Chlorosulphuric acid

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

The data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Repeated dose oral toxicity study of the test chemical

Author:

Kilgour et al

Year:

2002

Bibliographic source:

JOURNAL OF APPLIED TOXICOLOGY

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Principles of method if other than guideline:

The study was performed to evaluate the levels of toxicity caused by the test chemical in rats through the inhalation route of exposure

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Sulphuric acid

EC Number:

231-639-5

EC Name:

Sulphuric acid

Cas Number:

7664-93-9

Molecular formula:

H2O4S

IUPAC Name:

Acide Sulfurique

Test material form:

liquid

Details on test material:

- Name of test material: Sulfuric acid
- IUPAC name: Dihydrogensulfate
- Molecular formula: H2O4S
- Molecular weight: 98.0778 g/mol
- Substance type: Inorganic

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Alpk:APfSD strain

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Alderley Park, Macclesfield, Cheshire, UK
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: No data
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: Animals were housed in multiple rat racks at five per cage total initially
- Diet (e.g. ad libitum): Food ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period: 1-2 weeks

DETAILS OF FOOD AND WATER QUALITY: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30–70%
- Air changes (per hr): at least 15 air changes per hou
- Photoperiod (hrs dark / hrs light): 12 h per day of fluorescent ligh

IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Resevoir chambers
- Method of holding animals in test chamber: No data
- Source and rate of air: Glass concentric jet atomizer
- Method of conditioning air: No data
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber: Humidified dilution air was added, where appropriate, to maintain a humidity as close as possible to 50% inside the chambers.
- Air flow rate: Air flows were monitored and recorded frequently using variable area flowmeters and were altered as necessary to maintain the target concentrations and maintain a minimum of 12 air changes per hour
- Air change rate: No data
- Method of particle size determination: The aerodynamic particle size distribution of each test atmosphere was measured using a Marple Cascade Impactor (supplied by Schaeffer Instruments Ltd, Wantage, Oxon, UK) that aerodynamically separates airborne particles into predetermined size ranges. The amount of aerosol by weight in each size range was then used to calculate the mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD)
- Treatment of exhaust air: No data

TEST ATMOSPHERE
- Brief description of analytical method used: The particulate concentration of each test atmosphere, close to the animals’ breathing zone, was measured gravimetrically and analytically at least twice during each exposure. This was done by drawing each test atmosphere at a known flow rate, for a known time, through a 25-mm diameter polyvinyl chloride (PVC) GN-4 filter housed in a Delrin open-faced filter holder (both filters and holders were supplied by Gelman Sciences Ltd, Northampton, UK). The filter was weighed before and after the sample was taken and the gravimetric concentration was calculated by dividing the weight gain by the total volume of atmosphere sampled.

Analytical concentrations of sulphuric acid were determined by extracting the material collected on the filters and stages with water (1 × 5 ml wash) and isopropyl alcohol (IPA, 3 × 5 ml washes) and titrating the resulting solution with 0.005 M barium perchlorate, using Thorin as an indicator. A sharp colour change from yellow to salmon pink signified the end-point

- Samples taken from breathing zone: Yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle: Air
- Composition of vehicle: No data
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle: 0, 0.2, 1.0 or 5.0 mg/m3
- Lot/batch no. of vehicle (if required):
- Purity of vehicle: No data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The particulate concentration of each test atmosphere, close to the animals’ breathing zone, was measured gravimetrically and analytically at least twice during each exposure. This was done by drawing each test atmosphere at a known flow rate, for a known time, through a 25-mm diameter polyvinyl chloride (PVC) GN-4 filter housed in a Delrin open-faced filter holder (both filters and holders were supplied by Gelman Sciences Ltd, Northampton, UK). The filter was weighed before and after the sample was taken and the gravimetric concentration was calculated by dividing the weight gain by the total volume of atmosphere sampled.

Duration of treatment / exposure:

28 days

Frequency of treatment:

5 days/week

Doses / concentrations

Remarks:

0, 0.2, 1.0 or 5.0 mg/m³ air
Achieved concentrations: 0, 0.3, 1.38 and 5.52 mg/m3

No. of animals per sex per dose:

10 females / group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): After an acclimatization period of 1–2 weeks, rats were assigned randomly to experimental groups by a method that takes into account the bodyweight of the animals
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: Yes, Recovery groups of five females were exposed to 0 or 5.0 mg m−3 for 28 days and retained without further treatment for either 4 or 8 weeks to monitor recovery
- Section schedule rationale (if not random): No data

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data
- Time schedule: No data
- Cage side observations checked in table [No.?] were included. No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were recorded daily immediately prior to exposure

BODY WEIGHT: Yes
- Time schedule for examinations: The bodyweight of each rat was recorded before the first exposure, once a week thereafter and prior to termination

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY: No data
- 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: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined:

