Ozone

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2000

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

No guideline followed, experimental research study

GLP compliance:

no

Test type:

other: experimental research study

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ozone

EC Number:

233-069-2

EC Name:

Ozone

Cas Number:

10028-15-6

Molecular formula:

O3

IUPAC Name:

trioxygen

Test material form:

gas

Specific details on test material used for the study:

Ozone was generated from a tube filled with aluminum chips with two electrodes inside and through which a high-voltage current circulated.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Two hundred and ten male Wistar rats weighing 180 to 450 g (young: 47 days; mature: 540 days; old: 900 days) were used. The animals were individually housed in acrylic boxes with free access to food (Nutricubo; Purina, USA) and water, kept in a clear air room, and separated into two experiments.

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

A closed chamber with a diffuser connected to a variable-flux ozone generator (5 lt/seg) was used. Ozone was generated from a tube filled with aluminum chips with two electrodes inside and through which a high-voltage current circulated. Some of the oxygen of the air that circulated around the tube was converted into ozone. Ozone level production was proportional to the current intensity and the air flux. (The ozone generator was fed with ambient air). A PCI Ozone and Control System Monitor approved by E.P.A quantified the ozone concentration inside the chamber throughout the experiment.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

A PCI Ozone and Control System Monitor approved by E.P.A quantified the ozone concentration inside the chamber throughout the experiment.

Concentrations:

0.7-0.8 ppm
see details on study design

No. of animals per sex per dose:

Experimnet 1:50 rats per age group young, adult and old. For each age group: 5 sub-groups of 10 animals/ treatment.
Experiment 2: 30 rats per age group (young and old). For each age group: 5 sub-groups of 6 animals/treatment.

Control animals:

yes

Details on study design:

Experiment one.
One hundred and fifty young, mature, and old animals were divided into three experimental blocks according to the age of the subjects. Each block was formed by five groups. Each group received one of the following treatments: group 1 (control), group 2 (taurine), group 3 (ozone exposure), group 4 (taurine before ozone exposure), and group 5 (taurine after ozone exposure).
One hour after the 4 h ozone exposure session had finished, all groups were trained in one-trial passive avoidance conditioning and tested 10 min and 24 h later. As a control, motor activity was measured before submitting the group to a passive avoidance test. The motor activity in the rats of the control group, exposed to clean air, was taken as 100% activity and compared to that of the animals exposed to ozone. We measured motor activity of each animal for 10 min, at 4 min and 24 h after ozone exposure had finished.
Each animal was placed in the safety compartment for 10 s. Then the sliding door was lifted and the time required for the animal to cross the threshold to the shock compartment was recorded (acquisition latency). If an animal required more than 100 s to cross to the other side, it was dropped from the experiment and substituted with another rat exposed to the same conditions. Once the animal crossed with all four paws into the next compartment, the door was closed and a 3-mA footshock was delivered for 5 s. Then the door was opened and the time required for the animal to return to the safe compartment was measured (escape latency). The animal remained there for 30 s before being returned to its individual housing. Both 10 min (short-term memory) and 24 h (long-term memory) after this training procedure, a retention test was performed. The animal was again placed in the safety compartment for 10 s, the door was opened, and the time that the animal remained in the safety compartment before entering the shock compartment was recorded (retention latency). The test session ended when the animal either entered the shock compartment or remained in the safe compartment for 600 s.

Experiment two. Sixty rats were divided into two experimental blocks according to the age of the subjects (young and old). Each block was randomly divided into five groups of six rats each and received the same treatments used for the memory tests. One hour after ozone exposure, animals were sacrificed by decapitation, the hippocampus, striatum, frontal cortex, and cerebellum of both hemispheres were dissected on ice and weighed immediately. Each tissue sample was homogenized in PBS 1 : 20 and stored at -70 C until the day of the assay to measure lipid peroxidation. Homogenates of each sample were centrifuged. The supernatant was removed, and the enzyme reagent (ascorbic oxidase and lipoprotein lipase) was added. The mixture was incubated for 5 min at a temperature of 30 C and the cromogen reagent (10-N-methylcarbamoyl-3,7-dimethylamino-10-H-phenothiazine (MCDP)) was added. This mixture was incubated for 10 min at a temperature of 30 C. Finally, absorbance was measured at 675 nm in a spectrophotometer. A two-point callibration curve was made using a saline blank (0 nmol/ml) and the 50 nmol/ml cumene hydroperoxides standard provided with the kit. The results were calculated using the following
equation, whose linear range for this assay is between 2.0 to 300 nmol/ml:

LPO [nmol/ml] = [(Es-Eb) x 50.0/(Estd-Eb)]

where Es is sample absorbance, Estd is absorbance of 50 nmol/ml standard, and Eb is blank absorbance. This procedure was applied to each selected brain region in every group of rats.

Drugs:
Synthetic taurine (2-aminoethanesulfonic acid) from Sigma was used. Taurine was administered ip at 43 mg/kg, 5 min either after or before acute exposure to ozone in a single dose. As a control, saline solution was used.

Lipid peroxidation.
Rats were sacrificed, and lipid peroxidation levels were quantified using the K-ASSAY LPO kit of Kamiya Biomedical Co., which quantifies lipid peroxides using, ascorbic oxidase, lipoprotein lipase, lyophilized chomogen reagent, buffer, and hemoglobin.

