Flue dust, portland cement

Genetic toxicity: in vivo

Administrative data

Endpoint:

genetic toxicity in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-2016 to 01-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals 489. In Vivo Mammalian Alkaline Comet Assay, adopted 29 July 2016

GLP compliance:

yes

Type of assay:

other: This study was conducted in accordance with the following guideline: OECD Guideline for the Testing of Chemicals 489. In Vivo Mammalian Alkaline Comet Assay, adopted 29 July 2016.

Test material

Test material information

Test material form:

solid: particulate/powder

Details on test material:

Batch 01-2016

Test animals

Species:

rat

Sex:

male

Administration / exposure

Route of administration:

inhalation: dust

Vehicle:

The animals were exposed to the test atmosphere in nose-only exposure units, in an illuminated
laboratory room different from the room where the animals were housed. Animals of groups 2,
3 and 4 were exposed in inhalation chambers consisting of a cylindrical stainless steel column,
surrounded by a transparent cylinder (a modification of the design of the chamber made by ADG
Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom). The column had a
volume of 55.6 L and consisted of a top assembly with the entrance of the unit, a mixing section,
two rodent tube sections and at the bottom the base assembly with the exhaust port. Each
rodent tube section had 20 ports for animal exposure. Negative control animals (group 1) were
exposed to clean air in a polypropylene nose-only inhalation chamber with a volume of 48.2 L
manufactured by P. Groenendijk Kunststoffen BV, which was very similar in construction to the
stainless steel chambers described above.
Empty ports were used for test atmosphere sampling (for analysis of the actual concentration
and particle size) and measurement of oxygen, carbon dioxide, temperature and relative
humidity. The animals were secured in plastic animal holders (Battelle), positioned radially
through the outer cylinder around the central column. The remaining ports were closed. Only
the nose of the rats protruded into the interior of the column. Habituation to the restraint in the
animal holders was performed because in our experience habituation does not help to reduce
possible stress (Staal et al., 2012).
In our experience, the animal’s body did not exactly fit in the animal holder which always results
in some leakage from high to low pressure side. By securing a positive pressure in the central
column and a slightly negative pressure in the outer cylinder, which encloses the entire animal
holder, dilution of test atmosphere by air leaking from the animals’ thorax to the nose was
avoided.
The unit was illuminated externally by normal laboratory fluorescent tube lighting. The total air
flow through the unit was at least 1 liter/min for each rat. The air temperature and relative
humidity in the unit were maintained at 22 ± 3˚C and between 30 and 70%, respectively

Details on exposure:

The inhalation equipment was designed to expose the rats to a continuous supply of fresh test
atmosphere. A schematic diagram of the generation and exposure system is presented in
Figure 1. The test atmosphere was generated using a turntable dust feeder (Reist and Taylor,
2000) and an eductor (Fox Valve Development Corp., Dover, NJ, USA; Cheng et al., 1989). The
compartment of the dust feeder containing the test material was flushed with a stream of dry
compressed air (about 2.5-3.5 L/min) to avoid humidification of the test material. The test
material was aerosolized in the eductor, which was supplied with a flow (controlled using a
reducing valve) of dry compressed air. The resulting aerosol was led through a glass cyclone,
which was used to remove the largest particles from the aerosol, and was subsequently
introduced at the top inlet of the exposure chamber. The eductors were calibrated by measuring
the total air flow at a range of driving air pressures of the eductors encompassing the driving
pressures used during the study. The driving air pressure was used to monitor the total flow. A
bypass stream of humidified compressed air (measured by mass view meter; Bronkhorst Hi Tec,
Ruurlo, The Netherlands) was added at the top of the exposure chamber to ensure a relative
humidity above 30%. The resulting test atmosphere was directed downward and led to the noses
of the animals. At the bottom of the unit the test atmosphere was exhausted.
The exposure chamber for the negative control animals (group 1) was supplied with a stream
of humidified compressed air only, which was controlled by a reducing valve and measured by
mass view meter (Bronkhorst Hi Tec).
The animals were placed in the exposure unit after stabilization of the test atmospheres
. Test atmosphere generation and animal exposure were performed in
an illuminated laboratory at room temperature.
During the exposure period of this study, the animals were exposed to test atmosphere also
generated for a range finding study (study 20862/01, preceding a sub-chronic inhalation toxicity
study conducted according to OECD guideline 413) which is not finished yet.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air

Remarks:

reference dose

Doses / concentrations 2

Dose / conc.:

40 mg/m³ air (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/m³ air (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/m³ air (nominal)

No. of animals per sex per dose:

6

Control animals:

yes

Examinations

Tissues and cell types examined:

liver and lung cells

Applicant's summary and conclusion

Conclusions:

Under the conditions used in this study, it is concluded that the test material Flue Dust (REACH)
2016 did not induce primary DNA damage in liver and lung (first site of contact) cells of male
rats after inhalation exposure up to 1000 mg/m3 for 6 hours per day, 5 days per week, over a
2-week study period.
Based on the clinical observations, effects on body weight and results obtained in the 14 day
range finder (14d RF study 20862/01: reduced food consumption (mid & high dose), increased
lung weights (mid & high dose), concentration related changes in bronchoalveolar lavage
parameters indicative of tissue damage and inflammation (all dose levels), histopathological
findings in the nose, larynx and lungs in the form of degeneration, ulceration, inflammation of
the epithelium, collagenisation and fibrotic phenomena (mid & high dose), the highest dose is
considered to be the maximum tolerable dose and treatment with higher concentrations was
expected to result in mortality.
In the current comet assay the liver was evaluated to detect the genotoxic potential of any
systemically available components or fraction of the test substance, whereas the lung was
evaluated to detect the genotoxic potential of the components or fraction at the ‘first site of
contact’.

