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Genetic toxicity: in vivo

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Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
The publication presented herein shows significant methodological and reporting deficiencies. The authors tested only one concentration, which precludes an assessment of a potential concentration-response relationship. However, the concentration tested (50 mg/m³) could be considered as limit concentration, which induce a local inflammatory response in the lung in a shorter 90-days dose range-finding study (Konduru, N. et al., 2014). The authors did not report on the analysis of the test concentration in the exposure and whether the target concentration was achieved. Exposure of the target tissue was not demonstrated. The number of cells scored for DNA damage was lower (100 vs. 150 per animal) than recommended by the current test guideline (OECD TG 489, 2016). Information on scoring, acceptability, and evaluation criteria is not provided. The authors stated that the proportion of DNA in tail in was slightly above the historical control data, which is not in line with acceptability criteria provided by the current test guideline. Historical control data is not provided. No information on body weights at study initiation, during the study, and at the end of the study. The authors did not state whether the test groups were randomised. Information on hedgehog occurrence and potential cytotoxicity missing. It is unclear whether slides were coded and scored blinded. No information is given whether the proportion of DNA was evaluated by calculation of the mean (animal) of the medians (slides). Based on these findings the reference is considered to be not reliable [RL-3].
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
The study had several experimental and reporting deficiencies: females investigated only; one concentration level tested only. equilibration time of the chamber concentration not stated; exposure conditions during inhalation not stated; timing of measuring particle size distribution and particle concentration not stated; information on measuring the concentration in the breathing zone of the animals missing; post-exposure time too long; clinical signs, food consumption, haematology and clinical chemistry not recorded/investigated; apparently incomplete historical control data.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
1981-05-12
Deviations:
yes
Remarks:
please refer to the field "Rationale for reliability incl. deficiencies" above
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
other: Wistar Han
Details on species / strain selection:
not specified
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Sulzfeld, Germany)
- Age at study initiation: 5 or 7 weeks of age
- Housing: groups of up to 5 animals in a polysulfon cage (H-Temp [PSU], TECNIPLAST, Germany) with a floor area of about 2065 cm2 with access to wooden gnawing blocks
- Diet (ad libitum): GLP certified diet (Kliba laboratory diet, Provimi Kliba SA, Kaiseraugst, Basel Switzerland)
- Water (ad libitum)
- Acclimation period: to adapt to the exposure conditions, the animals were acclimatized to exposure conditions over two days (3 and 6 hours, respectively).

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 30 - 70%
- Air changes: 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
2.3 µm
Geometric standard deviation (GSD):
2
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus/Method of holding animals in test chamber: animals were exposed while in wire cages that were located in a stainless-steel whole-body inhalation chamber (V = 2.8 m3 or V = 1.4 m3). Up to two animals per wire cage type DK III (BECKER & Co., Castrop-Rauxel, Germany) were exposed in the whole body exposure chamber.

- System of generating particulates/aerosols: test item aerosols were produced by dry dispersion of powder pellets with a brush dust generator using compressed air at 1.5 m3/h (developed by the Technical University of Karlsruhe in cooperation with BASF, Germany). The dust aerosol was diluted by conditioned air passed into the whole-body inhalation chambers. The control group was exposed to conditioned clean air. The desired concentrations were achieved by varying the feeding speed of the powder pellet or by varying the rotation speed of the brush. Based on a comprehensive technical trial, atmospheric concentrations within the chambers were found to be homogenous (particle count concentration: 92752 particle/cm3; particle count median diameter 326 nm). Nevertheless, exposure cages were rotated within each chamber daily for the 4-week.

- Treatment of exhaust air: the aerosols were passed into the inhalation chambers with the supply air and were removed by an exhaust air system with 20 air changes per hour. For the control animals, the exhaust air system was adjusted in such a way that the amount of exhaust air was lower than the filtered clean, supply air (positive pressure) to ensure that no laboratory room air reaches the control animals. For the BaSO4-exposed rats, the amount of exhaust air was higher than the supply air (negative pressure) to prevent contamination of the laboratory as a result of potential leakages from the inhalation chambers.

- Method of particle size and concentration determination: generated aerosols were continuously monitored by scattered light photometers (VisGuard). Particle concentrations in the inhalation chambers were analyzed by gravimetric measurement of air filter samples. Particle size distribution was determined gravimetrically by cascade impactor analysis using eight stages Marple Personal Cascade Impactor (Sierra-Anderson, USA). In addition, a light-scattering aerosol spectrometer (WELAS 2000) was used to measure particle sizes from 0.24 to 10 μm. To measure particles in the submicrometer range, a scanning mobility particle sizer (SMPS 5.400) was used. The sampling procedures and measurements to characterize the generated aerosols were previously described (Ma-Hock et al., 2007)*.

*Reference:
- Ma-Hock L, Gamer AO, Landsiedel R, Leibold E, Frechen T, Sens B, Linsenbuehler M, Van Ravenzwaay B: Generation and characterization of test atmospheres with nanomaterials. Inhal Toxicol 2007, 19:833–848.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The target concentration of 50 mg/m3 was achieved and maintained throughout the inhalation exposures. Particle size distribution of aeroslized test item was in the respirable range of rats.

Please also refer to the field "Details on inhalation exposure".
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6 hours/day on 5 consecutive days/week
Dose / conc.:
50 mg/m³ air (nominal)
Remarks:
actual concentration: 46.2 ± 5.9 mg/m3
No. of animals per sex per dose:
15 female rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period: 35 days (number of animals: 15)
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Not specified
DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: before and every week throughout the duration of the experiments

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified
HAEMATOLOGY: Not specified
CLINICAL CHEMISTRY: Not specified
URINALYSIS: Not specified
NEUROBEHAVIOURAL EXAMINATION: Not specified
IMMUNOLOGY: Not specified

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: 1 day and 35 days (recovery group) after the end of exposure
- Dose groups that were examined: treatment group and control group
- Number of animals: five animals/group
- Method and parameters checked: after euthanasia, the lungs were lavaged twice in situ with 22 mL/kg body weight (4 to 5 mL) of normal saline. The recovered volume ranged from 8 to 10 mL per animal. Aliquots of bronchoalveolar lavage (BAL) were used for determinations of total protein concentration, total cell count, differential cell count and enzyme activities. In the 4 week-exposure group and its control, BAL analysis was performed twice (1 and 35 days after the end of exposure). Lavaged lung tissue and aliquots of the BAL fluid (1 mL) were stored at −80°C and used for determination of barium content.

Total BAL cell counts were determined with an Advia 120 (Siemens Diagnostics) hematology analyzer. Differential cell counts were made on Wright stained cytocentrifuge slide preparations. Using a Hitachi 917 (Roche Diagnostics) reaction rate analyzer, levels of BAL total protein and activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT) and N-acetyl-β-glucosaminidase (NAG) were measured. Inflammatory cytokines (MCP-1, IL-8/CINC-1, M-CSF, osteopontin) in BAL were measured using ELISA test kits as described previously (Ma-Hock et al., 2007)*.

*Reference:
- Ma-Hock L, Gamer AO, Landsiedel R, Leibold E, Frechen T, Sens B, Linsenbuehler M, Van Ravenzwaay B: Generation and characterization of test atmospheres with nanomaterials. Inhal Toxicol 2007, 19:833–848.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

After 4 weeks of exposure, necropsy and histopathology were performed on selected rats at 1 day and 34 days after the end of exposure. Gross and histopathological examination of the lungs and extrapulmonary organs were
performed on ten rats per group. The animals were euthanized by cutting the abdominal aorta and vena cava under sodium pentobarbital anesthesia. The following organs were weighed: adrenal glands, brain, heart, ovaries, uterus with cervix, kidney, liver, lungs, spleen, thymus, thyroid glands. The lungs were intratracheal-instilled with neutral buffered 10% formalin at 30 cm water pressure. All other organs were fixed in the same fixative. The organs and tissues were trimmed, paraffin embedded and sectioned according to RITA trimming guides for inhalation studies (Kittel et al., 2004; Morawietz et al., 2004; Ruehl-Fehlert et al., 2003)*. Paraffin sections were stained with hematoxylin and eosin. Extrapulmonary organs and the respiratory tract, comprised of the nasal cavity (four levels), larynx (three levels), trachea (transverse and longitudinal with carina), lungs (five lobes), and mediastinal and tracheobronchial lymph nodes, were examined by light microscopy.

*References:
- Kittel B, Ruehl-Fehlert C, Morawietz G, Klapwijk J, Elwell MR, Lenz B, O'Sullivan
MG, Roth DR, Wadsworth PF: Revised guides for organ sampling and trimming in rats and mice–part 2. A joint publication of the RITA and NACAD groups. Exp Toxicol Pathol 2004, 55:413–431.