HAEMATOLOGY: No data
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

CLINICAL CHEMISTRY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

URINALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. No data

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

IMMUNOLOGY: No data
- Time schedule for examinations: No data
- How many animals: No data
- Dose groups that were examined: No data
- Parameters checked in table [No.?] were examined. No data

OTHER: Cell proliferation was investigated in animals terminated after 28 days of exposure. Two different techniques were used, depending on the region of the respiratory tract being examined. Bromodeoxyuridine (BrdU) labelling and subsequent immunolocalization of BrdU-containing nuclei were used for the assessment of cell proliferation in the lung. For this technique, five designated rats per group, per time point, were implanted subcutaneously with osmotic minipumps (Alzet 2ML1) for delivery of BrdU 7 days before termination (minipumps containing BrdU at a concentration of 15 mg ml−1).

For the nasal cavity and larynx, pulse labelling with tritiated thymidine followed by autoradiography was used. Five designated rats per group, per time point, were
injected intraperitoneally with tritiated thymidine at a dose level of 1 µCi g−1 bodyweight ∼1 h prior to scheduled termination.

At termination of both the main study and recovery groups, rats were killed by an overdose of halothane with subsequent exsanguination by cardiac puncture. All animals were subjected to a full examination or postmortem, involving an external observation and careful internal examination of all organs and structures. The
lung, trachea and small intestine were examined in situ and fixed in 10% neutral-buffered formol–saline or an appropriate fixative (the lung after inflation with 10%
neutral-buffered formol–saline).

After fixation, lung tissue was trimmed and processed to paraffin wax blocks and sections made and stained to reveal BrdU-positive nuclei. The heads from all designated animals were removed, excess skin and muscle was removed, the brain was excised and the nasal cavity was 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 the different epithelial cell types and accessory nasal structures. The sections taken represent sections 1, 6, 8, 15/17, 23 and 28. The six sections were exposed to nuclear emulsion (Ilford K2) for 8–10 weeks. Sections were developed and examined by light microscopy.

The larynx was removed from all animals, fixed in 10% neutral-buffered formalin for 24 h, trimmed and processed to paraffin wax blocks. Three standard sections
of larynx were prepared, these being taken at the level of the base of the epiglottis through the seromucinous glands (level 1), through the ventral pouch (level 2)
and through the cricoid cartilage (level 3), to include all different epithelial cell types of the larynx and underlying seromucinous glands (Bahnemann et al., 1995). Sections of larynx were exposed to nuclear emulsion (Ilford K2) for 8–10 weeks before being developed and examined by light microscopy.

Statistics:

All data (except cell proliferation data) were evaluated using analysis of variance and/or covariance for each specified parameter using the MIXED procedure of the
SAS Institute (1989) and were carried out separately for the main study and for recovery animals.

Cell proliferation data was analysed using ARTEMIS statistical methods (two-sided Student’s t-test).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No adverse clinical symptoms were seen due to the exposure apart from minor effects seen commonly due to the restraint used for nose only exposure, e.g. wet fur, snout stains.

Mortality:

not specified

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects on bodyweight in any group at any time point

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

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no effects on lung weight in any group at any time point

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic abnormalities in any of the animals at the scheduled termination times.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no compound-related histopathological changes in the lung or nasal cavity after 28 days of exposure at any concentration. Significant histological changes were observed in the larynx.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Squamous metaplasia in the ventral epithelium at level 1 was evident and again was concentration-related in severity. Following exposure to 5.52 mg m−3 sulphuric acid, this effect was evident in some animals at level 2, lateral to the ventral pouch Parakeratosis was also seen in some animals at this level of the larynx. Following exposure to 0.30 mg m−3 sulphuric acid, some animals (6/10) showed minimal squamous metaplasia at level 1.

After 4 weeks of recovery, animals exposed to 5.52 mg m−3 test chemical showed evidence of squamous epithelial metaplasia at level 1 of the larynx. This lesion was less severe following 4 weeks of recovery than it was at the end of the 28-day exposure period, but there were no signs of further resolution after 8 weeks of recovery

Other effects:

no effects observed

Description (incidence and severity):

There were no exposure-related changes in labelling index in either the terminal bronchioles or central acinar alveoli of the lung or any of the levels of the nasal cavity at either time point examined: reduction in tritiated thymidine uptake in the olfactory epithelium at level 5 following exposure to 0.30 mg m−3 sulphuric acid for 28 days was the only statistically significant group mean difference from control values; the lack of dose response suggests that this was not biologically relevant.

Comparison of BrdU and tritiated thymidine labelling in the larynx showed that the magnitude of increase in labelling compared with controls was greater when
autoradiography of tritiated thymidine-labelled tissue was used.

