Gallium arsenide

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-08-01 to 2016-09-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Test species and strain: B6C3F1 mice
- Reason for selection of the test species:It is 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.
- 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

Type of inhalation exposure:

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

ca. 1.1 - ca. 1.5 µm

Details on inhalation exposure:

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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples were analyzed by gravimetric measurement. This method gives solid particle concentrations in the inhalation atmospheres. The gravimetric method was validated in technical trial run. The constancy of concentrations in the inhalation atmospheres was surveyed continuously with scattered light photometers.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
0 mg/m3
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
10 mg/m3
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
75 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

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 exposure): 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

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.

- Rationale 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.

Positive control:

not applicable

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly thereafter until one day prior to gross necropsy

FOOD CONSUMPTION: determined twice weekly and calculated as mean food consumption in grams per animal and day.

WATER CONSUMPTION: determined weekly and calculated as mean water consumption in grams per animal and day.

RECTAL TEMPERATURE: determined in all main group animals prior to exposure period and approach of the end of the exposure

CLINICAL PATHOLOGY:
Blood was taken from the retrobulbar venous plexus in the morning from overnight fasted animals without anesthesia for blood gases measurement and hematology. Afterwards, the animals were immediately 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). After intra-testicular temperature measurement both brachial vessels were opened and the dripping was collected for clinical chemistry. The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence.
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The following examinations were carried out in 6 male animals per test group after 4 weeks or 14 weeks.

SPERM PARAMETERS: Immediately after necropsy and organ weight determination the right testis and cauda epididymidis were taken from all male animals. Sperm motility examinations were carried out immediately after necropsy in a randomized sequence, as well as sperm morphology. For sperm head count, the right testis and right cauda epididymidis were deep frozen at -20°C until evaluation.

ORGAN BURDEN:
Satellite groups 1 male mice designated for organ and tissue distribution of Ga and As after 4 weeks exposure. Satellite groups 2 male mice 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.
Lungs, lung associated lymph nodes and epididymides were stored. They may be analyzed on request of the sponsor.

INTRA-TESTICULAR TEMPERATURE: During anesthesia the right intra-testicular temperature was measured with an appropriate probe thermometer

Sacrifice and pathology:

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).

PATHOLOGY
Main group 1 (4 week exposure) and 2 (14 weeks exposure).
- Organ weights: Adrenal glands, Brain, Cauda epididymis, Epididymides, Heart, Kidneys, Liver, Lung, Prostate, Spleen, Seminal vesicles incl. coagulating gland, Testes, Thymus
- Histopathology: All gross lesions, Bone marrow (femur), Epididymidis, left, Kidneys, Lungs (5 lobes), Lymph nodes (mandibular, tracheobronchial/
mediastinal), Spleen, Sternum with marrow, Testis, left

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 (twosided) 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

Clinical signs:

no effects observed

Description (incidence and severity):

During the pre-exposure period all animals were free of clinical signs and findings different from normal. During the exposure period, fracture of one lower teeth was noted in animals No. 28 from study day 96 onward. In animal No. 60, gasping and respiration sound were noted on study days 42 and 43, in each case after exposure. Other animals of all groups showed no clinical signs and findings different from normal.
The teeth fracture is likely to be due to mechanical force. The respiration sound and gasping observed only in one animal of the low concentration group, only on two days after exposure.
Due to the lack of concentration response relationship, the transient clinical signs were considered incidental and not substance-related.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weight of the animals exposed to 10 or 75 mg/m³ GaAs were not different from the concurrent control animals.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The significantly increased food consumption of animals either to 10 mg/m³ or to 75 mg/m³ GaAs were considered due to food spreading by the animals.
In animals exposed to 75 mg/m³, food consumptions decreased between day 79 through to day 89. As this finding was only observed in high concentration group animals at the end of the exposure period, it was considered substance-related. As no significant decrease of body weight and body weight changes were observed in these animals during this time period, the decreased food consumption was considered biologically not relevant.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, non-treatment-related

Description (incidence and severity):

The decreased water consumption was considered incidental, because it was only observed in animals exposed to the low concentration of 10 mg/m³ GaAs designated for sacrificing after 14 weeks exposure, not in the animals exposed to the same concentration, but designated for sacrificing after 4 weeks exposure.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

