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Diss Factsheets

Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Study lacks sufficient experimental details and does not assess parameters of a guideline study.

Data source

Reference Type:
Effect of inhalation of low doses of crocidolite and fibrous gypsom on the glutathione concentration and γ-glutamyl transpeptidase activity in macrophages and bronchoalveolar lavage fluid.
Clouter A, Houghton CE, Hibbs LR & Hoskins JA.
Bibliographic source:
Inhalation Toxicology 10: 3 - 14

Materials and methods

Test guideline
according to guideline
other: no data
not applicable
Principles of method if other than guideline:
Rats were exposed to an aerosol of anhydrous calcium sulfate fibres for 3 weeks for 6 h/day, 5 days/ wk at a concentration of approximately 10 mg m-3 in a nose-only inhalation system.
GLP compliance:
not specified
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
Calcium sulfate
EC Number:
EC Name:
Calcium sulfate
Cas Number:
Molecular formula:
calcium sulfate
Details on test material:
- Name of test material: anhydrous calcium sulfate, A-30.

Test animals

Fischer 344
Details on test animals or test system and environmental conditions:
- Source: Harlan Olac., Bicester, Oxon.
- Age at study initiation: no data
- Weight at study initiation: 130 - 150 g
- Fasting period before study: no data
- Housing: felxible film isolator
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: no data

- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
other: no data
Remarks on MMAD:
MMAD / GSD: No data
Details on inhalation exposure:
- Exposure apparatus: nose-only system
- Method of holding animals in test chamber: no data
- Source and rate of air: no data
- Method of conditioning air: no data
- System of generating particulates/aerosols: no data
- Temperature, humidity, pressure in air chamber: no data
- Air flow rate: no data
- Air change rate: no data
- Method of particle size determination: air was drawn through a filter system during exposure at 1 L/min for up to 6 h and the fibre concentration in the aerosol was measured gravimetrically.
- Treatment of exhaust air: no data

- Brief description of analytical method used:
- Samples taken from breathing zone: yes

- Justification for use and choice of vehicle: no data
- Composition of vehicle: no data
- Type and concentration of dispersant aid: no data
- Concentration of test material in vehicle: no data
- Lot/batch no. of vehicle: no data
- Purity of vehicle: no data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
During inhalation, fibre samples were taken from the aerosol, both isoaxially and isokinetically under conditions in which the sampling probe had little effect on the flow before it entered the exposure tower. Twice a day the aerosol breathed by the animals was sampled by replacing one animal at random with a filter system during the exposure period. Air was drawn through the filter at 1 L/min for up to 5 h and the fibre concentration was measured gravimetrically.
Duration of treatment / exposure:
3 weeks
Frequency of treatment:
6 h/day for 5 days/week
Doses / concentrations
Doses / Concentrations:
10 mg m-3
no data
No. of animals per sex per dose:
36 males only
Control animals:
not specified
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment: no data
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: no data
- Section schedule rationale: no data
Positive control:
No data


Observations and examinations performed and frequency:
Alveolar macrophages and "concentrated" bronchoalveolar lavage fluid (cBALF) from 12 animals were prepared by filling the lungs, via the trachaea, with 0.15 M saline (8 mL) by means of syringe attached to the Luer port cannula. The fluid was withdrawn (usually about 4 mL) and made up to 8 mL) with fresh saline. This was then reintroduced into the lung and withdrawn. The resulting fluid was centrifuged at 300 x g for 20 min to remove the free cell population (mainly macrophages - data not provided). The supernatant ("concentrated" acellular BAL, cBALF) was removed and the macrophages resuspended in phosphate-buffered saline. Aliquots of cBALF and macrophages were stored at -80 ºC. Prior to assay, cell samples were thawed and sonicated for 2 min to ensure lysis and the following biochemical measurements were made on the samples: protein, non-protein sulfhydryl (mainly glutathione) and γ-glutamyl transpeptidase (γ-GT). The numbers of macrophages per alveolus were counted in haematoxylin and eosin (H & E) stained fixed sections and in the lavage fluid using a haemocytometer.
Sacrifice and pathology:
The lungs from 6 animals in each group were removed and fixed with 10 % buffered formalin. Lungs from the remaining 12 animls in each group were perfused with saline before removal. Paraffin blocks were made for histological study. The post caval lobes from a minimum of 2 animals in each group were tied off and kept for fibre analysis. The samples were dried with acetone and plasma ashed.
Calcium levels in lung tissue from gypsum-treated animals were measured in the ashed postcaval lobe by inductively coupled plasma spectroscopy (ICP at a wavelength of 393.366 nm.
Other examinations:
No data
Data was normalised to control values and the significance of findings were determined using a two-tailed Student's t-test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
no mortality observed
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:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Details on results:
No gysum fibres were found in any of the samples analyzed desite measuring airborne levels of, on average, 15 mg•m-3. Analysis of calcium sulfate levels in ashed lung, in an attempt to estimate lung burden, was made but calcium levels in animals from all groups, including controls were not significantly different from each other. Even animals that were killed immediately post-exposure did not have raised calcium levels. However, hiostological sections showed the presence of fibres in the lungs, proving that there were enough respirable fibres in the aerosol to reach the alveoli (data not shown). It is possible that the method of solubilizing the lung may have interfered with the calcium assay or that the amount of calcium remaining in the lung was below detection limits.
The number of macrophages per alveolus did not increase overall following any treatment (see table) although there were some localised areas containing a high number of macrophages, notably around the acinar region. The number of macrophages isolated by lavage was on average 0.5 - 0.6E06 and this did not alter significantly, regardless of treatment.
There were no significant changes in protein concentration in any of the samples following treatment.
There was a significant decrease in extracellular GGT after recovery following calcium sulfate exposure. Under all conditions the γ-GT levels did not differ significantly from the controls. An increase in glutathione (NPSH) levels was seen in cBALF after calcium sulfate exposure. Macrophages from the calcium sulfate-treated animals lost almost all of their NPSH.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Number of macrophages per alveolus counted from formalin-fixed sections


Number of macrophages per alveolus (± SD)



(n = 10)

Sacrificed after

4 week recovery

(n = 10)

Air controls

0.22 (± 0.13)

0.18 (± 0.06)


0.33 (± 0.10)

0.2 (± 0.02)

Protein levels in cBALF and macrogphages from animals exposed to calcium sulfate or air


Protein levels

Sacrificed immediately

Sacrificed after

4 week recovery









Air controls

0.92 (± 0.2)

0.21 (± 0.07)

0.51 (± 0.2)

1.27 (± 0.4)


1.39 (± 0.4)

0.53 (± 0.2)

0.59 (± 0.3)

0.36 (± 0.1)

Applicant's summary and conclusion

The number of macrophages per alveolus did not increase overall following any treatment.
Executive summary:

This study summary was provided for the registration of calcium sulfate.