection periods; NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), changed daily
- Water: ad libitum, softened tap water (Richland municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI),changed weekly
- Acclimation period: 14 days quarantine

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ~23 - 25
- Humidity (%): 55 +/- 15
- Air changes: 15/hour
- Photoperiod: 12 hours dark/light cycle

Route of administration:

inhalation: aerosol

Vehicle:

other: air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the study laboratory designed the stainless-steel inhalation exposure chambers so that uniform vapour concentrations could be maintained throughout the chambers when catch pans were in place. The total active mixing volume of each chamber was 1.7 m³.
- System of generating particulates/aerosols: the aerosol generator consisted of a drum, body, and cap. The drum rotated at 60° increments, with set time intervals between drum rotation. Rotation of the drum was controlled by a compressed-air -driven valve driver (VICI Valco Instument Co., Houston, TX). Output of the generator was regulated by adjusting the rotation cadence. The aerosol passed through the distribution line to the exposure chambers, where it was diuted with filtered air to the proper exposure concentration.

- Method of particle size determination: the particle size distribution in each chamber was determined during pre-study testing and monthly during the 2-year study using a Mercer-style seven-stage impactor. The stages (glass coverslips lightly sprayed with silicone) were analysed by ICP/MS. The relative mass collected on each stage was analysed by probit analysis. The mass median aerodynamic particle diameter and the geometric standard deviation of each set of samples were estimated.

- Chamber aerosol concentrations were monitored with real-time aerosol monitors (RAMs) that used 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 instrument responds to particles 0.1 to 20 µm in diameter; the geometric diameter of gallium arsenide aerosol approached the minimum of this range. Each RAM was calibrated by correlating the measured voltage with gallium arsenide concentrations determined by analyzing exposure chamber samples collected on fiberglass filters (Teflon-coated Pallflex, Pallflex Corp., Putum, CT). Filter samples were dissolved in nitric acid and analyzed for gallium arsenide using inductively coupled plasma/mass spectroscopy (ICP/MS). RAMs were calibrated twice monthly during the 2-year studies. During the 2-year studies, calibration was verified by ICP/MS analysis of filter samples collected every other day (control chambers) or daily.
- Uniformity of aerosol concentration was evaluated every 3 months. Chamber concentration uniformity was acceptable throughout the studies

Duration and frequency of treatment / exposure:

2 years, 6 hours/day, 5 days /week

Remarks:

Doses / Concentrations:
0, 0.01, 0.1, 1 mg GaAs/m³

No. of animals per sex per dose / concentration:

Month 1, 2, 4, 6, 12, and 18: 5 males (0, 0.1, and 1 mg/m³)
Month 2, 12, and 18: 4 or 5 males (0.01 mg/m3)

Control animals:

yes

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale: in a preceding 90-day study no no-effect level was achieved for the lung.
Based on the increased severities of lung proteinosis and at least a 2-fold increase in lung weights in males and females, exposure concentrations of 10 mg/m³ or greater were considered sufficiently severe to preclude their use in a 2-year study. Because a no-effect level was not achieved for the lung, the lowest exposure concentration for rats in the 2-year stady was set at the lowest concentration that the chamber particle monitor could monitor continuously with accuracy.
Therefore the gallium arsenide exposure concentrations selected for the 2-year inhalation study in rats were 0.01, 0.1 and 1.0 mg/m³.

Details on dosing and sampling:

Lung burden study:
The lungs, testes, blood, and serum from male rats were evaluated at:
Month 1, 2, 4, 6, 12, and 18: 5 males (0, 0.1, and 1 mg/m³)
Month 2, 12, and 18: 4 or 5 males (0.01 mg/m³)

- Method type(s) for identification: atomic absorption spectrometry
- Limits of quantification: 0.20 µg Ga/g lung, 1.45 µg As/g lung
0.005 µg Ga/g blood, 2.68 µg As/g blood
0.042 µg Ga/g serum, 0.086 µg As/g serum
0.051 µg Ga/g testes, 0.126 µg As/g testes

Statistics:

Survival analyses:
- 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. All reported P values for the survival analyses are two sided.

Analysis of neoplasm and non-neoplastic lesion incidences:
- 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.

Analysis of continuous variables:
- Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
- Blood and bone marrow hematology, clinical chemistry, urinalysis, spermatid, and epididymal spermatozoal data, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere.s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (William's or Shirley's test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett.s or Dunn.s test).
- Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations.

Details on absorption:

Lung weights increased in all male rats exposed to 0.1 or 1 mg/m³ throughout the study when compared to chamber controls and the 0.01 mg/m³ group. In addition, lung weights of these rats continued to increase to a greater extent throughout the study than did lung weights of the chamber controls and the 0.01 mg/m³ group. The percentages of gallium and arsenic in the lung relative to the total lung burden were similar at all exposure concentrations throughout the study because the deposition and clearance rates in the lung for gallium and arsenic were similar within each exposed group.

Lung burden for gallium and arsenic increased with increasing exposure concentration. The lung burdens increased with increasing exposure concentration over time in all exposed groups. It appears that a steady state lung burden was achieved for the 0.1 and possibly the 1.0 mg/m³ groups, although the lung burdens at 18 months were low.
Lung burdens did not increase proportionally with exposure concentration over time. Lung burdens normalized to exposure concentration would be expected to remain constant across all exposure concentrations if the toxicinetics were linear; however, gallium and arsenic normalized lung burdens in the 1.0 mg/m³ group were considerably lower than those observed in the 0.01 or the 0.1 mg/m³ group. Although deposition rates increased proportionally to exposure concentration, the lung clearance half-lives for the 1.0 mg/m³ group were considerably less than those for the 0.1 or the 0.01 mg/m³ group.

Half-lives for gallium in the lung were 133, 96, and 37 days for the 0.01, 0.1, and 1.0 mg/m³ groups, respectively. Arsenic lung half-lives were similar.

The gallium arsenide clearance half-live for the 1.0 mg/m³ group from the 2-year study was similar to the clearance half-live for the 1.0 mg/m³ group from the 14-week study. Accordingly extended exposure for 2 years had no effect on the clearance rate and, therefore, the clearance rate was dependent on the lung burden. Increased clearance at 1.0 mg/m³ was likely due to an increase in alveolar macrophages.