After four weeks of compound administration in males of test group 12 (75 mg/m3) mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were decreased. Hemoglobin values were slightly decreased in males of test group 12 (75 mg/m3; median – 7% compared to controls) although not statistically significantly because of a higher variation in the control individuals. MCV was already lower in males of test group 1 (10 mg/m3), but in these individuals no measured red blood cell parameter (i.e., hemoglobin, hematocrit and red blood cell (RBC) counts) was changed and therefore the alteration of MCV in this test group was
regarded as incidental and not treatment-related.
After 14 weeks of inhalation red blood cell (RBC) counts were increased and hemoglobin values, MCV, MCH as well as mean corpuscular hemoglobin concentration (MCHC) were decreased in males of test group 22 (75 mg/m3). MCV and MCH were already decreased in mice of test group 21 (10 mg/m3), but in this test group no measured red blood cell parameter (i.e., hemoglobin, hematocrit and RBC counts) was changed and therefore the alterations of MCV and MCH in this test group were regarded as incidental and not treatment-related.
After 14 weeks exposure, absolute and relative eosinophil counts of test group 21 (10 mg/m3) animals were higher compared to controls but the change was not dose-dependent and therefore it was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

After four weeks of inhalation exposure only blood for four parameters (alanine aminotransferase (ALT), urea, albumin and cholesterol) could be sampled due to technical issues. None of the parameters was changed in the test groups compared to controls.
After 14 weeks of inhalation in males of test group 22 (75 mg/m3) alkaline phosphatase (ALP) activities were lower and urea values were higher compared to controls. ALP activities were not dose-dependently changed, as mean and median of test group 22 were higher than that of test group 21. Therefore, this alteration was regarded as incidental and not treatment-related.
Urea increase was the only changed clinical chemistry parameter and therefore this change was regarded as treatment-related but not adverse (ECETOC Technical Report No. 85, 2002).

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute organ weights in main group 1 animals (4 week exposure):
When compared with control group 0 (=100%), the mean absolute weights of lungs were significantly increased in test group 11 (10 mg/m^3) and in test group 12 (75 mg/m^3). When compared with control group 0 (=100%), the mean absolute weights of Seminal vesicles were significantly increased in test group 12 (75 mg/m^3). All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights in main group 1 animals (4 week exposure):
When compared with control group 0 (=100%), the mean relative organ weights of liver in test group 12 (75 mg/m^3) and the mean relative organ weights of lungs in test group 11 (10 mg/m^3) and in test group 12 (75 mg/m^3) were significantly increased.

The absolute and relative weight increase of the lungs had a histopathological correlate and was regarded as treatment-related and adverse. The significant relative weight increase of the liver in males of test group 12 correlated with histopathological findings that were considered treatment-related but not adverse. A conclusive interpretation of the weight increases of the seminal vesicles in test group 12 cannot be made because no histopathology of this organ was performed. However, no weight deviations of this organ were seen after 14 weeks of treatment. Thus, the weight change after 4 weeks was regarded as transient and not adverse.

Absolute organ weights in main group 2 animals (14 week exposure):
The absolute and relative weight changes in cauda epididymis, epididymides, lungs and testes had a histopathological correlate and were regarded as treatment-related and adverse.

All other mean absolute and relative weight parameters did not show significant differences when compared to the control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Main group 1 (4 week exposure)
In test group 12 (75 mg/m³), the lung of 5 out of 6 animals showed a grey discoloration, which was considered treatment-related. The acinar pattern in the liver of one male correlated with fatty change and was regarded as treatment-related. Black discolorations were noted in the contents of jejunum and cecum of animals in all test groups including control animals. This finding is characteristic of melena, a well-known background lesion found in mice of this strain, associated with upper gastrointestinal bleeding of mucosal erosions.
All other findings occurred either individually or were equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
In main group 1 animals (4 weeks exposure), the mediastinal/tracheobronchial lymph nodes were activated with secondary follicles and increased number of plasmocytes at 75 mg/m3 (minimal to slight) (4 out of 6 animals).