Executive summary:

The test material Flue Dust (REACH) 2016 was examined for its potential to cause primary DNA damage, such as single and double strand DNA breaks, alkali labile sites and incomplete repair sides, in rat liver and lung cells using the comet assay after inhalation exposure. Male rats (n=5) were exposed to three concentrations of the test material or clean air (negative control) for 6 hours per day, 5 days per week, over a 2-week study period (10 exposure days in total), with the last 6 hour exposure on the day before scheduled sacrifice. In addition, the animals were exposed for 2 hours on the day of sacrifice. This exposure regimen meets the OECD guideline 489 requirements to sample 2-6 h and 16-26 h after exposure in the same animal. The highest concentration was based on the results of an acute inhalation toxicity study with the test material and was intended to result in toxic effects but not death or severe suffering. The lowest concentration was intended to produce little or no evidence of toxicity. Positive control animals for liver and lung were dosed once orally (by gavage) with 2-acetylaminofluorene (2-AAF, 50 mg/kg-bw) 12-16 h prior to sacrifice or dosed once intraperitoneally with methyl methanesulfonate (MMS, 40 mg/kg-bw) 2-6 h prior to sacrifice, respectively. The target concentrations were accurately achieved as demonstrated by the results of gravimetric analysis of the test atmospheres. The overall mean actual concentrations (± standard deviation) during exposure were 42.3 (± 5.3), 204 (± 12) and 1018 (± 75) mg/m3 for the low-, mid- and high-concentration groups, respectively. The aerodynamic particle size of the aerosol was 1.96 (± 0.11), 1.68 (± 0.05) and 2.02 (± 0.46) μm MMAD (mass median aerodynamic diameter) for the low-, mid- and-high concentration test atmospheres, i.e. well within the recommended range of 1 - 3 μm. All animals survived until scheduled sacrifice. No treatment-related clinical abnormalities were observed in the negative control and positive control groups and in the low and mid concentration groups (groups 1, 2, 3, 5 & 6). In the high concentration group (group 4), effects on respiration were observed in the form of sniffing, grunting and dyspnea. In addition, shallow breathing at a decreased rate was observed during exposure to the high concentration. There were no statistically significant effects on mean body weight following treatment with the low and mid concentration when compared to the clear air group. Treatment with the highest concentration resulted in a lower mean body weight, which was statistically significant at days 3 and 10 when compared to the negative control group. Shortly after the end of the 2-hour exposure to the test material, liver cells (hepatocytes) were isolated by liver perfusion, followed by collection of the lung tissue and mincing to obtain lung cells and preparation of comet slides. The percentage viability of the isolated hepatocytes was 81%, 80%, 80% and 77% for the negative control (clean air), low, mid and high concentration respectively, whereas the percentage viability of the isolated lung cells was 100%, 100%, 99%, 100% and 100%, respectively. Based on the observed viability, all cell suspensions were considered suitable for the comet assay. Tail intensity (i.e. the percentage DNA in the ‘tail’ of the comet) was used as a measure for DNA damage. Fifty cells per slide and three slides per animal were analyzed (i.e. in total 150 cells per animal per tissue). The median of each slide was calculated and the mean of the three medians was calculated per animal. Finally, the group mean of the individual animal values was calculated. The positive control substances 2-AAF and MMS demonstrated a statistical significant increase in tail intensity compared to the negative control (clean air) for liver and lung, Triskelion Report | V20861 | Draft | 02 June 2017 6/54 respectively. Mean tail intensity of the negative control (clean air) was within the historical range for liver, but was just outside the historical range for lung. Since the tail intensity was within ranges found in recent literature and was in line with the preferred range for liver according to OECD guideline 489 (i.e. not exceeding 6%), the negative control data of the lung were accepted. Therefore, the comet assay was considered valid. Tail intensity of the test material Flue Dust (REACH) 2016 was comparable to the negative control (clean air) and did not demonstrate a statistically significant increase in tail intensity in liver cells at any of the concentrations tested and in lung cells at the mid and highest concentrations tested. Tail intensity of the test material Flue Dust (REACH) 2016 was statistically significantly increased compared to the negative control (clean air) at the lowest concentration tested in lung cells (p=0.0389). Since this increase was observed at the lowest concentration only and was not dose-related, the response was considered not biologically relevant. Based on the clinical observations, effects on body weight and results obtained in the 14d RF (study 20862-01: reduced food consumption (mid & high dose), increased lung weights (mid & high dose), concentration related changes in bronchoalveolar lavage parameters indicative of tissue damage and inflammation (all dose levels), histopathological findings in the nose, larynx and lungs in the form of degeneration, ulceration, inflammation of the epithelium, collagenisation and fibrotic phenomena (mid & high dose), the highest dose is considered to be the maximum tolerable dose and treatment with higher concentrations was expected to result in mortality. In the current comet assay the liver was evaluated to detect the genotoxic potential of any systemically available components or fraction of the test substance, whereas the lung was evaluated to detect the genotoxic potential of the components or fraction at the ‘first site of contact’. Under the conditions used in this study, it is concluded that the test material Flue Dust (REACH) 2016 did not induce primary DNA damage in liver and lung cells of male rats after inhalation exposure for 6 hours per day, 5 days per week, over a 2-week study period.