The interpretation of the analytical results is difficult: in contrast to fundamental analytical experiences the standard deviations are higher with higher Gallium concentrations. This seems to be the reason why no statistically significant differences were indicated in the tables of the study report.
There seems to be an increase of the Gallium concentrations above the background level of the control animals in the 1 mg GaAs/m³ group and at the end of the study in the blood of the 0.1 mg GaAs/m³ group.
Increased mean Gallium concentrations in the serum were only found in the 1 mg GaAs/m³ group after 6 months and later.
In the testes increased mean Gallium concentrations were found after 12 months and later in the 0.1 mg GaAs/m³ group and after 2 months and later in the 1 mg GaAs/m³ group.
As expected arsenic was detected in whole blood where it is preferentially bound to erythrocytes. Mean Arsenic concentrations in whole blood were greater than those of chamber controls only in the 1.0 mg/m³ group where they were approximately 2-fold higher, but this was not a statisically significant difference. The concentration of arsenic in whole blood was small relative to the concentration of arsenic in the lung. This indicates that there was no accumulation of either gallium or arsenic in these tissues.

Metabolites identified:

no

Details on metabolites:

no data

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results Half-times for gallium in the lung were 133, 96, and 37 days for the 0.01, 0.1, and 1.0 mg GaAs/m³ groups, respectively. Arsenic lung half-times were similar.
The interpretation of the analytical results is difficult because of the large standard deviations. No significant statistically significant differences are indicated in the tables. Therefore differences of the means cannot be interpreted as biological effects.
The Gallium concentrations in blood, serum, and testes were small relative to the concentrations of gallium and arsenic in the lung. This indicates that there was no accumulation of either gallium or arsenic in these tissues. As expected arsenic was detected in whole blood where it is preferentially bound to erythrocytes.
Only in the 1.0 mg/m³ group arsenic concentrations in whole blood were greater than those of chamber controls. They were approximately 2-fold higher and without a statistical significance.

Executive summary:

In a 2 -year toxicity study with inhalation exposure of male rats to concentrations of 0, 0.01, 0.1, 1 mg GaAs/m³, additional animals were treated for the characterisation of the lung burden.

Lung weights increased in all male rats exposed to 0.1 or 1 mg/m³ throughout the study when compared to chamber controls and the 0.01 mg/m³ group. Lung burden for gallium and arsenic increased with increasing exposure concentration. It appears that a steady state lung burden was achieved for the 0.1 and possibly the 1.0 mg/m³ groups after 6 months, although the lung burdens at 18 months were low.

Half-lives for gallium in the lung were 133, 96, and 37 days for the 0.01, 0.1, and 1.0 mg GaAs/m³ groups, respectively. Arsenic lung half-lives were similar.

The interpretation of the analytical results is difficult because of the large differences of the standard deviations. This seems to be the reason why no statistically significant differences were indicated in the tables of the study report. Therefore differences of the means cannot be interpreted directly as biological effects.

The gallium concentrations in blood, serum, and testes were small relative to the concentrations of gallium and arsenic in the lung. This indicates that there was no accumulation of either gallium or arsenic in these tissues. As expected arsenic was detected in whole blood where it was preferentially bound to erythrocytes. Arsenic concentrations in whole blood were greater than those of chamber controls only in the 1.0 mg/m³ group where they were approximately 2-fold higher but not statistically significantly different.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 1989 to June 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP toxicity study conducted under the US National Toxicology Program (NTP)

Reason / purpose for cross-reference:

reference to same study

Objective of study:

other: lung and tissue burden

Guideline:

other: The 14-week study was conducted in compliance with FDA Good Laboratory Practice Regulations (21 CFR, Part 58). In addition, as records from the 14-week study were submitted to the NTP Archives, this study was auditied retrospectively.

Principles of method if other than guideline:

Lung and tissue burden study.The 14-week study was conducted in compliance with FDA Good Laboratory Practice Regulations (21 CFR, Part 58). In addition, as records from the 14-week study were submitted to the NTP Archives, this study was auditied retrospectively by an independent quality assurance contractor. Seperate audits covered completeness and accuracy of the pathology data, pathology speciems, final pathology tables, and a draft of this NTP Technical Report. Audit procedures and findings are presented in the reports and are on file at NIEHS. The audit findings were reviewed and assessed by NTP staff, and all comments were resolved or otherwise addressed during the preparation of this Technical Report.

GLP compliance:

yes

Radiolabelling:

no

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA)
- Age at study initiation: approx. 7 weeks old (4 weeks old on receipt)
- Weight at study initiation: range of means: 130-135 g (males); 105-111 g (females)
- Housing: individually; stainless steel wire bottom (Lab Products, Inc. Harford Systems Division, Aberdeen, MD), changed weekly
- Diet: ad libitum, except during exposure and urine collection periods; NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), changed daily
- Water: ad libitum, softened tap water (Richland municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), changed weekly
- Acclimation period: 19 to 20 days quarantine

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ~23
- Humidity (%): 55 +/- 15
- Air changes: 15/hour
- Photoperiod: 12 hours dark/light cycle
No further details are given.

IN-LIFE DATES: From: 6 (males) or 7 (females) March 1989 To: 6 (males) or 7 (females) June 1989

Route of administration:

inhalation: aerosol

Vehicle:

other: air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the study laboratory designed the stainless-steel inhalation exposure chambers so that uniform vapour concentrations could be maintained throughout the chambers when catch pans were in place. The total active mixing volume of each chamber was 1.7 m³.
- System of generating particulates/aerosols: the gallium arsenide aerosol generation and delivery system had five basic components: a flexible-brush dust feed mechanism developed at the study laboratory, a Trost Model GEM-T air-impact mill, a cyclone separator, an aerosol charge neutraliser, and an aerosol distribution system. The flexible-brush dust feed mechanism employed a hopper into which the dry powder was poured. The hopper was reloaded with additional gallium arsenide at regular intervals throughout each day's exposure period. Aerosol passed through the charge neutraliser into the distribution line. At each chamber location, a vacuum pump drew aerosol from the distribution line into the chamber inlet, where the aerosol was further diluted with HEPA-filtered air to the appropriate concentration.
- Temperature, humidity in air chamber: 23-25°C, 55% +/- 15%
- Air change rate: 15 air changes per hour
- Method of particle size determination: the particle size distribution in each chamber was determined during pre-study testing and monthly during the 14-week study using a Mercer-style seven-stage impactor. The stages (glass coverslips lightly sprayed with silicone) were analysed by ICP/MS. The relative mass collected on each stage was analysed by probit analysis. The mass median aerodynamic particle diameter and the geometric standard deviation of each set of samples were estimated.