Main group 2 (14 weeks exposure)
All animals of test group 22 (75 mg/m³) had a grey discoloration of the lungs and a reduced size of the testes. Only one animal showed reduced size of the epididymides. All of these findings had a histopathological correlate and were considered treatment-related. All other findings occurred spontaneously in the control group and were considered to be incidental.
In main group 2 animals (14 weeks exposure), lymph nodes were still activated at 75 mg/m3 suggesting a continuous immunologic stimulus(minimal). Black particles, most likely representing GaAs, were visualized in macrophages at 10 mg/m3, whereas at 75 mg/m3, the number of macrophages and their particle content was slightly increased, representing an increased clearing of foreign material from the lung. Since no cellular toxicity or other alterations were detected, these changes were regarded as treatment-related but not adverse.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology of main group 1 animals (4 weeks exposure):
Treatment-related findings were observed in the liver, lungs and mediastinal/tracheobronchial lymph nodes. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
LIVER:
Compared to the controls, the liver of animals in test group 12 (75 mg/m³) showed an increased incidence and grading of the cytoplasmic microvesicular pattern of hepatocytes. Furthermore, the microvesiculation was accentuated centrilobularly.
LUNG:
Alveolar proteinosis was characterized by accumulation of material, which varied from light eosinophilic granules to bright eosinophilic compact globular masses in the alveolar lumina. This proteinaceous material was PAS positive for the detection of glycoproteins. Test group 11 (10 mg/ m³) and test group 12 (75 mg/m³) showed an increased incidence of alveolar proteinosis.
Very few black refracting particles (grade 1) of irregular shape and size (< 1μm) were visualized at 200x to 400x magnification. They most likely represent GaAs, and were seen admixed within the proteinaceous material and in the histiocytosis (macrophages with phagocytosed particles and eosinophilic material) in test group 11 (10 mg/ m³) and test group 12 (75 mg/m³).
Bronchi, bronchioles and terminal bronchioles showed in some animals minimal to slight epithelial hypertrophy/hyperplasia (test group 12 (75 mg/m³)). In one animal, multifocal hypertrophy of alveolar type II pneumocytes was seen.
LYMPH NODES:
Lympho-reticulocellular hyperplasia was shown in test group 12 (75 mg/m³). Lympho-reticulocellular hyperplasia was characterized by increased cellularity and the presence of secondary follicles accompanied by increased number of cord plasmocytes.


Histopathology of main group 2 animals (14 weeks exposure):
Treatment-related findings were observed in the lungs, mediastinal/tracheobronchial lymph nodes, left testis, left epididymis and spleen.
All other findings occurred either individually or were equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
LUNG:
Compared to main group 1 (4 weeks treatment), histopathological findings were quantitatively increased. The alveolar proteinosis increased in severity by affecting a greater number of alveoli (grade 3 to 4). Bright eosinophilic compact masses that almost completely filled the alveolar lumina predominated over the granular eosinophilic material. Both types stained PAS positive for the detection of glycoproteins. Black particles within the eosinophilic masses were more abundant in the high concentration group. Histiocytosis, characterized by macrophages filled with eosinophilic material and black particles was minimal in both concentration groups. The epithelial hypertrophy/hyperplasia affected bronchi, bronchioles and terminal bronchioles. Alveoli showed type II cell hypertrophy in the high concentration group.
LYMPH NODES:
Lympho-reticulocellular hyperplasia, characterized by increased lymphoid cellularity, and/or secondary follicles, and/or plasmocytosis was minimal. The particle storage grade 1 was characterized by the presence of very few macrophages storing very few particles; in grade 2,
the number of macrophages and particles engulfed by the macrophages was slightly increased.
LEFT TESTIS:
Degeneration grade 1 was characterized by the presence of delayed spermatid release and abnormal residual bodies in very few stage VIII tubules. Grade 2 was characterized by few more tubules with additional stage IX to XI tubules with step 16 spermatid retention at the tubular base, and architectural disorganization of the germinal epithelium. The most severe degeneration was characterized by multifocal to coalescent (grade 4) or diffuse (grade 5) tubular atrophy, characterized by depletion of germinal epithelium, with only few spermatogonia, Sertoli cells and spermatocytes lining the tubules.
LEFT EPIDIDYMIDIS:
In association with the testicular findings, cellular debris in the epididymidis ducts increased from test group 21 to 22, with all animals of test group 22 showing aspermia. One of them had additional ductal atrophy, characterized by generalized narrowing of the ductal lumina, increased epithelial height, and focal infolding of the epithelium in the proximal cauda.
SPLEEN:
Compared to the control animals, a minimal extramedullary hematopoiesis was present at the high concentration group.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Sperm parameters:
After four weeks of inhalation no treatment-related effects were observed concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis.
After 14 weeks of exposure in males of test group 22 (75 mg/m3) all found sperms were nonmotile and the sperm head counts in the testis and the cauda epididymidis were decreased. Because of the low counts of sperms in this test group the sperm morphology of only one animal could be assessed, with 79 % abnormal sperms found. At 10 mg/m3, the percentage of abnormal sperms was already increased with most of the sperms having abnormal hooks or headless sperms whereas the sperm head count in the cauda epididymidis was decreased in mice of test group 21.