- Morawietz G, Ruehl-Fehlert C, Kittel B, Bube A, Keane K, Halm S, Heuser A,
Hellmann J: Revised guides for organ sampling and trimming in rats and
mice–part 3. A joint publication of the RITA and NACAD groups. Exp Toxicol Pathol 2004, 55:433–449.

- Ruehl-Fehlert C, Kittel B, Morawietz G, Deslex P, Keenan C, Mahrt CR, Nolte
T, Robinson M, Stuart BP, Deschl U: Revised guides for organ sampling and trimming in rats and mice–part 1. Exp Toxicol Pathol 2003, 55:91–106.
Statistics:
Body weight differences were compared between test item-exposed and control groups using Dunnett’s test. Bronchoalveolar lavage cytology, enzyme and cell mediator data were analyzed by non-parametric one-way analysis of variance using the Kruskal-Wallis test (two-sided). If the resulting p value was equal or less than 0.05, a pair-wise comparison of each test group with the control group was performed using the Wilcoxon test or the Mann–Whitney U-test. Comparison of organ weights was performed by nonparametric one-way analysis using the two-sided Kruskal–Wallis test, followed by a two-sided Wilcoxon test for the hypothesis of equal medians.
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Endocrine findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Details on results:
BODY WEIGHTS
Body weights showed no significant change.

HISTOPATHLOGICAL FINDINGS
No morphological changes were detected by histopathology in the lungs and extrapulmonary organs.

Please also refer to section "Overall remarks, attachments".

BRONCHOALVEOLAR LAVAGE (BAL)
After 4 weeks of exposure, neutrophils were statistically significantly increased compared to concurrent controls one day after the end of exposure (control group: 0.007 ± 0.003 million; treatment group: 0.021 ± 0.010 million; p ≤ 0.05). However, these values were within the historical control range in previous studies. Cytokine levels of monocyte chemoattractant protein-1 (MCP-1; control group: 14.0 ± 0.0 pg/mL; treatment group: 54.7 ± 14.3 pg/mL; p ≤ 0.05) and cytokine-induced neutrophil chemoattractant-1 (CINC-1; control group: 104.2 ± 26.7 pg/mL; treatment group: 158.7 ± 22.4 pg/mL; p ≤ 0.05) were elevated. All BAL parameters elevated at 1 day post-exposure returned to control levels in the 4-week exposure group at 35 days. Lastly, the treatment group did not show a statistically significantly difference in total protein BAL total cell, gamma glutamyl transferase (GGT) and alkaline phosphatase (ALP) compared to the control.

Please also refer to section "Overall remarks, attachments".

Remarks on result:
other: see "Remarks"
Remarks:
Body weights showed no significant change and no morphological changes were detected by histopathology in the lungs and extrapulmonary organs. Furthermore, bronchoalveolar lavage (BAL) showed that neutrophils were statistically significantly increased compared to concurrent controls one day after the end of exposure. However, these values were within the historical control range in previous studies. Cytokine levels of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) were elevated in the BAL of treated animals. All BAL parameters elevated at 1 day post-exposure returned to control levels in the 4-week exposure group at 35 days.
Critical effects observed:
not specified
Conclusions:
In the current repeated dose inhalation toxicity study, barium sulfate nanoparticles (primary particle size = 25 nm) were administered via whole body inhalation to a group of 15 female Wistar Han rats at a concentration of 50 mg/m3. (actual concentration: 46.2 ± 5.9 mg/m3). The substance was administered 6 hours/day on five consecutive days/week for a duration of 4 weeks. A control group receiving filtered air only was run concurrently. Animals were investigated 1 day or 35 days after the end of treatment period.

No test item-related effects were observed on the body weight of the animals. Furthermore, no morphological changes were detected by histopathology in the lungs and extrapulmonary organs (adrenal glands, brain, heart, ovaries, uterus with cervix, kidney, liver, lungs, spleen, thymus, thyroid glands , nasal cavity, larynx, trachea, and mediastinal and tracheobronchial lymph nodes).
After 4 weeks of exposure, a bronchoalveolar lavage showed that neutrophils were statistically significantly increased compared to concurrent controls one day after the end of exposure (control group: 0.007 ± 0.003 million; treatment group: 0.021 ± 0.010 million; p ≤ 0.05). However, these values were within the historical control range in previous studies. Cytokine levels of monocyte chemoattractant protein-1 (MCP-1; control group: 14.0 ± 0.0 pg/mL; treatment group: 54.7 ± 14.3 pg/mL; p ≤ 0.05) and cytokine-induced neutrophil chemoattractant-1 (CINC-1; control group: 104.2 ± 26.7 pg/mL; treatment group: 158.7 ± 22.4 pg/mL; p ≤ 0.05) were elevated in the bronchoalveolar lavage of treated animals. All bronchoalveolar lavage parameters elevated at 1 day post-exposure returned to control levels in the 4-week exposure group at 35 days.

This reference had several reporting and experimental deficiencies, which do not allow an independent review about the exposure-effect correlation:
The study includes an investigation of females only, whereas the OECD guideline 412 foresees testing of males and females. Therefore, no conclusion can be drawn on males regarding the substance. Furthermore, the guideline foresees that at least three dose level and a concurrent control should be used. In this study, one concentration level of 46 ± 5.9 mg/m3 was only tested. The usage of one concentration level precludes the possibility to demonstrate any dose-related response and the determination of a No-Observed-Adverse Effect Concentration (NOAEC). In addition, the authors did not state, if the exposure of the animals started after equilibration of the chamber concentration. It could be that in the beginning of starting the equipment the concentration has not reached yet the level that is suppose to be tested. Therefore, the animals might have gotten less substance administered as assumed. Furthermore, the authors did not state the exposure conditions during inhalation administration (e.g. temperature, humidity, oxygen level). The timing of measuring particle size distribution and particle concentration was not stated as well as information on measuring the concentration in the breathing zone of the animals was missing.

According to the OECD guideline, the post-exposure time should be 14 days to provide information on reversibility, or persistence or delayed occurrence of toxic effects. The post-exposure time in the current study lasted 35 days, which is two and half times longer as requested by the guideline. Clinical signs, food consumption, haematology and clinical chemistry were not recorded/investigated as foreseen be the guideline. Therefore, systemic effects caused by the substance cannot fully be ruled out. Lastly, it was mentioned that historical control was used for interpretation of the neutrophils, however, it is unclear if the remaining data was also interpreted using historical control data. Historical control data is useful to concluded, if the data lies within the biological variation of a species. In conclusion, the study is considered as not reliable (RL = 3) based on the shortcomings. However, the study can be used in a weight of evidence approach for repeated dose toxicity via inhalation route.
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
The study had several experimental and reporting deficiencies: females tested only; number of animals/group too low; one concentration level tested only; exposure period too short; no justification for 5 days/week treatment; clinical signs, mortality, detailed clinical examination, ophthalmology, food consumption, food efficiency, haematology, clinical chemistry, urinalysis, gross pathology, organ weights and histopathology not recorded/investigated; historical control data and individual data missing.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
2009-09-07
Deviations:
yes
Remarks:
Please refer to the field "Rationale for reliability".
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
Wistar
Remarks:
Wister Han
Details on species / strain selection:
not specified
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Sulzfeld, Germany)
- Age at study initiation: 5 or 7 weeks of age
- Housing: groups of up to 5 animals in a polysulfon cage (H-Temp [PSU], TECNIPLAST, Germany) with a floor area of about 2065 cm2 with access to wooden gnawing blocks
- Diet (ad libitum): GLP certified diet (Kliba laboratory diet, Provimi Kliba SA, Kaiseraugst, Basel Switzerland)
- Water (ad libitum)
- Acclimation period: to adapt to the exposure conditions, the animals were acclimatized to exposure conditions over two days (3 and 6 hours, respectively).

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 30 - 70%
- Air changes: 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
1.9 µm
Geometric standard deviation (GSD):
2.1
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus/Method of holding animals in test chamber: animals were exposed while in wire cages that were located in a stainless-steel whole-body inhalation chamber (V = 2.8 m3 or V = 1.4 m3). Up to two animals per wire cage type DK III (BECKER & Co., Castrop-Rauxel, Germany) were exposed in the whole body exposure chamber.

- System of generating particulates/aerosols: test item aerosols were produced by dry dispersion of powder pellets with a brush dust generator using compressed air at 1.5 m3/h (developed by the Technical University of Karlsruhe in cooperation with BASF, Germany). The dust aerosol was diluted by conditioned air passed into the whole-body inhalation chambers. The control group was exposed to conditioned clean air. The desired concentrations were achieved by varying the feeding speed of the powder pellet or by varying the rotation speed of the brush. Based on a comprehensive technical trial, atmospheric concentrations within the chambers were found to be homogenous (particle count concentration: 77992 particle/cm3; particle count median diameter 304 nm). Nevertheless, exposure cages were rotated within each chamber weekly for the 13-week group.