- Chamber aerosol concentrations were monitored with real-time aerosol monitors (RAMs) that used 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 instrument responds to particles 0.1 to 20 µm in diameter; the geometric diameter of gallium arsenide aerosol approached the minimum of this range. Each RAM was calibrated by correlating the measured voltage with gallium arsenide concentrations determined by analyzing exposure chamber samples collected on fiberglass filters. Filter samples were dissolved in nitric acid and analyzed for gallium arsenide using inductively coupled plasma/mass spectroscopy (ICP/MS). RAMs were calibrated one to two times weekly during the 14-week studies. Additional filter samples were collected on days not dedicated to RAM calibration during the 14-week studies for gravimetric analysis of chamber concentrations as an additional check of monitor operation.
- Uniformity of aerosol concentration in the 14-week studies was evaluated prior to the start of the studies without animals present and once during each of the studies with animals present in the exposure chambers. Chamber concentration uniformity was acceptable throughout the studies

Duration and frequency of treatment / exposure:

90 days, 6 hours/day, 5 days /week

Remarks:

Doses / Concentrations:
0, 0.1, 1, 10, 37, 75 mg GaAs/m³

No. of animals per sex per dose / concentration:

Lung from male rats were evaluated at three time points.
Day 23: 10 males (o mg/m³) or 6 males (0.1, 1, 10, 37, and 75 mg/m³)
Day 45: 2 males (o mg/m³) or 4 males (0.1, 1, 10, 37, and 75 mg/m³)
Day 93: 4 males (o mg/m³) or 3 males (0.1, 1, 10, 37, and 75 mg/m³)

Control animals:

yes

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale: in a preceding 16-day study (range-finding), the severity of alveolar proteinosis increased with increasing exposure concentration and was considered the primary reason for the concomitant increased lung weights. The proteinosis and lung weights were markedly increased in the 75 and 150 mg/m3 groups and represented the upper exposure limits for the 14-week study. Because effects were similar between the 75 and 150 mg/m3 groups, 75 mg/m3 was selected as the high exposure concentration for the 14-week study. Because a no-effect level was not achieved for the lung and the effects observed at 37 mg/m3 were similar to but less severe than those in the 75 mg/m3 group, the three lower concentrations for the 14-week study were spaced by a factor of ten.
- Rationale for animal assignment (if not random): randomly into groups of approximately equal initial mean body weights

Details on dosing and sampling:

Lung burden study:
The lungs from male rats were evaluated at three time points.
Day 23: 10 males (o mg/m³) or 6 males (0.1, 1, 10, 37, and 75 mg/m³)
Day 45: 2 males (o mg/m³) or 4 males (0.1, 1, 10, 37, and 75 mg/m³)
Day 93: 4 males (o mg/m³) or 3 males (0.1, 1, 10, 37, and 75 mg/m³)P
- Method type(s) for identification: atomic absorption spectrometry
- Limits of detection and quantification: no data

Statistics:

Survival analyses:
- 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. All reported P values for the survival analyses are two sided.

Analysis of neoplasm and non-neoplastic lesion incidences:
- 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.

Analysis of continuous variables:
- Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
- Blood and bone marrow hematology, clinical chemistry, urinalysis, spermatid, and epididymal spermatozoal data, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Jonckheere.s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams. or Shirley.s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett.s or Dunn.s test).
- Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across exposure concentrations.

Details on absorption:

Lung weights increased with increasing exposure concentrations in males exposed to 1 mg/m³ and greater on days 23 and 45 and in all exposed groups at week 14.
In addition, lung weights in exposed rats continued to increase to a greater extent throughout the study than did chamber control lung weights. The percentages of gallium and arsenic in the lung relative to the total lung burden of gallium arsenide were similar at all exposure concentrations throughout the study because the deposition and clearance rates in the lung for gallium and arsenic were similar within the exposed groups.
Lung burden for gallium and arsenic increased with increasing exposure concentration, and each increased thoughout the study; therefore, steady state lung burdens were not achieved for any exposure concentration. When lung burdens were normalized to exposure concentration, they were inversely proportional to the exposure concentration.
Although lung deposition rates increased proportionally to exposure concentration, lung clearance half-times actually decreased as exposure concentration increased, indicating a possible increase in lung clearance mechanisms at the higher concentrations. Thus the more rapid elimination rate at higher exposure concentrations accounts for the subproportional retained lung burdens that were observed as exposure concentration increased.

Metabolites identified:

yes

Details on metabolites:

no data

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results The percentages of gallium and arsenic in the lung were similar at all exposure concentrations throughout the study. Lung clearance half-times decreased as exposure concentration increased, indicating a possible increase in lung clearance mechanisms.
In a 90-day toxicity study with inhalative administration of GaAs to male rats, additional animals were treated for the characterisation of the lung burden.
Lung weights increased with increasing exposure concentrations in males exposed to 1 mg/m³ and greater on days 23 and 45 and in all exposed groups at week 14.
In addition, lung weights in exposed rats continued to increase to a greater extent throughout the study than did chamber control lung weights. The percentages of gallium and arsenic in the lung relative to the total lung burden of gallium arsenide were similar at all exposure concentrations throughout the study because the deposition and clearance rates in the lung for gallium and arsenic were similar within the exposed groups.
Lung burden for gallium and arsenic increased with increasing exposure concentration, and each increased thoughout the study; therefore, steady state lung burdens were not achieved for any exposure concentration. When lung burdens were normalized to exposure concentration, they were inversely proportional to the exposure concentration.
Although lung deposition rates increased proportionally to exposure concentration, lung clearance half-times actually decreased as exposure concentration increased, indicating a possible increase in lung clearance mechanisms at the higher concentrations. Thus the more rapid elimination rate at higher exposure concentrations accounts for the subproportional retained lung burdens that were observed as exposure concentration increased.

Executive summary:

In a 90-day toxicity study with inhalation exposure of male rats to concentrations of 0, 0.1, 1, 10, 37, 75 mg GaAs/m³, additional animals were treated for the characterisation of the lung burden.

Lung weights increased with increasing exposure concentrations in males exposed to 1 mg/m³ and above on days 23 and 45 and in all exposed groups at week 14 (day 90) including the lowest exposure concentration of 0.1 mg/m³.

In addition, lung weights in exposed rats continued to increase to a greater extent throughout the study than did chamber control lung weights.

The percentages of gallium and arsenic in the lung relative to the total lung burden of gallium arsenide were similar at all exposure concentrations throughout the study because the deposition and clearance rates in the lung for gallium and arsenic were similar within the exposed groups.

Lung burden for gallium and arsenic increased with increasing exposure concentration, and each increased thoughout the study; therefore, steady state lung burdens were not achieved for any exposure concentration.

When lung burdens were normalized to exposure concentration, they were inversely proportional to the exposure concentration. Although lung deposition rates increased proportionally to exposure concentration, lung clearance half-times actually decreased as exposure concentration increased, indicating a possible increase in lung clearance mechanisms at the higher concentrations.Thus, the more rapid elimination rate at higher exposure concentrations accounts for the subproportional retained lung burdens that were observed as exposure concentration increased.