Details on results:

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:
At 10 mg/m³ none of the elements were detected in blood, spleen and testis. At 75 mg/m³, trace amounts of Ga could be detected in spleen and testis, whereas As content was still below the detection limit. Considering the low amount of blood per animal, the methods used to analyze As and Ga may be not sensitive enough to detect these elements in the blood.
As and Ga content in blood, spleen, and testis after 14 weeks exposure:
In blood, only trace amounts of As and Ga were detected at 10 and 75 mg/m³. The content of As was lower than that of Ga. In spleen and testis, As was not detectable, whereas Ga could be found in a concentration-related manner.

VENOUS BLOOD GAS ANALYSIS
After four weeks of exposure base excess (BE, median -85% compared to controls), calculated hydrogen carbonate concentration (cHCO3, median -19 %) as well as partial oxygen (pO2, median – 11%) and partial carbon dioxide pressure (pCO2, median -11%) in venous blood of males of test group 12 (75 mg/m3) were decreased although only BE was statistically significantly changed. BE was already lower in males of test group 11 (10 mg/m3; medians - 47% not statistically significant) but in this test group this was the only changed parameter of the blood gas analysis and therefore it was regarded as treatment-related but not adverse (ECETOC Technical Report No 85, 2002).
After 14 weeks of inhalation in males of test group 22 (75 mg/m3) BE (medians -24%), cHCO3 (medians -10%) and pCO2 (medians – 9%) were still lower compared to controls, although not statistically significantly.

Dose descriptor:

LOAEC

Effect level:

10 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Dose descriptor:

LOAEC

Effect level:

10 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Increased percentage of abnormal sperms and decreased sperm head counts in the cauda epididymidis

Dose descriptor:

LOAEC

Effect level:

75 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

haematology

Critical effects observed:

yes

Lowest effective dose / conc.:

75 mg/m³ air (nominal)

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

10 mg/m³ air (nominal)

System:

male reproductive system

Organ:

cauda epididymis

testes

other: Increased percentage of abnormal sperms and decreased sperm head counts in the cauda epididymidis

Treatment related:

yes

Conclusions:

Inhalative exposure to GaAs (MMAD 1.1-1.5 µm) 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. Analysis of the elements gallium and arsenic revealed trace amounts of these elements in blood, spleen and testis starting at 75 mg/m³ after 4 weeks exposure in spleen and testis.
Basically, the overall hematological, histopathological and sperm findings reported by NTP (2000) were reproduced.

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. Mortality, clinical findings, body weight, food consumption, water consumption, and rectal temperature of the mice were determined during the study. Parameters of clinical pathology, intra-testicular temperature and histopathology were evaluated after 4 weeks and 14 weeks. Clinical pathological parameters comprised hematology, clinical chemistry (including hemoglobin in serum), blood gas measurements and sperm parameters. A complete necropsy including gross pathological evaluation and weighing of selected organs was performed. Histological examinations comprised bone marrow, epididymis, kidneys, liver, lungs, lymph nodes (mandibular, tracheobronchial, mediastinal), spleen, sternum with marrow and testis (left).

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). The target concentrations were reached.

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. Analysis of the elements gallium and arsenic revealed trace amounts of these elements in blood, spleen and testis starting at 75 mg/m³ after 4 weeks exposure in spleen and testis.

Basically, the overall hematological, histopathological and sperm findings reported by NTP (2000) were reproduced.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Version / remarks:

OECD 413

GLP compliance:

yes (incl. QA statement)

Type of method:

in vivo

Test material

Test material information

Constituent 1

Reference substance name:

gallium arsenide - Mechanically destroyed material not marketed.

IUPAC Name:

gallium arsenide - Mechanically destroyed material not marketed.

