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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is published, but was performed largely in compliance with a recognised guideline and is GLP compliant.

Data source

Referenceopen allclose all

Reference Type:
Responses in the respiratory tract of rats following exposure to sulphuric acid aerosols for 5 or 28 days
Kilgour JD, Foster J, Soames A, Farrar DG & Hext PM
Bibliographic source:
J Appl Toxicol 22(6): 687-395
Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
: Exposure for 5 or 28 days; pathology limited to the respiratory tract
Principles of method if other than guideline:
The study was performed to OECD 412 with the addition of a 5-day exposure. Pathology was limited to the respiratory tract.
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
Sulphuric acid
EC Number:
EC Name:
Sulphuric acid
Cas Number:
Molecular formula:
sulfuric acid
Details on test material:
Animals were exposed to sulphuric acid mist.

Test animals

other: ALPK:APfSD (Wistar)
Details on test animals or test system and environmental conditions:
Rats were housed 5 per cage, in multiple rat racks suitable for animals of this strain and the weight range expected during the course of the study.
The rats were transferred to clean cages and racks as necessary during the studyBoth temperature and relative humidity were measured and recorded daily and the recorded values were within the specified ranges.
Diet (CT1) supplied by Special Diet Services Limited, Witham, Essex, UK and mains water, supplied by an automatic system, were available ad libitum, except during exposure.
Each batch of diet was routinely analysed for composition and for the presence of contaminants. Water was also periodically analysed for the presence of contaminants. No contaminants were found to be present in the diet or water at levels considered to be capable of interfering with the purpose or outcome of the study. The animals were housed under the experimental conditions for 13 days at CTL, prior to the start of the study (animals arrived 12 November 1998; first exposure 25 November 1998). The study was divided into 2 replicates (randomised blocks), each containing 10 cages, one per treatment group. Computer-generated, random number permutations were used to allocate the cages within each replicate to an experimental group.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: Particle sizes were 0.62, 0.83 and 0.94 um, respectively.
Details on inhalation exposure:
The rats were exposed nose-only to the test atmospheres. Animals were restrained in polycarbonate tubes supplied by Battelle, Geneva, Switzerland. These were inserted into a PERSPEX exposure chamber. The chamber was covered with a stainless steel cone and stood on a stainless steel base. The atmosphere was shown to be acceptably stable over approximately 30 minutes before exposure of the test animals. During this period the holes of the exposure chamber were plugged. The animals were exposed for 6 hours per day, 5 days per week, for a period of 28 days.Test atmospheres were generated using a glass concentric - jet atomiser to generate atmospheres into 2 reservoir chambers (each fitted with a cyclone), one serving the exposure chambers for groups 4&5 and 6&7, and another serving exposure chambers for groups 8, 9 & 10. Clean, dry air (dried and filtered using equipment supplied by Atlas-Copco, Sweden) was passed through the atomiser at nominal flow rates (given below) for each group respectively and carried the atmosphere to each of the exposure chambers (internal volume of 36.8 litres), in order to achieve a minimum of 12 air changes per hour. Air flows were monitored and recorded at approximately 30 minute intervals using variable area flowmeters (KDG Flowmeters, Burgess Hill, Sussex, UK) and were altered as necessary to maintain the target concentrations.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The particulate concentration of each test atmosphere, close to the animals’ breathing zone, was measured gravimetrically at least twice during exposure. This was done by drawing each test atmosphere, at a known flow rate, for a known time, through a 25mm diameter, polyvinyl chloride (PVC) GN-4 filter housed in a Delrin open-faced filter holder (both filters and holders supplied by Gelman Sciences Limited, Northampton, UK). The filter was weighed before and after the sample was taken. The aerodynamic particle size distribution of each test atmosphere was measured at least once during exposure period, using a Marple Cascade Impactor (supplied by Schaeffer Instruments Limited, Wantage, Oxon, UK) which aerodynamically separates airborne particles into pre-determined size ranges. The amount of aerosol, by weight, in each size range, was then used to calculate the aerodynamic particle size distribution of the aerosol. Using a microcomputer, the data were transformed using a log/probit transform and a linear regression derived from the cumulative data.
Using this regression line, the mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated.
Duration of treatment / exposure:
Animals were exposed for 6 hours/day; 5 days/week for 5 or 28 days.
Frequency of treatment:
Animals were exposed for 6 hours/day; 5 days/week for 5 or 28 days.
Doses / concentrationsopen allclose all
Doses / Concentrations:
0.00, 0.2, 1.0, 5.0 mg/m3
nominal conc.
Doses / Concentrations:
0.00, 0.30. 1.38, 5.52 mg/m3
analytical conc.
No. of animals per sex per dose:
10 females
Control animals:
yes, concurrent no treatment