Statistics:

The behavioral and biochemical data were statistically analyzed with nonparametric statistical methods, according to Siegel (1991). Since these data were unlikely to be normally distributed, we chose the Kruskal-Wallis analysis of variance to measure the differences among all groups. The Mann-Whitney-U test was used to compare the control and experimental groups.

Results and discussion

Preliminary study:

Not applicable

Mortality:

no data

Clinical signs:

other: no data

Body weight:

no data

Gross pathology:

no data

Other findings:

Young rats.
Ozone exposure damages short-term and long-term memory. These effects were reversed with taurine when it was applied after ozone exposure (O+T). However, when taurine was applied before exposure it increased the memory alteration caused by ozone (T+O). A larger effect of taurine was observed in short-term memory tests.

Mature rats.
In this experiment results showed a no significant tendency for ozone (O) to impair memory and a recovery by taurine application after ozone exposure (O+T).

Old rats.
Results showed that ozone exposure (O) caused short-term and long-term memory alterations and that taurine alone improved both (T). Taurine blocked memory impairment when it was applied after ozone exposure (O+T). When taurine was applied before exposure (T+O) shortterm memory was improved and longterm memory was impaired.

Lipid peroxidation by brain are:

Frontal cortex.
Results show that when young rats received taurine (T), ozone (O), or taurine before ozone (T+O), lipid peroxidation levels increased. Only in the group that received taurine after ozone (O+T) was the lipid peroxidation caused by ozone exposure decreased. Old rats had increased lipid peroxidation levels in the control group (C) as compared to young rats. Opposite to the results in young rats, taurine and ozone decreased the lipid peroxidation levels. The highest lipid peroxidation levels were found in frontal cortex when taurine was administererd before ozone exposure in old rats (T+O).

Hippocampus.
Results indicate that young rats had increased in lipid peroxidation levels when exposed to taurine (T), ozone (O), or taurine before ozone exposure (T+O); in old rats the highest lipid peroxidation levels were found in this structure when rats were exposed to ozone (O). Taurine applied after ozone blocked these effects (O+T) in both groups.

Striatum.
Data obtained in young rats showed that taurine (T) and ozone exposure (O) caused increased lipid peroxidation levels. When taurine was applied before or after ozone exposure it blocked these effects. In old rats the control group (C) and the groups receiving taurine (T), ozone (O), and taurine applied after ozone exposure (O+T) presented similar lipid peroxidation levels.

Cerebellum.
Results show that young rats presented increased lipid peroxidation levels when they received taurine alone (T), were exposed to ozone (O), or received taurine before ozone exposure (T+O). When taurine was applied after ozone exposure effects on lipid peroxidation were blocked (O+T). In old rats the control group (C) and the groups that received taurine before (T+O) or after (O+T) ozone exposure presented higher lipid peroxidation levels.However in those that received taurine (T) or were exposed to ozone (O), lipid peroxidation levels were lower than in the control group.

Applicant's summary and conclusion

Interpretation of results:

Category 1 based on GHS criteria

Conclusions:

This scientific publication provides supportive information that exposure for 4 hours to 0.7-08 ppm ozone caused memory alteration and increased lipid peroxidation levels in rats of different age groups. Short-term and Long-term memory changes in passive avoidance conditioning caused by ozone depend on the age of the rats. The changes in lipid peroxidation levels caused by ozone depend on age and brain region. Taurine after ozone attenuate the effect on memory, the effect size is age dependent..

Executive summary:

In this experimental research study two experiments were conducted to determine the antioxidant effects of taurine on changes in memory and lipid peroxidation levels in brain caused by exposure to ozone. In the first experiment, 150 rats were separated into three experimental blocks (young, mature, and old) with average groups (10 animals) each and received one of the following treatments: control, taurine, ozone, taurine before ozone, and taurine after ozone. Ozone exposure was 0.7 - 0.8 ppm for 4 h and taurine was administered ip at 43 mg/kg, after or before ozone exposure. Subsequently, rats were tested in passive avoidance conditioning. In the second experiment, samples from frontal cortex, hippocampus, striatum, and cerebellum were obtained from 60 rats (young and old, 6 animals/group), using the same treatments with 1 ppm ozone. Results show both an impairment in short-term and long-term memory with ozone and an improvement with taurine after ozone exposure, depending on age. In contrast to young rats, old rats showed peroxidation in all control groups and an improvement in memory with taurine. When taurine was applied before ozone, high peroxidation levels have been found in the frontal cortex of old rats and the hippocampus of young rats; in the striatum, peroxidation caused by ozone was blocked when taurine was applied either before or after ozone exposure. In conclusion, ozone exposure caused memory alteration and increased lipid peroxidation levels. Short-term and long-term memory changes in passive avoidance conditioning caused by ozone depend on the age of the rats. The changes in lipid peroxidation levels caused by ozone depend on age and brain region.

Furthermore, different brain regions have different levels of vulnerability to oxidative stress and different responses to the antioxidant effects of taurine. The antioxidant effect of taurine appears when there is a previous oxidative stress state.

This scientific publication meets basic scientific principle and is therefore used for supporting information in as weight of evidence approach regarding the acute toxicity of ozone.