Following exposure to 1.38 and 5.52 mg m−3, a treatment- and dose-related increase in cell proliferation was seen in the ventral epithelium at level 1 of the larynx after both 5 and 28 days of exposure, although the increases were larger at the earlier time point . No increases in cell proliferation were seen at level 2 or 3 after 5 or 28 days of exposure. No increases in cell proliferation were seen at any level in the larynx following both 5 and 28 days of exposure to 0.3 mg m−3 test chemical.

Effect levels

Dose descriptor:

NOAEC

Effect level:

5.52 mg/m³ air

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No significant adverse effects were noted

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Exposure of rats for up to 28 days to 1.38 and 5.52 mg/m3 sulphuric acid aerosols resulted in no effect in the nasal passages or lungs and squamous metaplasia in a limited region of the larynx. Hence the no observed adverse effect concentration is considered to be 5.52 mg/m3.

Executive summary:

Repeated dose inhalation toxicity study was performed to determine the toxic nature of the test chemical. Groups of 10 female animals were exposed for 6 h per day to target concentrations of 0, 0.2, 1.0 or 5.0 mg m−3 sulphuric acid for 5 days a week for 28 days. Recovery groups of five females were exposed to 0 or 5.0 mg/m3 for 28 days and retained without further treatment for either 4 or 8 weeks to monitor recovery. Prior to the start of the study, all rats were examined to ensure that they were physically normal and exhibited normal activity. During exposure they were observed frequently, and at the end of the daily exposure period each rat was examined. Detailed clinical observations were recorded daily immediately prior to exposure. During the recovery period the animals were checked daily and detailed observations were recorded weekly, recording any changes in clinical condition or behaviour. The bodyweight of each rat was recorded before the first exposure, once a week thereafter and prior to termination. Test atmospheres with target concentrations of 0.2, 1.0 and 5.0 mg m−3 analysed sulphuric acid were generated using a glass concentric jet atomizer to generate the atmospheres into two reservoir chambers (each fitted with a cyclone), one serving the exposure chambers for groups exposed to 0.2 and 1.0 mg m−3 and another serving exposure chambers for groups exposed to 5.0 mg m−3 . Clean, dry air (dried and filtered using equipment supplied by Atlas-Copco, Sweden) was passed through the atomizer to carry the atmosphere to each of the reservoir chambers and subsequently, after appropriate dilution, to the exposure chambers (internal volume = 36.8 l). Air flows were monitored and recorded frequently using variablearea flowmeters and were altered as necessary to maintain the target concentrations and maintain a minimum of 12 air changes per hour. Humidified dilution air was also added, where appropriate, to maintain a humidity as close as possible to 50% inside the chambers. There were no effects on bodyweight or lung weight in any group at any time point, and no adverse clinical symptoms were seen due to the exposure apart from minor effects seen commonly due to the restraint used for noseonly exposure, e.g. wet fur, snout stains. There were no macroscopic abnormalities in any of the animals at the scheduled termination times. Squamous metaplasia in the ventral epithelium at level 1 was evident and again was concentration-related in severity. Following exposure to 5.52 mg m−3 sulphuric acid, this effect was evident in some animals at level 2, lateral to the ventral pouch Parakeratosis was also seen in some animals at this level of the larynx. Following exposure to 0.30 mg m−3 sulphuric acid, some animals (6/10) showed minimal squamous metaplasia at level 1. After 4 weeks of recovery, animals exposed to 5.52 mg m−3 test chemical showed evidence of squamous epithelial metaplasia at level 1 of the larynx. This lesion was less severe following 4 weeks of recovery than it was at the end of the 28-day exposure period, but there were no signs of further resolution after 8 weeks of recovery. There were no exposure-related changes in labelling index in either the terminal bronchioles or central acinar alveoli of the lung or any of the levels of the nasal cavity at either time point examined: reduction in tritiated thymidine uptake in the olfactory epithelium at level 5 following exposure to 0.30 mg m−3 sulphuric acid for 28 days was the only statistically significant group mean difference from control values; the lack of dose response suggests that this was not biologically relevant. Comparison of BrdU and tritiated thymidine labelling in the larynx showed that the magnitude of increase in labelling compared with controls was greater when autoradiography of tritiated thymidine-labelled tissue was used. Following exposure to 1.38 and 5.52 mg m−3, a treatment- and dose-related increase in cell proliferation was seen in the ventral epithelium at level 1 of the larynx after both 5 and 28 days of exposure, although the increases were larger at the earlier time point . No increases in cell proliferation were seen at level 2 or 3 after 5 or 28 days of exposure. No increases in cell proliferation were seen at any level in the larynx following both 5 and 28 days of exposure to 0.3 mg m−3 test chemical. Exposure of rats for up to 28 days to 1.38 and 5.52 mg/m3 sulphuric acid aerosols resulted in no effect in the nasal passages or lungs and squamous metaplasia in a limited region of the larynx. Hence the no observed adverse effect concentration is considered to be 5.52 mg/m3.