- Treatment of exhaust air: the aerosols were passed into the inhalation chambers with the supply air and were removed by an exhaust air system with 20 air changes per hour. For the control animals, the exhaust air system was adjusted in such a way that the amount of exhaust air was lower than the filtered clean, supply air (positive pressure) to ensure that no laboratory room air reaches the control animals. For the BaSO4-exposed rats, the amount of exhaust air was higher than the supply air (negative pressure) to prevent contamination of the laboratory as a result of potential leakages from the inhalation chambers.

- Method of particle size and concentration determination: generated aerosols were continuously monitored by scattered light photometers (VisGuard). Particle concentrations in the inhalation chambers were analyzed by gravimetric measurement of air filter samples. Particle size distribution was determined gravimetrically by cascade impactor analysis using eight stages Marple Personal Cascade Impactor (Sierra-Anderson, USA). In addition, a light-scattering aerosol spectrometer (WELAS 2000) was used to measure particle sizes from 0.24 to 10 μm. To measure particles in the submicrometer range, a scanning mobility particle sizer (SMPS 5.400) was used. The sampling procedures and measurements to characterize the generated aerosols were previously described (Ma-Hock et al., 2007)*.

*Reference:
- Ma-Hock L, Gamer AO, Landsiedel R, Leibold E, Frechen T, Sens B, Linsenbuehler M, Van Ravenzwaay B: Generation and characterization of test atmospheres with nanomaterials. Inhal Toxicol 2007, 19:833–848.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The target concentration of 50 mg/m3 was achieved and maintained throughout the inhalation exposures. Particle size distribution of aeroslized test item was in the respirable range of rats.

Please also refer to the field "Details on inhalation exposure".
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day on 5 consecutive days/week
Dose / conc.:
50 mg/m³ air (nominal)
Remarks:
actual concentration: 50.1 ± 5.6 mg/m3
No. of animals per sex per dose:
15 female rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the result of the short-term study with 4 weeks of exposure (please refer to section 7.5.2 Repeated dose toxicity: inhalation: NANO_w_Konduru_2014_28 days), the long-term study was started at the same concentration
of 50 mg/m3 BaSO4.
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Not specified
DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: before and every week throughout the duration of the experiments

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified
HAEMATOLOGY: Not specified
CLINICAL CHEMISTRY: Not specified
URINALYSIS: Not specified
NEUROBEHAVIOURAL EXAMINATION: Not specified
IMMUNOLOGY: Not specified

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: 1 day and 35 days (recovery group) after the end of exposure
- Dose groups that were examined: treatment group and control group
- Number of animals: five animals/group
- Method and parameters checked: after euthanasia, the lungs were lavaged twice in situ with 22 mL/kg body weight (4 to 5 mL) of normal saline. The recovered volume ranged from 8 to 10 mL per animal. Aliquots of bronchoalveolar lavage (BAL) were used for determinations of total protein concentration, total cell count, differential cell count and enzyme activities. In the 13 week-exposure group and its control, BAL analysis was performed at 1 day post-exposure. Lavaged lung tissue and aliquots of the BAL fluid (1 mL) were stored at −80°C and used for determination of barium content.

Total BAL cell counts were determined with an Advia 120 (Siemens Diagnostics) hematology analyzer. Differential cell counts were made on Wright stained cytocentrifuge slide preparations. Using a Hitachi 917 (Roche Diagnostics) reaction rate analyzer, levels of BAL total protein and activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT) and N-acetyl-β-glucosaminidase (NAG) were measured. Inflammatory cytokines (MCP-1, IL-8/CINC-1, M-CSF, osteopontin) in BAL were measured using ELISA test kits as described previously (Ma-Hock et al., 2007)*.

*Reference:
- Ma-Hock L, Gamer AO, Landsiedel R, Leibold E, Frechen T, Sens B, Linsenbuehler M, Van Ravenzwaay B: Generation and characterization of test atmospheres with nanomaterials. Inhal Toxicol 2007, 19:833–848.
Sacrifice and pathology:
GROSS PATHOLOGY: Not specified
HISTOPATHOLOGY: Not specified

Statistics:
Body weight differences were compared between test item-exposed and control groups using Dunnett’s test. Bronchoalveolar lavage cytology, enzyme and cell mediator data were analyzed by non-parametric one-way analysis of variance using the Kruskal-Wallis test (two-sided). If the resulting p value was equal or less than 0.05, a pair-wise comparison of each test group with the control group was performed using the Wilcoxon test or the Mann–Whitney U-test.
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Endocrine findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
BRONCHOALVEOLAR LAVAGE
Rats exposed for 13 weeks showed statistically significant increases in BAL total cells (control group: 0.610 ± 0.20 million; treatment group: 0.836 ± 0.15 million; p ≤ 0.05) and neutrophils (control group: 0.016 ± 0.0006 million; treatment group: 0.204 ± 0.175 million; p ≤ 0.05) compared to control. Cytokine levels of monocyte chemoattractant protein-1 (MCP-1; control group: 24.2 ± 8.4pg/mL; treatment group: 176.7 ± 126.1 pg/mL; p ≤ 0.05) and cytokine-induced neutrophil chemoattractant-1 (CINC-1; control group: 93.7 ± 18.7 pg/mL; treatment group: 223.8 ± 125.7 pg/mL; p ≤ 0.05) were statistically significant elevated. Furthermore, rats exposed for 13 weeks showed statistically significantly higher gamma glutamyl transferase (GGT; control group: 41 ± 7 nkat/L; treatment group: 64 ± 13 nkat/L; p ≤ 0.05) and alkaline phosphatase (ALP; control group: 0.51 ± 0.10 µkat/L; treatment group: 0.87 ± 0.21 µkat/L; p ≤ 0.05) levels than their corresponding controls.
Details on results:
BODY WEIGHTS
Body weights showed no significant change.

BRONCHOALVEOLAR LAVAGE (BAL)
The treatment group did not show a statistically significantly difference in total protein compared to the control.

Please also refer to section "Overall remarks, attachments".
Remarks on result:
other: see "Remarks"
Remarks:
Body weights showed no significant change. Furthermore, rats exposed for 13 weeks showed statistically significant increases in bronchoalveolar lavage (BAL) total cells and neutrophils compared to control. Cytokine levels of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) were statistically significant elevated in the BAL of treated animals. Also, rats exposed for 13 weeks showed statistically significantly higher gamma glutamyl transferase (GGT) and alkaline phosphatase (ALP) levels in their BAL than their corresponding controls. Lastly, the treatment group did not show a statistically significantly difference in BAL total protein compared to the control.
Critical effects observed:
not specified
Conclusions:
In the current repeated dose inhalation toxicity study, barium sulfate nanoparticles (primary particle size = 25 nm) were administered via whole body inhalation to a group of 15 female Wistar Han rats at a concentration of 50 mg/m3. (actual concentration: 50.1 ± 5.6 mg/m3). The substance was administered 6 hours/day on five consecutive days/week for a duration of 13 weeks. A control group receiving filtered air only was run concurrently. Animals were investigated one day after the end of treatment period.

No test item-related effects were observed on the body weight of the animals. Furthermore, rats exposed for 13 weeks showed statistically significant increases in bronchoalveolar lavage (BAL) total cells (control group: 0.610 ± 0.20 million; treatment group: 0.836 ± 0.15 million; p ≤ 0.05) and neutrophils (control group: 0.016 ± 0.0006 million; treatment group: 0.204 ± 0.175 million; p ≤ 0.05) compared to control. Cytokine levels of monocyte chemoattractant protein-1 (MCP-1; control group: 24.2 ± 8.4pg/mL; treatment group: 176.7 ± 126.1 pg/mL; p ≤ 0.05) and cytokine-induced neutrophil chemoattractant-1 (CINC-1; control group: 93.7 ± 18.7 pg/mL; treatment group: 223.8 ± 125.7 pg/mL; p ≤ 0.05) were statistically significant elevated in the BAL of treated animals.Also, rats exposed for 13 weeks showed statistically significantly higher gamma glutamyl transferase (GGT; control group: 41 ± 7 nkat/L; treatment group: 64 ± 13 nkat/L; p ≤ 0.05) and alkaline phosphatase (ALP; control group: 0.51 ± 0.10 µkat/L; treatment group: 0.87 ± 0.21 µkat/L; p ≤ 0.05) levels in their BAL than their corresponding controls. Lastly, the treatment group did not show a statistically significantly difference in BAL total protein compared to the control.