Lung burden analysis indicated that the percentages of gallium and arsenic in the lung were similar at all exposure concentrations throughout the study. Deposition and clearance rates in the lung for both gallium and arsenic were similar.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2015-08-01 to 2016-09-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Objective of study:

other: Subchronic Inhalation Toxicity, 90-day Study

Qualifier:

according to guideline

Guideline:

other: OECD 413

Version / remarks:

Subchronic Inhalation Toxicity- 90-day Study with determination of organ burden after 4 weeks and 14 weeks exposure.

Principles of method if other than guideline:

- Principle of test: according to OECD 413

- Short description of test conditions:
Male B6C3F1 mice were whole body exposed to GaAs at exposure concentrations of 10 and 75 mg/m³ for 6 hours per day, 5 days per week, for a duration of either 4 weeks or 14 weeks.

The animals designated for clinical chemical and histological examinations after 4 weeks exposure were assigned to groups 10 (control group), 11 (10 mg/m³) and 12 (75 mg/m³). The animals examined for clinical chemical and histological examinations after 14 weeks exposure were assigned to groups 20 (control group), 21 (10 mg/m³) and 22 (75 mg/m³). In addition, the animals designated for determination of organ burden after 4 weeks exposure were assigned to test groups 30 (control group), 31 (10 mg/m³) and 32 (75 mg/m³), those lung burden animals after 14 weeks exposure were assigned to test groups 40 (control group), 41 (10 mg/m³) and 42 (75 mg/m³).

- Parameters analysed / observed:
Blood, serum, spleen and testes were analyzed at Competence Center Analytics, BASF SE (Ludwigshafen, Germany) for content of Ga and As.

GLP compliance:

yes

Radiolabelling:

no

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

- Reason for selection of the test species:
The mouse is a frequently used laboratory animal, and there is extensive experience with this species. The mouse is also proposed as suitable test animal by OECD and the EPA. The National Toxicology Program (NTP) performed several studies in B6C3F1 mice to characterize the toxic profile of the test substance. As a mechanistic study, this study is to be performed with the same strain as in the
NTP studies.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Test species and strain: B6C3F1 mice
- Age when supplied; sex: 7 - 8 weeks, male
- Age at start of exposure 8 - 9 weeks
- Supplier: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Arrival in the testing facility: 06 Jan 2015
- Identification: The animals were identified individually by tattooing the respective animal number into the ears (serial)
- All animals were randomized before the start of the pre-exposure period (according to weight).
Only animals free from clinical signs of disease were used for the study.

ENVIRONMENTAL CONDITIONS
- Air conditions: Temperature 20 to 24°C, relative humidity 30 to 70%. 15 air changes per hour. Deviations from these ranges did not occur.
- Illumination period: 06.00 a.m. - 06.00 p.m. light, 06.00 p.m. - 06.00 a.m. dark
- Type of cage / No. of animals per cage: Polycarbonate cages type M II with mesh wire tops, supplied by BECKER & Co., Castrop-Rauxel, Germany / 1 animal
- During Exposure: Wire cages , type DK I (BECKER & Co., Castrop-Rauxel, Germany) / 1 animal
- Enrichment: PLEXX mouse tunnel (red, transparent) and nest building material Nestlets NES 3600 (PLEXX b.v.; Elst, Netherlands).
- Type of diet: Kliba laboratory diet, mouse/rat maintenance “GLP”, 10 mm pellets, Provimi Kliba SA, Kaiseraugst, Basel Switzerland.
- Bedding: Dust-free wooden bedding
- Watering: Drinking water ad libitum
- Acclimatization: During the acclimatization period the animals were accustomed to the surroundings of the study and to the diet.

Route of administration:

inhalation: dust

Details on exposure:

Type of inhalation exposure: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Generator systems:
Solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany)
Mixing tube, stainless steel (BASF SE, Ludwigshafen, Germany)
- Generation procedure:
Dust aerosol was generated by means of brush generators using compressed air. The so generated aerosols were mixed with conditioned air and passed into the inhalation systems. The concentrations were adjusted by varying piston feed and brush rotation speed. The control group was exposed to conditioned air.

Homogeneity of the preparation during aerosol generation was guaranteed by means of technical measures.

Duration and frequency of treatment / exposure:

6 hours/day, 5 days/week, 14 weeks

Dose / conc.:

0 mg/m³ air (analytical)

Dose / conc.:

10 mg/m³ air (analytical)

Dose / conc.:

75 mg/m³ air (analytical)

No. of animals per sex per dose / concentration:

Main group 1 (male mice designated for clinical pathology and histopathology after 4 weeks): group 10 (0 mg/m^3) n=6, group 11 (10 mg/m^3) n=6, group 12 (75 mg/m^3) n=6

Main group 2 (male mice designated for clinical pathology and histopathology after 14 weeks): group 20 (0 mg/m^3) n=6, group 21 (10 mg/m^3) n=6, group 22 (75 mg/m^3) n=6

Satellite group 1 (male mice designated for organ and tissue distribution of Ga and As after 4 weeks exposure): group 30 (0 mg/m^3) n=1, group 31 (10 mg/m^3) n=5, group 32 (75 mg/m^3) n=5

Satellite group 2 (male mice designated for organ and tissue distribution of Ga and As after 14 weeks): group 40 (0 mg/m^3) n=1, group 41 (10 mg/m^3) n=5, group 42 (75 mg/m^3) n=5

Control animals:

yes

Positive control reference chemical:

not applicable

Details on study design:

- DOSE SELECTION RATIONALE:
By request of the sponsor, the following concentrations were selected for the present study based on the results of NTP (2000):
75 mg/m³: As high concentration causing toxic effects in lung, hematology, testicular effects and changes of sperm parameters.
10 mg/m³: As low concentration causing predominantly effects in the lung and to a minor degree also effects on the erythron and on sperm parameters.

- RATIONAL FOR SELECTING SATELLITE GROUPS: Animals designated for organ and tissue distribution of Ga and As

- POST-EXPOSURE OBSERVATION: The clinical condition of the test animals was recorded once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days. During exposure only a group wise examination was possible.


CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days, once a day on saturday, sunday, public holidays

SACRIFICE
Animals were deeply anesthetized by an intraperitoneal injection of a mixture of pentobarbital (e.g. Narcoren®, dose: 100 mg/kg body weight) and heparin (e.g. Heparin, Serva, Heidelberg, dose: 4mL/kg body weight).

Details on dosing and sampling:

FOOD ANALYSES:
The food used in the study was assayed for chemical and microbial contaminants. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p 27354 (EPA), served as the guideline for maximum tolerable contaminants. According to recommendations of the GV-SOLAS, the total amount of bacteria should not exceed 1*105 per g food.
The food was analyzed for Ga and As. Aliquots of the food were analyzed at Competence Center Analytics, BASF SE (Ludwigshafen, Germany) for content of Ga and As. The examination was performed under responsibility of the study director of the respective test facility without a GLP status.