Observations and examinations performed and frequency:
For examination of effects in the lung, 5 designated rats per group, per time point, were implanted subcutaneously with osmotic minipumps (Alzet 2ML1) for delivery of BrdU 7 days before termination. The minipumps contained BrdU at a concentration of 15mg/ml.
At post mortem, the lungs (tracheas attached but larynx removed) were excised and weighed, prior to inflation with 10% neutral buffered formalin. The lungs were fixed in 10% neutral buffered formalin for 24 hours. The tissue was trimmed and processed to paraffin wax blocks and sections made and stained to reveal BrdU positive nuclei. Labelling indices (LI) were determined for the central acinar region and in the terminal bronchiole regions of the lung. LI were determined by counting the number of BrdU labelled cell nuclei and the total number of cell nuclei (labelled and non labelled). LI were then determined by dividing the number of BrdU labelled cells by the total number of labelled and non labelled cells, the result being expressed as a percentage.Labelling indices were determined for the small intestine. The small intestine has an inherently high rate of cell turnover and as such acts as a positive control for the BrdU immunostaining. Pulse labelling with tritiated thymidine, followed by autoradiography, was used for the assessment of rate of cell proliferation in the nasal passages; thymidine being incorporated into DNA during s-phase.
Five designated rats per group were injected intra-peritoneally with tritiated thymidine at a dose level of 1¿Ci/g bodyweight, approximately one hour prior to scheduled termination. At post mortem, the heads from all designated animals were removed, excess skin and muscle removed, the brain excised and the nasal cavity perfused with 10% formol saline through the nasopharynx. The head was then immersed in formol saline followed by decalcification with 20% formic acid. After processing, six standard sections were produced to include all different epithelial cell types and accessory nasal structures (Appendix I). The six sections were exposed to nuclear emulsion (Ilford K2) for 8-10 weeks. Sections were developed and examined by light microscopy. Labelling indices (LI) were determined for each of the 6 nasal passage levels. LI were determined by counting the number of thymidine labelled cell nuclei and the total number of cell nuclei (labelled and non labelled). LI were then determined by dividing the number of thymidine labelled cells by the total number of labelled and non labelled cells, the result being expressed as a percentage.
Where more than one section was examined from a tissue, results for each level of that tissue are reported separately.
Sacrifice and pathology:
AFrom all animals surviving to scheduled termination (day 29 for main study animals and after 4 or 8 weeks monitoring in recovery animals), the lungs were removed, trimmed free of extraneous tissue and weighed (with trachea attached but larynx removed, each pair of lungs weighed together). t post mortem the larynx was removed from all animals and fixed in 10% neutral buffered formalin for 24 hours. The tissues were trimmed and processed to paraffin wax blocks and three standard sections of larynx produced, taken at the level of the base of the epiglottis, through the ventral pouch and the cricoid cartilage to include all different epithelial cell types of the larynx and underlying seromucinous glands (Appendix I).
Standard sections of larynx from animals receiving BrdU were stained to reveal BrdU positive nuclei, while those from animals receiving thymidine, were exposed to nuclear emulsion (Ilford K2) for 8-10 weeks before being developed. For both the BrdU and thymidine animals, cell replication rates in the larynx were expressed as unit length labelling indices (ULLI).
In ULLI’s, the basement membrane length is substituted for the total cell count in the labelling index equation. ULLI’s were determined by counting the number of BrdU (and thymidine) labelled nuclei and dividing the result by the length of underlying basement membrane; ULLI’s being expressed as the number of BrdU (or thymidine) labelled cells per unit length (mm) of epithelium. The length of the basement membrane was determined using a Kontron Image Analyser attached to a Leitz light microscope.

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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed

Effect levels

Dose descriptor:
Effect level:
0.3 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
other: Findings at 0.3 mg/m3 were limited to minimal metaplastic change after 28 days, considered to be an adaptive response to a respiratory irritant.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

There were no deaths, no signs of toxicity, and no adverse effects on bodyweight or lung weight in any treatment group.  There were no macroscopic findings in animals killed at term.  Microscopically, no treatment related changes were seen in either the lung or the nasal cavity.   The major treatment related effect was squamous metaplasia of the ventral epithelium of level 1 of the larynx, the severity of which was concentration-dependant.  In the 5 day satellite study, the NOEL for this effect was 0.30mg/m3.  In the 28 day study, squamous metaplasia of the larynx was seen at all concentrations (including minimal squamous metaplasia in 3/10 animals exposed to 0.30mg/m3); the severity of this finding was directly related to exposure concentration.  After 4 and 8 weeks recovery following exposure to the highest concentration (5.52mg/m3) evidence of metaplasia remained, although it was less severe than that seen immediately following the 28-day exposure period.  No increases in cell proliferation were detected in either the lung or the nasal cavity at either 5 or 28 days; results were in agreement with the pathological assessment.  In the larynx, a treatment related increase in cell turnover was seen in the same region of level 1 as the exposure related pathological finding of squamous metaplasia, with a NOEL for this finding of 0.30 mg/m3 for both 5 and 28 days. 

Applicant's summary and conclusion

Following inhalation exposure to sulphuric acid mists, treatment-related findings were limited to histopathology and cell proliferation of the larynx, consistent with a local irritant effect of the substance
Executive summary:

Groups of female rats were exposed to aerosols of sulphuric acid (mists) at target concentrations of 0, 0.2, 1.0 or 5.0 mg/m3 for 6 hours a day, 5 days a week for 5 or 28 days. Additional groups exposed to 0 or 5.0 mg/m3 (nominal concentration) for 28 days were investigated following recovery periods of 4 or 8 weeks Effects of exposure were limited to the larynx.

Squamous metaplasia and significant cell proliferation was seen following exposure to 1.38 and 5.52 mg/m3 for 5 and 28 days; findings had decreased in severity following the recovery periods. Findings following exposure to 0.3 mg/m3 for 28 days were limited to minimal metaplasia (with no proliferation); no effects were apparent following exposure to 0.3 mg/m3 for 5 days. The LOAEC for this study is therefore considered to be 0.3 mg/m3.