This reference had several reporting and experimental deficiencies:
The study includes an investigation of females only, whereas the OECD guideline 453 foresees testing of both sexes. Due to the lack of investigating males, no conclusion can be drawn on males regarding the substance. Furthermore, the number of animals per group was too low in the current study (n = 15 female rats). The OECD guideline 453 requests at least 10 animals of each sex for the chronic toxicity phase and at least 50 animals of each sex for the carcinogenicity phase. In addition, the guideline foresees that at least three dose level and a concurrent control should be used. In this study, one concentration level of 50.1 ± 5.6 mg/m3 was only tested. The usage of one concentration level precludes the possibility to demonstrate any dose-related response and the determination of a No-Observed-Adverse Effect concentration (NOAEC).
The authors used an exposure period of 13 weeks, whereas the OECD guideline 453 normally foresees exposure periods of 12 months (chronic phase) or 24 months (carcinogenicity phase). The guideline includes the possibility of shorter durations, however, these are longer than 13 weeks. Furthermore, the authors did not justify why they administered the substance on 5 days/week only instead of 7 days/week.
Apparently, clinical signs, mortality, detailed clinical examination, ophthalmology, food consumption, food efficiency, haematology, clinical chemistry, urinalysis, gross pathology, organ weights and histopathology were not recorded/investigated as foreseen by the guideline. The authors conducted only bronchoalveolar lavage fluid analysis one day and 35 days after final treatment. Therefore, systemic effects caused by the substance cannot fully be ruled out due to the missing information on the previous stated parameters. Lastly, historical control data and individual data are missing. Historical control data is useful to concluded if the data lies within the biological variation of a species and individual data is useful to find outliners in the data. In conclusion, the study is considered as not reliable (RL = 3) based on the shortcomings. However, the study can be used in a weight of evidence approach for repeated dose toxicity via inhalation route.
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
The publication presented herein shows significant methodological and reporting deficiencies. The authors tested only one concentration, which precludes an assessment of a potential concentration-response relationship. However, the concentration tested (50 mg/m³) could be considered as limit concentration, which induce a local inflammatory response in the lung in a shorter 90-days dose range-finding study (Konduru, N. et al., 2014). The authors did not report on the analysis of the test concentration in the exposure and whether the target concentration was achieved. Exposure of the target tissue was not demonstrated. The number of cells scored for micronuclei via microscopy was lower (2000 vs. 4000 per animal) than recommended by the current test guideline (OECD TG 474, 2016). However, in the analysis via flow cytometry a total of 20,000 erythrocytes per animals were scored. Information on scoring, acceptability, and evaluation criteria is not provided. Historical control data is missing. No information on body weights at study initiation, during the study, and at the end of the study. The authors did not state whether the test groups were randomised. Based on these findings the reference is considered to be not reliable [RL-3].
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Cordelli, E. et al. (2017) evaluated barium sulfate (NM-220) for its clastogenic and aneugenic potential in female Wistar rats after whole body inhalation. Five rats were exposed to barium sulfate NM-220 aerosol at a concentration of 50 mg/m³ for 6 hours per day on 5 consecutive days/week for a total period of 6 months. A vehicle control (filtered clean air) group was run concurrently. Animals exposed to N-ethyl-N-nitrosourea via gavage served as positive control group. Peripheral blood was sampled 24 hours after the last exposure. The blood samples were prepared for the evaluation of the micronucleus frequency in reticulocytes via microscopical or flow cytometric analysis. For the microscopical analysis, cells were stained with acridine orange and two slides (1000 reticulocytes per slide) per animal were analysed via fluorescence microscopy. The preparation for the flow cytometric analysis were performed using a commercial kit. Reticulocytes and platelets were labelled via immunostaining. Afterwards, the DNA was stained using the kit-supplied staining solution. A total of 20,000 reticulocytes per animal were scored for the presence of micronuclei via flow cytometry.
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
not specified
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Sulzfeld, Germany)
- Age at study initiation: 5-7 weeks
- Weight at study initiation: not specified
- Assigned to test groups randomly: not specified
- Fasting period before study: not specified
- Housing: Groups of up to five animals in polysulfone cages (H-Temp (PSU); TECNIPLAST, Germany).
- Diet: Kliba laboratory diet (ProvimiKliba SA; Kaiseraugst, Basel Switzerland); no access during exposure, otherwise ad libitum
- Water: no access during exposure, otherwise ad libitum
- Acclimation period: Acclimatised to the study conditions in the whole-body inhalation chambers by exposure to fresh air for 2 days (6 h, each) before the onset of the test substance exposure period

ENVIRONMENTAL CONDITIONS
- Temperature: controlled (not further specified)
- Humidity: controlled (not further specified)
- Photoperiod: controlled (not further specified)
Route of administration:
inhalation: aerosol
Vehicle:
- Vehicle(s)/solvent(s) used: clean air
Details on exposure:
Information on exposure conditions were extracted from Konduru et al. (2014)*.

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- Exposure apparatus/Method of holding animals in test chamber: animals were exposed while in wire cages that were located in a stainless-steel whole-body inhalation chamber (V = 2.8 m³ or V = 1.4 m³). Up to two animals per wire cage type DK III (BECKER & Co., Castrop-Rauxel, Germany) were exposed in the whole body exposure chamber.

- System of generating particulates/aerosols: test item aerosols were produced by dry dispersion of powder pellets with a brush dust generator using compressed air at 1.5 m³/h (developed by the Technical University of Karlsruhe in cooperation with BASF, Germany). The dust aerosol was diluted by conditioned air passed into the whole-body inhalation chambers. The control group was exposed to conditioned clean air.

- Treatment of exhaust air: the aerosols were passed into the inhalation chambers with the supply air and were removed by an exhaust air system with 20 air changes per hour. For the control animals, the exhaust air system was adjusted in such a way that the amount of exhaust air was lower than the filtered clean, supply air (positive pressure) to ensure that no laboratory room air reaches the control animals. For the BaSO4-exposed rats, the amount of exhaust air was higher than the supply air (negative pressure) to prevent contamination of the laboratory as a result of potential leakages from the inhalation chambers.

- Method of particle size and concentration determination: generated aerosols were continuously monitored by scattered light photometers (VisGuard). Particle concentrations in the inhalation chambers were analysed by gravimetric measurement of air filter samples. Particle size distribution was determined gravimetrically by cascade impactor analysis using eight stages Marple Personal Cascade Impactor (Sierra-Anderson, USA). In addition, a light-scattering aerosol spectrometer (WELAS 2000) was used to measure particle sizes from 0.24 to 10 μm. To measure particles in the submicrometer range, a scanning mobility particle sizer (SMPS 5.400) was used. The sampling procedures and measurements to characterize the generated aerosols were previously described (Ma-Hock et al., 2007)*.

*Reference:
- Konduru, N., Keller, J., Ma-Hock, L., Gröters, S., Landsiedel, R., Donaghey, T. C., Brain, J. D., Wohlleben, W. and Molina, R. M. (2014) Biokinetics and effects of barium sulfate nanoparticles. Part. Fibre Toxicol., 11, 55.
- Ma-Hock, L., Gamer, A.O., Landsiedel, R., Leibold, E., Frechen, T., Sens, B., Linsenbuehler, M., Van Ravenzwaay, B. (2007) Generation and characterization of test atmospheres with nanomaterials. Inhal Toxicol 19:833-848.
Duration of treatment / exposure:
6 months
Frequency of treatment:
6 hours/day on 5 consecutive days/week
Post exposure period:
24 hours
Dose / conc.:
50 mg/m³ air
No. of animals per sex per dose:
five (treatment group) and seven (vehicle control group) female rats
Control animals:
yes, concurrent vehicle
Positive control(s):
N-ethyl-N-nitrosourea (ENU)
- Route of administration: gavage
- Doses / concentrations: Rats received three doses of 20 mg/kg bw (10 mL/kg bw) at 24-hour intervals. The micronucleus assay was conducted on blood sampled four days after the first administration.
Tissues and cell types examined:
peripheral blood erythrocytes (n=2000 per animal via microscopy; n=20,000 per animal via flow cytometry)
Details of tissue and slide preparation:
DOSE-RANGE FINDING STUDY
Dose-range findings, short term inhalation studies were conducted, in during which BaSO4 NM-220 did not induce any effects at 50 mg/m³ (Konduru et al., 2014; please refer to section 7.5.2).

TREATMENT
The inhalation study, as a part of which the present genotoxicity studies were performed, was conducted as 2-year study according to OECD TG 453 under good laboratory practice conditions. Animals were exposed to an aerosol concentration of 50 mg/m³ BaSO4 NM-220 for 6 h/day whole-body exposure) on 5 consecutive days/week (working days, only). Concordantly, a control group was exposed to clean, filtered air. On exposure days, each animal was inspected clinically once after the exposure period. Occurrence of mortality was monitored twice daily on working days and once daily on weekends and holidays.