DRINKING WATER ANALYSES
The drinking water was regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation served as the guideline for maximum tolerable contaminants.
Aliquot of tap water was analyzed for Ga and As. The sample was analyzed at Competence Center Analytics, BASF SE (Ludwigshafen, Germany) for content of Ga and As. The examination was performed under responsibility of the study director of the respective test facility without a GLP status.

ORGAN BURDEN:
Satellite groups 1 male mice were designated for organ and tissue distribution of Ga and As after 4 weeks exposure. Satellite groups 2 male mice were designated for organ and tissue distribution of Ga and As after 14 weeks exposure.
Blood, serum, spleen and testes were analyzed at Competence Center Analytics, BASF SE (Ludwigshafen, Germany) for content of Ga and As. The examination was performed under responsibility of the study director of the respective test facility without a GLP status.

Statistics:

Means, median and standard deviations were calculated.

In addition, the following statistical analyses were carried out:
- Body weight,body weight change, food and water consumption:
Comparison of each group with the control group was performed using DUNNETT test (two-sided) for the hypothesis of
equal means.
- Blood parameters:
For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
- Sperm parameters:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians; If only control and one dose group are measured, WILCOXON-test (one-sided)
without adjustment were used. For the percentage of abnormal sperms (ABNORMAL6_C) values < 6 % were set to 6 % (cut off 6 %). In case of exactly the same values of the dose group and the control, no statistical test is performed.
- Weight of the anesthetized animals and absolute and relative organ weights:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for equal medians.
- Temperature (rectal- and intratesticular):
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pai wise comparison of each dose group with the control group was performed using MANN-WHITNEY U-test (two-sided) for the equal medians

Details on distribution in tissues:

ORGAN BURDEN
The analysis of blood, spleen and testis of one control animal revealed that arsenic (As) and gallium (Ga) were both below the detection limit of 0.3 μg per sample.
As and Ga content in blood, spleen and testis after 4 weeks exposure were analysed. The values referred to μg per g tissue.
The sample weight of blood were between 0.11 g to 0.36 g, the weight of spleen ranged from 0.033 to 0.048 g and testis from 0.143 to 0.231 g.

4 WEEKS
- test group 31 (10 mg/m³)
blood: arsenic (As) and gallium (Ga) were both below the detection limit of 0.3 μg per sample
spleen: arsenic (As) and gallium (Ga) were both below the detection limit of 0.3 μg per sample
testis: arsenic (As) and gallium (Ga) were both below the detection limit of 0.3 μg per sample

test group 32 (75 mg/m³)
blood: arsenic (As) and gallium (Ga) were both below the detection limit of 0.3 μg per sample
spleen: As below the detection limit of 0.3 μg per sample; 12.2 [μg/g] Ga (mean)
testis: As below the detection limit of 0.3 μg per sample; 2.2 [μg/g] Ga (mean)

As and Ga content in blood, spleen and testis after 14 weeks exposure were analysed. The values refer to μg per g tissue. The sampleweight of blood was between 0.58 and 0.96 g, the weight of spleen ranged from 0.060 to 0.085 g and testis from 0.105 to 0.241 g. The blood samples were pooled group-wise, to increase the probability of detecting As and Ga.

14 WEEKS
- test group 41 (10 mg/m³)
blood: arsenic (As) and gallium (Ga) were both below the detection limit of 0.3 μg per sample
spleen: As below the detection limit of 0.3 μg per sample; 5.8 [μg/g] Ga (mean)
testis: As below the detection limit of 0.3 μg per sample; 1.7 Ga [μg/g] (mean)

test group 42 (75 mg/m³)
blood: arsenic (As) and gallium (Ga) were both below the detection limit of 0.3 μg per sample
spleen: As below the detection limit of 0.3 μg per sample; 26.9 [μg/g] Ga (mean)
testis: As below the detection limit of 0.3 μg per sample; 11.5 [μg/g] Ga (mean)

Metabolites identified:

no

Conclusions:

Organ burden was examined in animals exposed 4 weeks to GaAs. In control animal as well as in animals exposed to 10 mg/m³ GaAs, neither Ga nor As could be detected in blood, spleen and testis. In animals exposed to 75 mg/m³, Ga could be detected in blood of two single animals (0.8 and 0.9 μg/g blood). A very low amount of Ga could be determined in spleen (10.4 – 13.2 μg/g tissue) and testis (1.9 – 2.8 μg/g tissue), while As could not be detected in any tissues mentioned above.
After 14 weeks exposure, in pooled blood samples, trace amounts of As and Ga were found in a concentration-related manner, so was Ga in spleen and testis at higher concentrations. In pooled blood, As content was lower than that of Ga, and it was not found in spleen and testis.
The Ga and As ratio in blood is not consistent to the data achieved in the rat study (Ma-Hock 2016).

Executive summary:

The objective of the study was to assess the toxicity of the test substance Gallium arsenide (GaAs) after multiple exposures for a maximum of 14 weeks.

Male B6C3F1 mice were whole body exposed to GaAs at exposure concentrations of 10 and 75 mg/m³ for 6 hours per day, 5 days per week, for a duration of either 4 weeks or 14 weeks.

The target concentrations of 10 and 75 mg/m³ were reached.

A total of 15 cascade impactor measurements per concentration were performed, showing MMADs between 1.1 and 1.5 μm with a GSD between 2.0 and 2.6. The calculated mass fractions of particles below 3 μm aerodynamic size ranged between 83% and 92%.

The animals designated for determination of organ burden after 4 weeks exposure were assigned to test groups 30 (control group), 31 (10 mg/m³) and 32 (75 mg/m³), those lung burden animals after 14 weeks exposure were assigned to test groups 40 (control group), 41 (10 mg/m³) and 42 (75 mg/m³).

Inhalation exposure GaAs led to concentration-related alveolar proteinosis starting at 10 mg/m³ after 4 weeks exposure. Following 14 weeks exposure to 75 mg/m³, hypoxia was observed, as revealed by changes of several blood gas parameters. The hypoxia is most likely attributed to proteinosis in the lung. Moreover, in main group 2 animals exposed to 75 mg/m³ GaAs (14 weeks exposure) extra-medullary hematopoiesis in spleen, and degeneration of testis were observed. Both findings were consistent with data of hematological and sperm parameters, respectively. Effect in male reproductive system was only observed at 75 mg/m³, where proteinosis and hypoxia was present.

Organ burden was examined in animals exposed 4 weeks to GaAs. In control animal as well as in animals exposed to 10 mg/m³ GaAs, neither Ga nor As could be detected in blood, spleen and testis. In animals exposed to 75 mg/m³, Ga could be detected in blood of two single animals (0.8 and 0.9 μg/g blood). A very low amount of Ga could be determined in spleen (10.4 – 13.2 μg/g tissue) and testis (1.9 – 2.8 μg/g tissue), while As could not be detected in any tissues mentioned above.