Details on test material:

Name of test substance: Gallium arsenide (GaAs)
Test substance No.: 14/0492-3
Batch identification: W417/14 milled
CAS No.: 1303-00-0
Purity: As: 49 +/-2 Mass-%
Ga: 50 +/-2 Mass -%
Homogeneity: given (vis.)
Physical state/ appearance: Solid / grey
Storage conditions: Room temperature, dry storage

Particle size distribution of target concentration 10 mg/m^3 and 75 mg/m^3:
- Mass median aerodynamic diameter (MMAD): 1.1 - 1.5 μm
- Geometrical standard deviation (GSD): 2.0 - 2.6
- calculated mass fractions of particles below 3 μm aerodynamic size: 83% - 92%

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Test species and strain: B6C3F1 mice
- Reason for selection of the test species:It is 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.
- 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.

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

MMAD 1.1-1.5 µm

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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples were analyzed by gravimetric measurement. This method gives solid particle concentrations in the inhalation atmospheres. The gravimetric method was validated in technical trial run. The constancy of concentrations in the inhalation atmospheres was surveyed continuously with scattered light photometers.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air

Doses / concentrations 2

Dose / conc.:

10 mg/m³ air

Doses / concentrations 3

Dose / conc.:

75 mg/m³ air

No. of animals per sex per dose:

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 exposure): 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

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.

- Rationale 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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly thereafter until one day prior to gross necropsy

FOOD CONSUMPTION: determined twice weekly and calculated as mean food consumption in grams per animal and day.

WATER CONSUMPTION: determined weekly and calculated as mean water consumption in grams per animal and day.

RECTAL TEMPERATURE: determined in all main group animals prior to exposure period and approach of the end of the exposure

CLINICAL PATHOLOGY:
Blood was taken from the retrobulbar venous plexus in the morning from overnight fasted animals without anesthesia for blood gases measurement and hematology. Afterwards, the animals were immediately 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). After intra-testicular temperature measurement both brachial vessels were opened and the dripping was collected for clinical chemistry. The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence.
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The following examinations were carried out in 6 male animals per test group after 4 weeks or 14 weeks.

SPERM PARAMETERS: Immediately after necropsy and organ weight determination the right testis and cauda epididymidis were taken from all male animals. Sperm motility examinations were carried out immediately after necropsy in a randomized sequence, as well as sperm morphology. For sperm head count, the right testis and right cauda epididymidis were deep frozen at -20°C until evaluation.

ORGAN BURDEN:
Satellite groups 1 male mice designated for organ and tissue distribution of Ga and As after 4 weeks exposure. Satellite groups 2 male mice 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.
Lungs, lung associated lymph nodes and epididymides were stored. They may be analyzed on request of the sponsor.

INTRA-TESTICULAR TEMPERATURE: During anesthesia the right intra-testicular temperature was measured with an appropriate probe thermometer



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).

PATHOLOGY
Main group 1 (4 week exposure) and 2 (14 weeks exposure).
- Organ weights: Adrenal glands, Brain, Cauda epididymis, Epididymides, Heart, Kidneys, Liver, Lung, Prostate, Spleen, Seminal vesicles incl. coagulating gland, Testes, Thymus
- Histopathology: All gross lesions, Bone marrow (femur), Epididymidis, left, Kidneys, Lungs (5 lobes), Lymph nodes (mandibular, tracheobronchial/mediastinal), Spleen, Sternum with marrow, Testis, left

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 (twosided) 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

Results and discussion

Effect levels

Dose descriptor:

LOAEC

Effect level:

10 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Increased percentage of abnormal sperms and decreased sperm head counts in the cauda epididymidis

Observed effects

Group 12 (75 mg/m3, 4 weeks)
• Decreased hemoglobin, mean corpuscular hemoglobin volume (MCV) and mean corpuscular
hemoglobin content (MCH)
• Decreased partial oxygen pressure (pO2), partial carbon dioxide pressure (pCO2), base
excess (BE) and calculated hydrogen carbonate concentration (cHCO3) in venous blood
• Lungs
 Significant absolute (+86%) and relative (+79%) weight increase
 Proteinosis, alveolar: all animals (minimal to slight)
 Particles, alveolar, black: all animals (minimal)
 Histiocytosis, alveolar, with particles, black: all animals (minimal)
 Hypertrophy/hyperplasia, epithelial of bronchi, bronchioles and terminal bronchioles: 3 out of 6 animals (minimal to slight)
 Hypertrophy, type II pneumocyte: one out of 6 animals (minimal)
• Mediastinal/tracheobronchial lymph nodes
 Hyperplasia lympho-reticulocellular: 4 out of 6 animals (minimal to slight)