BLOOD SAMPLING
For the genotoxicity studies, blood was collected from five animals per group after 6-month test substance exposure. Blood sampling was performed 24 hours after the last exposure by retro-orbital venous plexus puncture under isoflurane anaesthesia. The blood samples were placed into K2EDTA tubes (Becton Dickinson, San José, CA, USA) and immediately shipped refrigerated (2-8°C) to the ENEA Laboratories (Italian National Agency for New Technologies, Energy and Sustainable Economic Development) Centro Ricerche Casaccia (Rome, Italy) for analysis.

MICRONUCLEUS TEST
- Microscopic analysis:
Acridine Orange (AO) was dissolved in distilled water at a concentration of 1 mg/mL. Pre-heated cleaned glass slides were stained with 5 μL of this solution, and 5 μL of peripheral blood were placed in the centre of AO-stained slides, covered with a coverslip and gently pressed. Slides were stored at 4°C and analysed within a few days. Two slides, 1000 RETs per slide, were analysed for each animal using a fluorescence microscope.
- Flow cytometric analysis:
Peripheral blood was processed according to the procedure described in the Rat Blood MicroFlow Micronucleus Analysis Kit® (Litron Laboratories). Briefly, within 24 hours from sampling, 30 μL blood were mixed with 175 μL anticoagulant solution and 180 μL of this suspension were immediately fixed in ultra-cold methanol and stored at -80°C. Prior to analysis, samples were washed and incubated with RNase, anti-CD71-FITC (anti-transferrin receptor antibody that labels RETs), and anti-CD61-PE (anti-platelet antibody that labels platelets) for 30 minutes at 4°C followed by 30 minutes at room temperature. Immediately before analysis, the samples were resuspended in an appropriate amount of kit-supplied DNA staining solution. The flow cytometer photomultiplier and compensation tubes were adjusted using a kit-supplied malaria-infested blood sample. This biological standard was used to set the optimal voltage for resolving parasitised (micronucleus-like) RETs, and hence the position of the quadrant delineating normochromatic RBCs and RETs with and without micronuclei. The samples were analysed using a flow cytometer. Twenty thousand RETs were analysed per blood sample. Again, the %RETs, identified as CD-71 positive cells, was recorded as an index for cytotoxicity.

*References:
- Konduru, N., Keller, J., Ma-Hock, L., Gröters, S., Landsiedel, R., Donaghey, T. C., Brain, J. D., Wohlleben, W. and Molina, R. M. (2014) Biokinetics and effects of barium sulfate nanoparticles. Part. Fibre Toxicol., 11, 55.
Evaluation criteria:
not specified
Statistics:
Comparison among group means was performed by one-way ANOVA and Dunnett’s test was used for post hoc comparison of treatments to controls. The paired Student’s t-test was applied to analyse the data obtained before and after treatment with ENU. Findings were assessed as treatment-related when their probability level was lower than 5% (P < 0.05).
Sex:
female
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
not determinable because of methodological limitations
Additional information on results:
TOXICITY
- During the 6 months of inhalation exposure recorded for the present study, treatment-related clinical signs were not observed in any of the rats.

MN ASSAY
- After 6-month exposure to 50 mg/m³ barium sulfate NM-220, the frequency of micronucleated reticulocytes remained unaltered, with the microscopic and flow cytometric evaluations providing consistent results (please refer to attachments: 'Results').
- The proportion of reticulocytes among total erythrocytes was similar to the concurrent vehicle control group (1.00 vs 1.12%) indicating absence of cytotoxicity.

VALIDITY OF THE ASSAY
- The percentage of micronucleated reticulocytes was statistically significantly increased on Day 4 after the onset of N-ethyl-N-nitrosourea (ENU) administration as assessed microscopically and by flow cytometry with both evaluation techniques providing similar numerical results. By flow cytometric analysis, the proportion of reticulocytes among total erythrocytes was decreased in the blood samples collected 4 days after the onset of ENU administration which further reflected the cytotoxic effect of this positive control. The data of ENU administration demonstrate the proficiency of the laboratory.
Conclusions:
Cordelli, E. et al. (2017) evaluated barium sulfate (NM-220) for its clastogenic and aneugenic potential in female Wistar rats after whole body inhalation. Five rats were exposed to barium sulfate NM-220 aerosol at a concentration of 50 mg/m³ for 6 hours per day on 5 consecutive days/week for a total period of 6 months. A vehicle control (filtered clean air) group was run concurrently. Animals exposed to N-ethyl-N-nitrosourea via gavage served as positive control group. Peripheral blood was sampled 24 hours after the last exposure. The blood samples were prepared for the evaluation of the micronucleus frequency in reticulocytes via microscopical or flow cytometric analysis. For the microscopical analysis, cells were stained with acridine orange and two slides (1000 reticulocytes per slide) per animal were analysed via fluorescence microscopy. The preparation for the flow cytometric analysis were performed using a commercial kit. Reticulocytes and platelets were labelled via immunostaining. Afterwards, the DNA was stained using the kit-supplied staining solution. A total of 20,000 reticulocytes per animal were scored for the presence of micronuclei via flow cytometry.

Whole body inhalation exposure to 50 mg/m³ barium sulfate NM-220 did not result in treatment-related clinical signs. However, in a preceding 90-day study, barium sulfate NM-220 induced local inflammatory responses, characterised i.a. by significant neutrophil influx in bronchoalveolar lavage fluid, under the same exposure conditions, (Konduru, N. et al., 2014; please refer to section 7.5.2)*. Female rats exposed for 6 months to barium sulfate NM-220 showed micronucleus frequencies, which were comparable or even lower, when compared to the vehicle control group using both microscopic and flow-cytometric analysis. The flow cytometric analysis revealed comparable proportions of reticulocytes among total erythrocytes in treated and control animals, demonstrating the absence of cytotoxicity. The positive reference mutagen, N-ethyl-N-nitrosourea, induced a distinct and statistically significant increase in the micronucleus frequency, when compared to pre-treatment samples. Thus, the sensitivity of the test system was demonstrated.

The publication presented herein shows significant methodological and reporting deficiencies.

The authors tested only one concentration, which precludes an assessment of a potential concentration-response relationship. However, the concentration tested (50 mg/m³) could be considered as limit concentration, which induce a local inflammatory response in the lung in a shorter 90-days dose range-finding study (Konduru, N. et al., 2014). The authors did not report on the analysis of the test concentration in the exposure and whether the target concentration was achieved. Exposure of the target tissue was not demonstrated. The number of cells scored for micronuclei via microscopy was lower (2000 vs. 4000 per animal) than recommended by the current test guideline (OECD TG 474, 2016). However, in the analysis via flow cytometry a total of 20,000 erythrocytes per animals were scored. Information on scoring, acceptability, and evaluation criteria is not provided. Historical control data is missing. No information on body weights at study initiation, during the study, and at the end of the study. The authors did not state whether the test groups were randomised.

Based on these findings the reference is considered to be not reliable [RL-3].
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
in vivo mammalian somatic cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not specified
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
The publication presented herein shows significant methodological and reporting deficiencies. The authors tested only one concentration, which precludes an assessment of a potential concentration-response relationship. However, the concentration tested (50 mg/m³) could be considered as limit concentration, which induce a local inflammatory response in the lung in a shorter 90-days dose range-finding study (Konduru, N. et al., 2014). The authors did not report on the analysis of the test concentration in the exposure and whether the target concentration was achieved. Exposure of the target tissue was not demonstrated. The number of animals per group is lower than recommended in the current draft test guideline (2020). Blood sampling was performed 1 day but not 28 days after the last exposure. Information on acceptability and evaluation criteria is not provided. Historical control data is not provided. No information on body weights at study initiation, during the study, and at the end of the study. The authors did not state whether the test groups were randomised. Based on these findings the reference is considered to be not reliable [RL-3].
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Cordelli, E. et al. (2017) examined the gene mutation potential of barium sulfate NM-220 using the mammalian erythrocyte Pig-a gene mutation assay. Five female Wistar rats were exposed to barium sulfate NM-220 aerosol at a concentration of 50 mg/m³ for 6 hours per day on 5 consecutive days/week for a total period of 6 months. A vehicle control (filtered clean air) group was run concurrently. Peripheral blood was sampled 24 hours after the last exposure. Animals exposed to N-ethyl-N-nitrosourea via gavage served as positive control group. The blood from the positive control group was sampled 30 days after the first administration. Erythrocyte were enriched in the blood samples and labelled with phycoerythrin (PE)-anti-CD59 and PE-anti-CD61, followed by coupling with anti-PE magnetic beads. The CD59-positive wild type red blood cells and CD61-positive platelets were separated from CD51-negative mutants by application of a magnetic field. The separated cell samples (‘post-column’) and a small fraction of unseparated cell samples (‘pre-column’) were stained using a fluorescent nucleic acid stain and a calibrated suspension of dye-containing reference beads. Afterwards, the cells were counted using flow cytometry. For each animal, the frequencies of Pig-a mutant red blood cells and reticulocytes were calculated from the cell equivalents analysed using the ratios of the reference dye-containing beads to total red blood cells and reticulocytes in the ‘pre-column’ sample and the ratios of reference dye-containing beads to enriched mutant total red blood cells and mutant reticulocytes in the ‘post-column’ sample. The cytotoxicity was determined by the proportion of reticulocytes (positive nucleic acid stain) among total erythrocytes.
GLP compliance:
yes
Type of assay:
other: mammalian erythrocyte Pig-a gene mutation assay
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
not specified
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Sulzfeld, Germany)
- Age at study initiation: 5-7 weeks
- Weight at study initiation: not specified
- Assigned to test groups randomly: not specified
- Fasting period before study: not specified
- Housing: Groups of up to five animals in polysulfone cages (H-Temp (PSU); TECNIPLAST, Germany).
- Diet: Kliba laboratory diet (ProvimiKliba SA; Kaiseraugst, Basel Switzerland); no access during exposure, otherwise ad libitum
- Water: no access during exposure, otherwise ad libitum
- Acclimation period: Acclimatised to the study conditions in the whole-body inhalation chambers by exposure to fresh air for 2 days (6 h, each) before the onset of the test substance exposure period