After 14 weeks exposure, in pooled blood samples, trace amounts of As and Ga were found in a concentration-related manner, so was Ga in spleen and testis at higher concentrations. In pooled blood, As content was lower than that of Ga, and it was not found in spleen and testis. The Ga and As ratio in blood is not consistent to the data achieved in the rat study (Ma-Hock 2016).

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2015-07-01 to 2016-08-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Objective of study:

other: Subchronic Inhalation Toxicity, 90-day Study

Qualifier:

according to guideline

Guideline:

other: OECD 413

Version / remarks:

Subchronic Inhalation Toxicity- 90-day Study with determination of organ burden after 4 weeks and 14 weeks exposure.

Principles of method if other than guideline:

- Principle of test: according to OECD 413

- Short description of test conditions:
Male Wistar rats were whole body exposed to GaAs at exposure concentrations of 10 and 75 mg/m³ for 6 hours per day, 5 days per week, for a duration of either 4 weeks or 14 weeks.
The animals designated for clinical chemical and histological examinations after 4 weeks exposure were assigned to groups 10 (control group), 11 (10 mg/m³) and 12 (75 mg/m³).
The animals examined for clinical chemical and histological examinations after 14 weeks exposure were assigned to groups 20 (control group), 21 (10 mg/m³) and 22 (75 mg/m³).
In addition, the animals designated for determination of organ burden after 4 weeks exposure were assigned to test groups 30 (control group), 31 (10 mg/m³) and 32 (75 mg/m³), those after 14 weeks exposure were assigned to test groups 40 (control group), 41 (10 mg/m³) and 42 (75 mg/m³).

- Parameters analysed / observed:
Blood, serum, spleen and testes were analyzed at Competence Center Analytics, BASF SE (Ludwigshafen, Germany) for content of Ga and As.

GLP compliance:

yes

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

- Reason for selection of the test species:
The rat is the most frequently used laboratory animal, and there is extensive experience with this species. The rat is also proposed as suitable test animal by OECD and the EPA. The National Toxicology Program (NTP) performed several studies in F344 rats to characterize the toxic profile of the test substance. As a mechanistic study, this study should be performed in rats of the same strain as in the NTP studies. However, F344 rats were no more commercially available in Europe. Therefore, Wistar strain was selected because a huge amount of historical control data is available for this strain. As the effects of interest were observed in male rats, only males were used in this study.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Test species and strain: Wistar rats, Crl:WI(Han)
- Age when supplied; sex: about 8 weeks, male
- Age at start of exposure about 9 weeks
- Supplier: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Arrival in the testing facility: 06 Jan 2015
- Identification: The animals were identified individually by tattooing the respective animal number into the ears (serial)
- All animals were randomized before the start of the pre-exposure period (according to weight).
Only animals free from clinical signs of disease were used for the study.

ENVIRONMENTAL CONDITIONS
- Air conditions: Temperature 20 to 24°C, relative humidity 30 to 70%. 15 air changes per hour. Deviations from these ranges did not occur.
- Illumination period: 06.00 a.m. - 06.00 p.m. light, 06.00 p.m. - 06.00 a.m. dark
- Type of cage / No. of animals per cage: Polysulfon cages (H-Temp [PSU]), floor area about 2065 cm2 (610x435x215 mm); supplied by TECNIPLAST, Germany / up to 6 animals
- During Exposure: Wire cages , type DK III (BECKER & Co.,Castrop-Rauxel, Germany) / up to 2 animals
- Enrichment:Wooden gnawing blocks (Type NGM E-022); Abedd  Lab. and Vet. Service GmbH Vienna, Austria and Play Tunnel, large (Art.14153); PLEXX b.v., Elst, Netherlands
- Type of diet: Kliba laboratory diet, mouse/rat maintenance “GLP”, 10 mm pellets, Provimi Kliba SA, Kaiseraugst, Basel Switzerland.
- Bedding: Dust-free wooden bedding
- Watering: Drinking water ad libitum
- Acclimatization: During the acclimatization period the animals were accustomed to the surroundings of the study and to the diet.

Route of administration:

inhalation: dust

Details on exposure:

Type of inhalation exposure: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Generator systems:
Solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany)
Mixing tube, stainless steel (BASF SE, Ludwigshafen, Germany)
- Generation procedure:
Dust aerosol was generated by means of brush generators using compressed air. The so generated aerosols were mixed with conditioned air and passed into the inhalation systems. The concentrations were adjusted by varying piston feed and brush rotation speed. The control group was exposed to conditioned air.

Homogeneity of the preparation during aerosol generation was guaranteed by means of technical measures.

Duration and frequency of treatment / exposure:

6 hours/day, 5 days/week, 14 weeks

Dose / conc.:

0 mg/m³ air (analytical)

Dose / conc.:

10 mg/m³ air (analytical)

Dose / conc.:

75 mg/m³ air (analytical)

No. of animals per sex per dose / concentration:

Main group 1 (male rats designated for clinical pathology and histopathology after 4 weeks): group 10 (0 mg/m^3) n=6, group 11 (10 mg/m^3) n=6, group 12 (75 mg/m^3) n=6

Main group 2 (male rats designated for clinical pathology and histopathology after 14 weeks): group 20 (0 mg/m^3) n=6, group 21 (10 mg/m^3) n=6, group 22 (75 mg/m^3) n=6

Satellite group 1 (male rats designated for organ and tissue distribution of Ga and As after 4 weeks exposure): group 30 (0 mg/m^3) n=1, group 31 (10 mg/m^3) n=5, group 32 (75 mg/m^3) n=5

Satellite group 2 (male rats designated for organ and tissue distribution of Ga and As after 14 weeks): group 40 (0 mg/m^3) n=1, group 41 (10 mg/m^3) n=5, group 42 (75 mg/m^3) n=5

Control animals:

yes

Positive control reference chemical:

not applicable

Details on study design:

- DOSE SELECTION RATIONALE:
By request of the sponsor, the following concentrations were selected for the present study based on the results of NTP (2000):
75 mg/m³: As high concentration causing toxic effects in lung, hematology, testicular effects and changes of sperm parameters.
10 mg/m³: As low concentration causing predominantly effects in the lung and to a minor degree also effects on the erythron and on sperm parameters.

- RATIONAL FOR SELECTING SATELLITE GROUPS: Animals designated for organ and tissue distribution of Ga and As

- POST-EXPOSURE OBSERVATION: The clinical condition of the test animals was recorded once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days. During exposure only a group wise examination was possible.


CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days, once a day on saturday, sunday, public holidays

SACRIFICE
Animals were deeply anesthetized by an intraperitoneal injection of a mixture of pentobarbital (e.g. Narcoren®, dose: 100 mg/kg body weight) and heparin (e.g. Heparin, Serva, Heidelberg, dose: 4mL/kg body weight).

Details on dosing and sampling:

FOOD ANALYSES:
The food used in the study was assayed for chemical and microbial contaminants. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p 27354 (EPA), served as the guideline for maximum tolerable contaminants. According to recommendations of the GV-SOLAS, the total amount of bacteria should not exceed 1*105 per g food.
The food was analyzed for Ga and As. Aliquots of the food were analyzed at Competence Center Analytics, BASF SE (Ludwigshafen, Germany) for content of Ga and As. The examination was performed under responsibility of the study director of the respective test facility without a GLP status.

DRINKING WATER ANALYSES
The drinking water was regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation served as the guideline for maximum tolerable contaminants.
Aliquot of tap water was analyzed for Ga and As. The sample was analyzed at Competence Center Analytics, BASF SE (Ludwigshafen, Germany) for content of Ga and As. The examination was performed under responsibility of the study director of the respective test facility without a GLP status.

ORGAN BURDEN:
Satellite groups 1 male rats were designated for organ and tissue distribution of Ga and As after 4 weeks exposure. Satellite groups 2 male rats were designated for organ and tissue distribution of Ga and As after 14 weeks exposure.
Blood, serum, spleen and testes were analyzed at Competence Center Analytics, BASF SE (Ludwigshafen, Germany) for content of Ga and As. The examination was performed under responsibility of the study director of the respective test facility without a GLP status.

Statistics:

Means, median and standard deviations were calculated.

In addition, the following statistical analyses were carried out:
- Body weight,body weight change, food and water consumption:
Comparison of each group with the control group was performed using DUNNETT test (two-sided) for the hypothesis of
equal means.
- Blood parameters:
For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
- Sperm parameters:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians; If only control and one dose group are measured, WILCOXON-test (one-sided)
without adjustment were used. For the percentage of abnormal sperms (ABNORMAL6_C) values < 6 % were set to 6 % (cut off 6 %). In case of exactly the same values of the dose group and the control, no statistical test is performed.
- Weight of the anesthetized animals and absolute and relative organ weights:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for equal medians.
- Temperature (rectal- and intratesticular):
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pai wise comparison of each dose group with the control group was performed using MANN-WHITNEY U-test (two-sided) for the equal medians

Details on distribution in tissues:

ORGAN BURDEN

The analysis of organ tissues and blood of one control animal revealed a low background level of arsenic (As), whereas gallium (Ga) could not be detected (detection limit 0.3 μg per sample).

As and Ga content in blood, spleen, serum and testis after 4 weeks and 14 weeks exposure were analysed. The values referred to μg per g tissue.
The sample weights of blood and serum were around 1 g, the weight of spleen ranged from 0.56 to 0.87 g and testis from 2.95 to 3.76 g.

4 WEEKS
- test group 31 (10 mg/m³)
blood: 36.6 [μg/g] (mean) As ; - Ga [μg/g]
spleen: 10.4 [μg/g] (mean) As; 3.0 [μg/g] Ga (mean)
serum: - As [μg/g]; 0.4 Ga [μg/g] (mean)
testis: 0.2 [μg/g] (mean) As ; 0.5 Ga [μg/g] (mean)

test group 32 (75 mg/m³)
blood: 181.5 [μg/g] (mean) As; 0.8 [μg/g] (mean) Ga
spleen: 44.9 [μg/g] (mean) As; 13.8 [μg/g] Ga (mean)
serum: - As [μg/g]; 1.5 [μg/g] Ga (mean)
testis: 1.1 [μg/g] (mean) As; 2.2 [μg/g] Ga (mean)


14 WEEKS
- test group 41 (10 mg/m³)
blood: 111.5 [μg/g] (mean) As ; 0.3 Ga [μg/g] (mean)
spleen: 41.1 [μg/g] (mean) As; 18.3 [μg/g] Ga (mean)
serum: - As [μg/g]; 0.5 Ga [μg/g] (mean)
testis: 0.6 [μg/g] (mean) As; 2.3 Ga [μg/g] (mean)

test group 42 (75 mg/m³)
blood: 244.4 [μg/g] (mean) As; 1.0 [μg/g] (mean) Ga
spleen: 91.3 [μg/g] (mean) As; 59.6 [μg/g] Ga (mean)
serum: - As [μg/g]; 1.7 [μg/g] Ga (mean)
testis: 2.8 [μg/g] (mean) As; 13.1 [μg/g] Ga (mean)

Metabolites identified:

no

Conclusions:

Organ burden was examined in animals exposed 4 and 14 weeks to GaAs. Higher organ burden was associated to the high exposure concentration of 75 mg/m³. The overall data showed a certain bio-availability of the test substance.

Executive summary:

The objective of the study was to assess the toxicity of the test substance Gallium arsenide (GaAs) after multiple exposures for a maximum of 14 weeks. Male Wistar rats were whole body exposed to GaAs at exposure concentrations of 10 and 75 mg/m³ for 6 hours per day, 5 days per week, for a duration of either 4 weeks or 14 weeks.

The target concentrations of 10 and 75 mg/m³ were reached. A total of 15 cascade impactor measurements per concentration were performed, showing MMADs between 1.1 and 1.5 μm with GSDs between 2.0 and 2.6. The calculated mass fractions of particles below 3 μm

aerodynamic size ranged between 83% and 92%.

The animals designated for determination of organ burden after 4 weeks exposure were assigned to test groups 30 (control group), 31 (10 mg/m³) and 32 (75 mg/m³), those after 14 weeks exposure were assigned to test groups 40 (control group), 41 (10 mg/m³) and 42 (75 mg/m³).

Inhalation exposure GaAs led to concentration-related alveolar proteinosis starting at 10 mg/m³ after 4 weeks exposure. Following 14 weeks exposure to 10 and 75 mg/m³, hypoxia was observed, as revealed by changes of several blood gas parameters. The hypoxia is most likely attributed to proteinosis in the lung. Moreover, extra-medullary haematopoiesis in spleen at 10 mg/m³, and degeneration of testis at 75 mg/m³ in animals exposed for 14 weeks were observed. Both findings were consistent with data of haematological and sperm parameters, respectively. In addition, a slight chronic progressive nephropathy and minimal glomerulopathy were observed in kidneys.

Organ burden was examined in animals exposed 4 and 14 weeks to GaAs. Higher organ burden was associated to the high exposure concentration of 75 mg/m³. The overall data showed a certain bio-availability of the test substance. High As content was observed in blood, but was not detected in serum, indicating As was associated to the blood cells. This finding is consistent with the published data that inorganic and organic arsenic compound do have high affinity to rat hemoglobin (Lu et al. 2004).