Group 22 (75 mg/m3, 14 weeks)
• Increased red blood cell (RBC) counts
• Decreased hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH) and mean corpuscular hemoglobin concentration (MCHC)
• Decreased partial carbon dioxide pressure (pCO2), base excess (BE) and calculated hydrogen
carbonate concentration (cHCO3) in venous blood
• Only non-motile sperms with increased percentage of abnormal sperms and decreased
sperm head counts in the testis and the cauda epididymidis
• Lungs
 Significant absolute (+116%) and relative (+149%) weight increase
 Proteinosis, alveolar: all animals (slight to severe)
 Particles, alveolar, black: all animals (minimal to slight)
 Histiocytosis, with particles, black: all animals (minimal)
 Hypertrophy/hyperplasia , epithelial of bronchi, bronchioles and terminal bronchioles: 2
out of 6 animals (slight)
 Hypertrophy, type II pneumocyte: all animals (minimal to slight)
• Mediastinal/tracheobronchial lymph node
 Hyperplasia lympho-reticulocellular: 4 out of 6 animals (minimal)
 Particle storage, black: all animals (minimal to slight)
• Testes
 Significant absolute (-61%) and relative (-58%) weight decrease
 Organ size reduced in all animals
 Left testis: Degeneration, tubular: all males (severe to massive)
• Epididymides
 Significant absolute (-33%) and relative (-27%) weight decrease
 Organ size reduced in one out of 6 animals
• Cauda epididymis
 Significant absolute (-28%) and relative (-22%) weight decrease
• Left epididymis
 Debris, cellular: all animals (slight)
 Aspermia: all animals
 Atrophy, ductal: one out of 6 animals (moderate)

Group 11 (10 mg/m3, 4 weeks)
• Lungs
 Significant absolute (+22%) and relative (+20%) weight increase
 Proteinosis, alveolar: all animals (minimal)
 Particles, alveolar, black: all animals (minimal)
 Histiocytosis, with particles, black: all animals (minimal)

Group 21 (10 mg/m3, 14 weeks)
• Increased percentage of abnormal sperms and decreased sperm head counts in the cauda epididymidis
• Lungs
 Significant absolute (+50%) and relative (+43%) weight increase
 Proteinosis, alveolar: all animals (slight to moderate)
 Particles, alveolar, black: all animals (minimal)
 Histiocytosis, with particles, black: all animals (minimal)
 Hypertrophy/hyperplasia , epithelial of bronchi, bronchioles and terminal bronchioles: 2 out of 6 animals (minimal)
• Mediastinal/tracheobronchial lymph node
 Particle storage, black: all animals (minimal)
• Left testis
 Degeneration, tubular: 4 out of 6 animals (minimal to slight)
• Epididymides
 Significant absolute (-15%) and relative (-19%) weight decrease
• Cauda epididymis
 Significant absolute (-23%) and relative (-27%) weight decrease
Left epididymis
 Debris, cellular: 4 out of 6 animals (minimal)

Applicant's summary and conclusion

Conclusions:

Inhalative exposure to GaAs (MMAD 1.1-1.5 µm) 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. Analysis of the elements gallium and arsenic revealed trace amounts of these elements in blood, spleen and testis starting at 75 mg/m³ after 4 weeks exposure in spleen and testis.
Basically, the overall hematological, histopathological and sperm findings reported by NTP (2000) were reproduced.

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. Mortality, clinical findings, body weight, food consumption, water consumption, and rectal temperature of the mice were determined during the study. Parameters of clinical pathology, intra-testicular temperature and histopathology were evaluated after 4 weeks and 14 weeks. Clinical pathological parameters comprised hematology, clinical chemistry (including hemoglobin in serum), blood gas measurements and sperm parameters. A complete necropsy including gross pathological evaluation and weighing of selected organs was performed. Histological examinations comprised bone marrow, epididymis, kidneys, liver, lungs, lymph nodes (mandibular, tracheobronchial, mediastinal), spleen, sternum with marrow and testis (left).

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). The target concentrations were reached.

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. Analysis of the elements gallium and arsenic revealed trace amounts of these elements in blood, spleen and testis starting at 75 mg/m³ after 4 weeks exposure in spleen and testis.

Basically, the overall hematological, histopathological and sperm findings reported by NTP (2000) were reproduced.