ENVIRONMENTAL CONDITIONS
- Temperature: controlled (not further specified)
- Humidity: controlled (not further specified)
- Photoperiod: controlled (not further specified)
Route of administration:
inhalation: aerosol
Vehicle:
- Vehicle(s)/solvent(s) used: clean air
Details on exposure:
Information on exposure conditions were extracted from Konduru et al. (2014)*.

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- Exposure apparatus/Method of holding animals in test chamber: animals were exposed while in wire cages that were located in a stainless-steel whole-body inhalation chamber (V = 2.8 m³ or V = 1.4 m³). Up to two animals per wire cage type DK III (BECKER & Co., Castrop-Rauxel, Germany) were exposed in the whole body exposure chamber.

- System of generating particulates/aerosols: test item aerosols were produced by dry dispersion of powder pellets with a brush dust generator using compressed air at 1.5 m³/h (developed by the Technical University of Karlsruhe in cooperation with BASF, Germany). The dust aerosol was diluted by conditioned air passed into the whole-body inhalation chambers. The control group was exposed to conditioned clean air.

- Treatment of exhaust air: the aerosols were passed into the inhalation chambers with the supply air and were removed by an exhaust air system with 20 air changes per hour. For the control animals, the exhaust air system was adjusted in such a way that the amount of exhaust air was lower than the filtered clean, supply air (positive pressure) to ensure that no laboratory room air reaches the control animals. For the BaSO4-exposed rats, the amount of exhaust air was higher than the supply air (negative pressure) to prevent contamination of the laboratory as a result of potential leakages from the inhalation chambers.

- Method of particle size and concentration determination: generated aerosols were continuously monitored by scattered light photometers (VisGuard). Particle concentrations in the inhalation chambers were analysed by gravimetric measurement of air filter samples. Particle size distribution was determined gravimetrically by cascade impactor analysis using eight stages Marple Personal Cascade Impactor (Sierra-Anderson, USA). In addition, a light-scattering aerosol spectrometer (WELAS 2000) was used to measure particle sizes from 0.24 to 10 μm. To measure particles in the submicrometer range, a scanning mobility particle sizer (SMPS 5.400) was used. The sampling procedures and measurements to characterize the generated aerosols were previously described (Ma-Hock et al., 2007)*.

*Reference:
- Konduru, N., Keller, J., Ma-Hock, L., Gröters, S., Landsiedel, R., Donaghey, T. C., Brain, J. D., Wohlleben, W. and Molina, R. M. (2014) Biokinetics and effects of barium sulfate nanoparticles. Part. Fibre Toxicol., 11, 55.
- Ma-Hock, L., Gamer, A.O., Landsiedel, R., Leibold, E., Frechen, T., Sens, B., Linsenbuehler, M., Van Ravenzwaay, B. (2007) Generation and characterization of test atmospheres with nanomaterials. Inhal Toxicol 19:833-848.
Duration of treatment / exposure:
6 months
Frequency of treatment:
6 hours/day on 5 consecutive days/week
Post exposure period:
24 hours
Dose / conc.:
50 mg/m³ air
No. of animals per sex per dose:
five (treatment group) and seven (vehicle control group) female rats
Control animals:
yes, concurrent vehicle
Positive control(s):
N-ethyl-N-nitrosourea (ENU)
- Route of administration: gavage
- Doses / concentrations: Rats received three doses of 20 mg/kg bw (10 mL/kg bw) at 24-hour intervals. The micronucleus assay was conducted on blood sampled 30 days after the first administration.
Tissues and cell types examined:
peripheral blood erythrocytes
Details of tissue and slide preparation:
DOSE-RANGE FINDING STUDY
Dose-range findings, short term inhalation studies were conducted, in during which BaSO4 NM-220 did not induce any effects at 50 mg/m³ (Konduru et al., 2014; please refer to section 7.5.2).

TREATMENT
The inhalation study, as a part of which the present genotoxicity studies were performed, was conducted as 2-year study according to OECD TG 453 under good laboratory practice conditions. Animals were exposed to an aerosol concentration of 50 mg/m³ BaSO4 NM-220 for 6 h/day whole-body exposure) on 5 consecutive days/week (working days, only). Concordantly, a control group was exposed to clean, filtered air. On exposure days, each animal was inspected clinically once after the exposure period. Occurrence of mortality was monitored twice daily on working days and once daily on weekends and holidays.

BLOOD SAMPLING
For the genotoxicity studies, blood was collected from five animals per group after 6-month test substance exposure. Blood sampling was performed 24 hours after the last exposure by retro-orbital venous plexus puncture under isoflurane anaesthesia. The blood samples were placed into K2EDTA tubes (Becton Dickinson, San José, CA, USA) and immediately shipped refrigerated (2-8°C) to the ENEA Laboratories (Italian National Agency for New Technologies, Energy and Sustainable Economic Development) Centro Ricerche Casaccia (Rome, Italy) for analysis.

PIG-A GENE MUTATION ASSAY
Within 72 hours after sampling, peripheral blood was processed according to the procedures described in the MutaFlowPLUS Kit® (Rat Blood; Litron Laboratories, Rochester, NY, USA) and as described by Dertinger et al. (2011)*. Approximately 80 μL of blood were mixed with 100 μL of anticoagulant solution. Most leukocytes and platelets were depleted by density-gradient centrifugation with Lympholyte-Mammal solution (Cedarlane, Burlington, NC) and the resulting erythrocyte-enriched samples were labelled with phycoerythrin (PE)-anti-CD59 and PE-anti-CD61 (from the MutaFlow kit), followed by labelling with anti-PE magnetic microbeads (Miltenyi Biotech, Auburn, CA, USA). After washing, cells were mixed with 1 mL buffer solution. A small fraction of the labelled cells and beads suspension was reserved as a ‘pre-column’ sample for subsequent flow cytometric analysis. The main part of the labelled cells and beads suspension was processed on an LS magnetic column (Miltenyi) mounted onto a quadroMACS magnet (Miltenyi). CD59-positive wild-type RBCs, and CD61-positive platelets were retained on the column and the flow through fraction, consisting of enriched CD59-deficient (Pig-a mutant) RBCs, was collected as a ‘post-column’ sample for subsequent flow cytometric analysis. Before the analyses, both the ‘pre-’ and ‘post-column’ samples were stained with a solution containing SYTO13® dye (from the MutaFlow kit) and CountBright™ absolute counting beads (Life Technologies, Carlsbad, CA, USA). An instrument calibration standard was prepared by creating a specimen consisting of ~ 50% wild-type and 50% mutant-mimicking cells processed in the same way as the experimental samples but not incubated with antibodies. The samples were analysed via flow cytometry. For each animal, the frequencies of Pig-a mutant RBCs and RETs were calculated from the cell equivalents analysed using the ratios of the CountBright beads to total RBCs and RETs in the ‘pre-column’ sample and the ratios of CountBright beads to enriched mutant total RBCs and mutant RETs in the ‘postcolumn’ sample. Additionally, the percentage of RETs (%RETs), identified as SYTO® 13 positive cells, was recorded as an index for cytotoxicity with reduced %RET indicating cytotoxicity.