Exposure to 10 mg/m³ GaAs for 14 weeks led to 3 times higher As concentration in blood than after 4 weeks. At the high concentration of 75 mg/m³ however, the As concentration in animals exposed for 14 weeks were only 1.35 times higher than those exposed for 4 weeks. This findings indicated a potential saturation of binding to hemoglobin at the high concentration of 75 mg/m³, but not at 10 mg/m³.

Traces of Ga was found both in blood and serum, with slightly higher concentration in serum than in blood. Whereas As content increased significantly with the course of the exposure, Ga content did not change much.

Both As and Ga were found in spleen. As content (by weight and by molarity) in spleen was higher than Ga, which may associated with the high affinity of arsenic compound to hemoglobin. Exposure for 14 weeks increased both As and Ga content in spleen.

In contrast to spleen and blood, only traces of As and Ga were found in testis, with higher Ga content than As. With increased exposure duration from 4 to 14 weeks, the content of both elements increased proportionally, indicating a certain accumulation potential.

The overall data of organ burden in blood, serum and testis were consistent to the reported data of NTP study after 2 year inhalation exposure.

Additional information

There are 17 studies dealing with the metabolism and kinetics of GaAs. Unfortunately 13 of them are of very doubtful quality so that their results are not reliable.

Two studies were performed together with other NTP studies, well documented, and performed under a quality assurance system: a 90-day (14 -week) rat (F344N) study and a 2-year rat (F344N) study, both with inhalative exposure:

These and the other NTP studies (NTP, 2000) with rats and mice revealed that after repeated inhalative exposure to a fine dust of GaAs a local reaction in the lung is triggered. This reaction can be described as pulmonary alveolar proteinosis leading to chronic active inflammation.

The toxicokinetic investigations revealed:

- Lung burden analyses for male rats in the 14-week and 2-year studies indicates that the percentages of Gallium and Arsenic in the lung relative to the total lung burden were similar at all exposure concentrations throughout the study.

- Lung burden for gallium and arsenic increased proportionally throughout the 14-week or 2-year studies.

- However lung clearance half-lives decreased with increasing exposure concentrations. The clearance half-live of 37 days for Gallium in the 1.0 mg GaAs/m³ group was considerably less than the 133 or 96 days for the 0.01 or 0.1 mg GaAs/m³ groups in the 2-year study, respectively, Arsenic half-lives were similar.

- In the 2-year toxicity study with inhalative exposure of male rats to concentrations of 0, 0.01, 0.1, 1 mg GaAs/m³ lung weights increased in all male rats exposed to 0.1 or 1 mg/m³ throughout the study when compared to chamber controls and the 0.01 mg/m³ group.

- Lung burden for Gallium and Arsenic increased with increasing exposure concentration. It appears that a steady state lung burden was achieved for the 0.1 and possibly the 1.0 mg/m³ groups after 6 months, although the lung burdens at 18 months were low.

- The Gallium concentrations in blood, serum, and testes were small relative to the concentrations of Gallium and Arsenic in the lung. This indicates that there was no accumulation of either Gallium or Arsenic in these tissues.

- As expected Arsenic was detected in whole blood where it is preferentially bound to erythrocytes. The means of the Arsenic concentrations in whole blood were greater than those of chamber controls only in the 1.0 mg/m³ group where they were approximately 2-fold higher, but this difference was not statistically significant.

- The blood levels of Gallium and Arsenic of the rats in the 0.01 or 0.1 mg GaAs/m³ groups were similar to those of the control animals.

(In the NTP-report a half-life of 1 to 2 hours is given for plasma gallium levels as a citation from studies of Dudley et al., 1949.

Unfortunately the original publication does not contain such a statement. All 5 cited publications of Dudley et al. give only very poor and insufficient data on the performed investigations. Therefore this literature is classed with the reliability 4 (not assignable) according to Klimisch et al., 1997, and is not used for the evaluation.)

The good consistency of the data between all the NTP studies supports the reliability of the results of the 2 toxicokinetic studies.

Two studies according to OECD 413 were performed by Ma-Hock 2016. Male Wistar rats and male B6C3F1 mice were whole body exposed to GaAs at exposure concentrations of 10 and 75 mg/m³ for 6 hours per day, 5 days per week, for a duration of either 4 weeks or 14 weeks.

Male B6C3F1 mice:

In B6C3F1control animals as well as in B6C3F1 animals exposed to 10 mg/m³ GaAs, neither Ga nor As could be detected in blood, spleen and testis. In animals exposed to 75 mg/m³, Ga could be detected in blood of two single animals (0.8 and 0.9 μg/g blood). A very low amount of Ga could be determined in spleen (10.4 – 13.2 μg/g tissue) and testis (1.9 – 2.8 μg/g tissue), while As could not be detected in any tissues mentioned above. After 14 weeks exposure, in pooled blood samples, trace amounts of As and Ga were found in a concentration-related manner, so was Ga in spleen and testis at higher concentrations. In pooled blood, As content was lower than that of Ga, and it was not found in spleen and testis. The Ga and As ratio in blood is not consistent to the data achieved in the rat study (Ma-Hock 2016).

Male Wistar rats:

Higher organ burden was associated to the high exposure concentration of 75 mg/m³. The overall data showed a certain bio-availability of the test substance. High As content was observed in blood, but was not detected in serum, indicating As was associated to the blood cells. This finding is consistent with the published data that inorganic and organic arsenic compound do have high affinity to rat hemoglobin (Lu et al. 2004).

Exposure to 10 mg/m³ GaAs for 14 weeks led to 3 times higher As concentration in blood than after 4 weeks. At the high concentration of 75 mg/m³ however, the As concentration in animals exposed for 14 weeks were only 1.35 times higher than those exposed for 4 weeks. This findings indicated a potential saturation of binding to hemoglobin at the high concentration of 75 mg/m³, but not at 10 mg/m³.

Traces of Ga was found both in blood and serum, with slightly higher concentration in serum than in blood. Whereas As content increased significantly with the course of the exposure, Ga content did not change much.

Both As and Ga were found in spleen. As content (by weight and by molarity) in spleen was higher than Ga, which may associated with the high affinity of arsenic compound to hemoglobin. Exposure for 14 weeks increased both As and Ga content in spleen.

In contrast to spleen and blood, only traces of As and Ga were found in testis, with higher Ga content than As. With increased exposure duration from 4 to 14 weeks, the content of both elements increased proportionally, indicating a certain accumulation potential.

The overall data of organ burden in blood, serum and testis were consistent to the reported data of NTP study after 2 year inhalation exposure.