*References:
- Konduru, N., Keller, J., Ma-Hock, L., Gröters, S., Landsiedel, R., Donaghey, T. C., Brain, J. D., Wohlleben, W. and Molina, R. M. (2014) Biokinetics and effects of barium sulfate nanoparticles. Part. Fibre Toxicol., 11, 55.
- Dertinger, S. D., Bryce, S. M., Phonethepswath, S. and Avlasevich, S. L. (2011) When pigs fly: immunomagnetic separation facilitates rapid determination of Pig-a mutant frequency by flow cytometric analysis. Mutat. Res., 721, 163-170.
Evaluation criteria:
not specified
Statistics:
Comparison among group means was performed by one-way ANOVA and Dunnett’s test was used for post hoc comparison of treatments to controls. The paired Student’s t-test was applied to analyse the data obtained before and after treatment with ENU. Findings were assessed as treatment-related when their probability level was lower than 5% (P < 0.05).
Sex:
female
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
not determinable because of methodological limitations
Additional information on results:
TOXICITY
- During the 6 months of inhalation exposure recorded for the present study, treatment-related clinical signs were not observed in any of the rats.

PIG-A GENE MUTATION ASSAY
- In the Pig-a gene mutation assay, the frequency of mutant red blood cells and reticulocytes remained unaltered after 6-month exposure to barium sulfate NM-220 (please refer to attachments: 'Results').
- An outlier was noticed in the vehicle control group that had some impact on the mean and standard deviation values. In this case, the statistical analysis was carried out without removing their values from their group. The outcome of the statistical comparison between treated and control groups would not have changed irrespectively of the inclusion or exclusion of the outlier.
- The proportion of reticulocytes among total erythrocytes remained unchanged after exposure to barium sulfate NM-220 thereby providing an indication that the test material does not elicit cytotoxic effects in red blood cell precursors

VALIDITY OF THE ASSAY
- The mutation frequencies were significantly increased in red blood cells and reticulocytes 30 days after the onset of ENU administration. The data of ENU administration demonstrate the proficiency of the laboratory.
Conclusions:
Cordelli, E. et al. (2017) examined the gene mutation potential of barium sulfate NM-220 using the mammalian erythrocyte Pig-a gene mutation assay. Five female Wistar rats were exposed to barium sulfate NM-220 aerosol at a concentration of 50 mg/m³ for 6 hours per day on 5 consecutive days/week for a total period of 6 months. A vehicle control (filtered clean air) group was run concurrently. Peripheral blood was sampled 24 hours after the last exposure. Animals exposed to N-ethyl-N-nitrosourea via gavage served as positive control group. The blood from the positive control group was sampled 30 days after the first administration. Erythrocyte were enriched in the blood samples and labelled with phycoerythrin (PE)-anti-CD59 and PE-anti-CD61, followed by coupling with anti-PE magnetic beads. The CD59-positive wild type red blood cells and CD61-positive platelets were separated from CD51-negative mutants by application of a magnetic field. The separated cell samples (‘post-column’) and a small fraction of unseparated cell samples (‘pre-column’) were stained using a fluorescent nucleic acid stain and a calibrated suspension of dye-containing reference beads. Afterwards, the cells were counted using flow cytometry. For each animal, the frequencies of Pig-a mutant red blood cells and reticulocytes were calculated from the cell equivalents analysed using the ratios of the reference dye-containing beads to total red blood cells and reticulocytes in the ‘pre-column’ sample and the ratios of reference dye-containing beads to enriched mutant total red blood cells and mutant reticulocytes in the ‘post-column’ sample. The cytotoxicity was determined by the proportion of reticulocytes (positive nucleic acid stain) among total erythrocytes.

Whole body inhalation exposure to 50 mg/m³ barium sulfate NM-220 did not result in treatment-related clinical signs. However, in a preceding 90-day study, barium sulfate NM-220 induced local inflammatory responses, characterised i.a. by significant neutrophil influx in bronchoalveolar lavage fluid, under the same exposure conditions, (Konduru, N. et al., 2014; please refer to section 7.5.2)*. According to the authors, barium sulfate NM-220 did not induce a statistically significant increase in the mutant frequency of both reticulocytes and red blood cells, when compared to the vehicle control group. The proportion of reticulocytes among total erythrocytes was comparable between the treatment and vehicle control groups. The positive reference mutagen, N-ethyl-N-nitrosourea, induced a distinct and statistically significant increase in the mutant frequencies in red blood cells and reticulocytes, when compared to pre-treatment samples. Thus, the sensitivity of the test system was demonstrated.

The publication presented herein shows significant methodological and reporting deficiencies.

The authors tested only one concentration, which precludes an assessment of a potential concentration-response relationship. However, the concentration tested (50 mg/m³) could be considered as limit concentration, which induce a local inflammatory response in the lung in a shorter 90-days dose range-finding study (Konduru, N. et al., 2014). The authors did not report on the analysis of the test concentration in the exposure and whether the target concentration was achieved. Exposure of the target tissue was not demonstrated. The number of animals per group is lower than recommended in the current draft test guideline (2020). Blood sampling was performed 1 day but not 28 days after the last exposure. Information on acceptability and evaluation criteria is not provided. Historical control data is not provided. No information on body weights at study initiation, during the study, and at the end of the study. The authors did not state whether the test groups were randomised.

Based on these findings the reference is considered to be not reliable [RL-3].

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The DNA damaging potential of barium sulfate NM-220 was evaluated in female Wistar rats after inhalation exposure by using the alkaline comet assay (Cordelli, E. et al., 2017). Five rats were exposed to barium sulfate NM-220 aerosol at a concentration of 50 mg/m³ for 6 hours per day on 5 consecutive days/week for a total period of 6 months. A vehicle control (filtered clean air) group was run concurrently. Animals exposed to N-ethyl-N-nitrosourea via gavage served as positive control group. Peripheral blood was sampled 24 hours after the last exposure. Afterwards, the blood was mixed with agarose, lysed, treated with alkaline solution, and subjected to electrophoresis. Subsequently, the cells were neutralised, fixed, and stained with ethidium bromide. A total of 100 comets per animal (from two different slides) were scored for the proportion of DNA in tail via fluorescence microscopy in combination with computerised image analysis.
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Barium sulfate
EC Number:
231-784-4
EC Name:
Barium sulfate
Cas Number:
7727-43-7
Molecular formula:
BaO4S
IUPAC Name:
barium sulfate
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
not specified

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
not specified
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Sulzfeld, Germany)
- Age at study initiation: 5-7 weeks
- Weight at study initiation: not specified
- Assigned to test groups randomly: not specified
- Fasting period before study: not specified
- Housing: Groups of up to five animals in polysulfone cages (H-Temp (PSU); TECNIPLAST, Germany).
- Diet: Kliba laboratory diet (ProvimiKliba SA; Kaiseraugst, Basel Switzerland); no access during exposure, otherwise ad libitum
- Water: no access during exposure, otherwise ad libitum
- Acclimation period: Acclimatised to the study conditions in the whole-body inhalation chambers by exposure to fresh air for 2 days (6 h, each) before the onset of the test substance exposure period

ENVIRONMENTAL CONDITIONS
- Temperature: controlled (not further specified)
- Humidity: controlled (not further specified)
- Photoperiod: controlled (not further specified)

Administration / exposure

Route of administration:
inhalation: aerosol
Vehicle:
- Vehicle(s)/solvent(s) used: clean air
Details on exposure:
Information on exposure conditions were extracted from Konduru et al. (2014)*.

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- Exposure apparatus/Method of holding animals in test chamber: animals were exposed while in wire cages that were located in a stainless-steel whole-body inhalation chamber (V = 2.8 m³ or V = 1.4 m³). Up to two animals per wire cage type DK III (BECKER & Co., Castrop-Rauxel, Germany) were exposed in the whole body exposure chamber.

- System of generating particulates/aerosols: test item aerosols were produced by dry dispersion of powder pellets with a brush dust generator using compressed air at 1.5 m³/h (developed by the Technical University of Karlsruhe in cooperation with BASF, Germany). The dust aerosol was diluted by conditioned air passed into the whole-body inhalation chambers. The control group was exposed to conditioned clean air.

- Treatment of exhaust air: the aerosols were passed into the inhalation chambers with the supply air and were removed by an exhaust air system with 20 air changes per hour. For the control animals, the exhaust air system was adjusted in such a way that the amount of exhaust air was lower than the filtered clean, supply air (positive pressure) to ensure that no laboratory room air reaches the control animals. For the BaSO4-exposed rats, the amount of exhaust air was higher than the supply air (negative pressure) to prevent contamination of the laboratory as a result of potential leakages from the inhalation chambers.

- Method of particle size and concentration determination: generated aerosols were continuously monitored by scattered light photometers (VisGuard). Particle concentrations in the inhalation chambers were analysed by gravimetric measurement of air filter samples. Particle size distribution was determined gravimetrically by cascade impactor analysis using eight stages Marple Personal Cascade Impactor (Sierra-Anderson, USA). In addition, a light-scattering aerosol spectrometer (WELAS 2000) was used to measure particle sizes from 0.24 to 10 μm. To measure particles in the submicrometer range, a scanning mobility particle sizer (SMPS 5.400) was used. The sampling procedures and measurements to characterize the generated aerosols were previously described (Ma-Hock et al., 2007)*.

*Reference:
- Konduru, N., Keller, J., Ma-Hock, L., Gröters, S., Landsiedel, R., Donaghey, T. C., Brain, J. D., Wohlleben, W. and Molina, R. M. (2014) Biokinetics and effects of barium sulfate nanoparticles. Part. Fibre Toxicol., 11, 55.
- Ma-Hock, L., Gamer, A.O., Landsiedel, R., Leibold, E., Frechen, T., Sens, B., Linsenbuehler, M., Van Ravenzwaay, B. (2007) Generation and characterization of test atmospheres with nanomaterials. Inhal Toxicol 19:833-848.
Duration of treatment / exposure:
6 months
Frequency of treatment:
6 hours/day on 5 consecutive days/week
Post exposure period:
24 hours
Doses / concentrations
Dose / conc.:
50 mg/m³ air
No. of animals per sex per dose:
five (treatment group) and seven (vehicle control group) female rats
Control animals:
yes, concurrent vehicle
Positive control(s):
N-ethyl-N-nitrosourea (ENU)
- Route of administration: gavage
- Doses / concentrations: Rats received three doses of 20 mg/kg bw (10 mL/kg bw) at 24-hour intervals. The micronucleus assay was conducted on blood sampled four days after the first administration.

Examinations

Tissues and cell types examined:
peripheral blood leukocytes (n=100 cells per animal)
Details of tissue and slide preparation:
DOSE-RANGE FINDING STUDY
Dose-range findings, short term inhalation studies were conducted, in during which BaSO4 NM-220 did not induce any effects at 50 mg/m³ (Konduru et al., 2014; please refer to section 7.5.2).

TREATMENT
The inhalation study, as a part of which the present genotoxicity studies were performed, was conducted as 2-year study according to OECD TG 453 under good laboratory practice conditions. Animals were exposed to an aerosol concentration of 50 mg/m³ BaSO4 NM-220 for 6 h/day whole-body exposure) on 5 consecutive days/week (working days, only). Concordantly, a control group was exposed to clean, filtered air. On exposure days, each animal was inspected clinically once after the exposure period. Occurrence of mortality was monitored twice daily on working days and once daily on weekends and holidays.

BLOOD SAMPLING
For the genotoxicity studies, blood was collected from five animals per group after 6-month test substance exposure. Blood sampling was performed 24 hours after the last exposure by retro-orbital venous plexus puncture under isoflurane anaesthesia. The blood samples were placed into K2EDTA tubes (Becton Dickinson, San José, CA, USA) and immediately shipped refrigerated (2-8°C) to the ENEA Laboratories (Italian National Agency for New Technologies, Energy and Sustainable Economic Development) Centro Ricerche Casaccia (Rome, Italy) for analysis.

COMET ASSAY
Within 24 hour after sampling, 30 μL of peripheral blood were used to perform the Comet assay as described by Singh et al. (1988)* with minor modifications. Briefly, blood was mixed with 0.7% (w/v) low-melting point agarose and sandwiched on microscope slides between a bottom layer of 1% (w/v) normal-melting point agarose and a top layer of 0.7% (w/v) low-melting point agarose. The slides were immersed overnight at 4°C in a lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris; pH 10) containing 10% DMSO and 1% Triton X-100. Upon completion of the lysis step, the slides were placed in a horizontal electrophoresis tank with fresh alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA; pH >13) and retained in the solution for 25 minutes at 4°C to allow for DNA unwinding and expression of the alkali-labile sites. Electrophoresis was carried out at 4°C for 25 minutes [27 V (1 V/cm) and 300 mA]. After electrophoresis, slides were neutralised in 0.4 M Tris (pH 7.4) and fixed in absolute ethanol. Slides were air-dried at room temperature. Immediately before scoring, slides were stained with 12 μg/mL ethidium bromide and examined with a fluorescence microscope. Slides were analysed by a computerised image analysis system (Delta Sistemi, Rome, Italy). A total of one hundred cells, from two different slides were scored for each animal. DNA damage was recorded as the percentage of total DNA in the Comet tail (% tail intensity).

*References:
- Konduru, N., Keller, J., Ma-Hock, L., Gröters, S., Landsiedel, R., Donaghey, T. C., Brain, J. D., Wohlleben, W. and Molina, R. M. (2014) Biokinetics and effects of barium sulfate nanoparticles. Part. Fibre Toxicol., 11, 55.
- Singh, N. P., McCoy, M. T., Tice, R. R. and Schneider, E. L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res., 175, 184-191.
Evaluation criteria:
not specified
Statistics:
Comparison among group means was performed by one-way ANOVA and Dunnett’s test was used for post hoc comparison of treatments to controls. The paired Student’s t-test was applied to analyse the data obtained before and after treatment with ENU. Findings were assessed as treatment-related when their probability level was lower than 5% (P < 0.05).

Results and discussion

Test results
Sex:
female
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
not determinable because of methodological limitations
Additional information on results:
TOXICITY
- During the 6 months of inhalation exposure recorded for the present study, treatment-related clinical signs were not observed in any of the rats.

COMET ASSAY
- No statistically significant increase of DNA damage over the concurrent vehicle controls was induced by inhalation exposure to barium sulfate NM-220.
- The proportion of DNA in tail was 1.83% (individual values: 2.15, 2.51, 1.86, 2.26, and 0.36%) and 1.79% (individual values: 2.95, 2.48, 2.60, 2.62, 0.36, 0.42, and 1.10%) in rats exposed to barium sulfate NM-220 and clean air, respectively.

VALIDITY OF THE ASSAY
- N-ethyl-N-nitrosourea (ENU) treatment elicited a significant increase in DNA damage, when compared to the pre-treatment control. The data of ENU administration demonstrate the proficiency of the laboratory.
- The comet assay vehicle control values were slightly higher than the historical control value of the laboratory which can be likely explained by the 24-h shipment interval between sampling and slide preparation.

Applicant's summary and conclusion

Conclusions:
The DNA damaging potential of barium sulfate NM-220 was evaluated in female Wistar rats after inhalation exposure by using the alkaline comet assay (Cordelli, E. et al., 2017). Five rats were exposed to barium sulfate NM-220 aerosol at a concentration of 50 mg/m³ for 6 hours per day on 5 consecutive days/week for a total period of 6 months. A vehicle control (filtered clean air) group was run concurrently. Animals exposed to N-ethyl-N-nitrosourea via gavage served as positive control group. Peripheral blood was sampled 24 hours after the last exposure. Afterwards, the blood was mixed with agarose, lysed, treated with alkaline solution, and subjected to electrophoresis. Subsequently, the cells were neutralised, fixed, and stained with ethidium bromide. A total of 100 comets per animal (from two different slides) were scored for the proportion of DNA in tail via fluorescence microscopy in combination with computerised image analysis.
Whole body inhalation exposure to 50 mg/m³ barium sulfate NM-220 did not result in treatment-related clinical signs. However, in a preceding 90-day study, barium sulfate NM-220 induced local inflammatory responses, characterised i.a. by significant neutrophil influx in bronchoalveolar lavage fluid, under the same exposure conditions, (Konduru, N. et al., 2014; please refer to section 7.5.2)*. In the alkaline comet assay, the proportion of DNA in tail observed in leukocytes from barium sulfate NM-220-treated rats was comparable to that obtained for the vehicle control group. The positive reference mutagen, N-ethyl-N-nitrosourea, induced a distinct and statistically significant increase in the proportion of DNA in tail, when compared to pre-treatment samples. Thus, the sensitivity of the test system was demonstrated.

The publication presented herein shows significant methodological and reporting deficiencies.

The authors tested only one concentration, which precludes an assessment of a potential concentration-response relationship. However, the concentration tested (50 mg/m³) could be considered as limit concentration, which induce a local inflammatory response in the lung in a shorter 90-days dose range-finding study (Konduru, N. et al., 2014). The authors did not report on the analysis of the test concentration in the exposure and whether the target concentration was achieved. Exposure of the target tissue was not demonstrated. The number of cells scored for DNA damage was lower (100 vs. 150 per animal) than recommended by the current test guideline (OECD TG 489, 2016). Information on scoring, acceptability, and evaluation criteria is not provided. The authors stated that the proportion of DNA in tail in was slightly above the historical control data, which is not in line with acceptability criteria provided by the current test guideline. Historical control data is not provided. No information on body weights at study initiation, during the study, and at the end of the study. The authors did not state whether the test groups were randomised. Information on hedgehog occurrence and potential cytotoxicity missing. It is unclear whether slides were coded and scored blinded. No information is given whether the proportion of DNA was evaluated by calculation of the mean (animal) of the medians (slides).

Based on these findings the reference is considered to be not reliable [RL-3].