Registration Dossier

Diss Factsheets

Toxicological information

Carcinogenicity

Currently viewing:

Administrative data

Description of key information

A total of 10 animal studies were reviewed here for data related to carcinogenicity of ozone, and overall, there is limited scientific evidence to indicate the carcinogenic potential of ozone in animals.

On the other hand, carcinogenicity of ozone in humans has been assessed by the US EPA as part of the "Integrated Science Assessment for Ozone and Related Photochemical Oxidants" (EPA 2013, pages 80-85). In this assessment, the authors carefully reviewed the epidemiological studies on the effects of ozone on human health and concluded that, albeit that ozone exposure may contribute to DNA damage, the evidence is inadequate to determine if a causal relationship exists between ambient ozone exposure and cancer. Since there was insufficient evidence for carcinogenic effects of ozone obtained from animal or human epidemiological studies, classification for carcinogenicity is considered not appropriate.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via inhalation route

Link to relevant study records

Referenceopen allclose all

Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.4200 (Carcinogenicity)
Principles of method if other than guideline:
The State of California and the Health Effects Institute (HEI) nominated ozone to the National Toxicology Program (NTP) for long-term studies in rats and mice to determine the potential toxicity and carcinogenicity of long-term ozone exposure. The studies reported here include exposures at the current EPA standard (0.12 ppm), the maximum concentration considered to be compatible with long-term survival (1.0 ppm) and an intermediate concentration (0.5 ppm). A second ozone inhalation study was included in which 0.5- and 1.0 ppm exposures were continued to 30 months based on lifetime studies in rats in which the majority of lung tumors developed after 24 months. A third study was included in which male rats were administered 2 concentrations of a known pulmonary carcinogen, 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK), and exposed to 0.5 ppm ozone to determine the co-carcinogenic potential of ozone.
GLP compliance:
yes
Specific details on test material used for the study:
Ozone was generated by corona discharge using an OREC model 03V5-ozonator (Ozone Research and Equipment Corp., Phoenix, AZ) with 100% oxygen. The 03 concentration in each chamber was monitored by a multiplexed Dasibi model 1003-AH or 1003-PC ultraviolet spectrophotometric analyser (Dasibi Environmental Corp., Glendale, CA). The monitor was calibrated by comparing it with a chemical specific, calibrated monitor (neutral-buffered potassium iodide method) simultaneously sampling the exposure chambers.
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female F344/N rats were obtained from Simonsen Laboratories (Gilroy, CA). Rats were quarantined for 14 days before the beginning of the study. Five male and five female rats were selected for bacterial culture and selected histopathology prior to the beginning of the study. Approximately 3 weeks after receipt, serology samples were collected for viral screening from up to seven male and seven female rats. Rats were approximately 6 weeks old at the beginning of the studies. The health of the animals was monitored during the studies according to the protocols of the NTP sentinel Animal Program.

All animals were housed individually. Water was available ad libitum, and feed (NIH-07 open formula meal diet, Zeigler Brothers, Inc., Gardners, PA; changed weekly) was available ad libitum except during exposure periods. Cage units were rotated vertically (2-year study) or horizontally (life time study) within each chamber weekly.

Environmental conditions:
Average temperature: 23.9 degrees centigrade;
Relative humidity: 40% to 70%;
Fluorescent light: 12 hours/day;
Room air: 12 to 18 changes/hour.

IN-LIFE DATES:
2 Year ozone study
From: 25 January 1990 to 24 January 1992.

Lifetime study
From: 26 October 1989 to 13 March 1992.

2 Year ozone/NKK study
Ozone - From: 28 November 1989 to 27 November 1991.
NKK - From: 27 November 1989 to 13 April 1990.
Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Animals were exposed in H-2000 chambers. The animals were hold single in stainless steel, wire bottom cages. The build-up of vapour concentration in the chamber at the beginning of exposure to 90% of its final stable concentration (T90) and the decay of concentration at the end of exposure to 10% (T10) were measured prior to the start of each study in chambers with a full complement of mature F344/N rats. These tests were done in conjunction with the pre-start tests for the ozone study. A T90 value of 30 minutes was used based on the experimental data. The T10 value ranged from 5 to 11 minutes.
Chamber concentrations were monitored using an ultraviolet spectrophotometric analyser (Dasibi Model 1003-AH or Dasibi Model 1003-PC systems). Samples were directed to the ozone monitor through computer controlled, multiplexed Teflon valves. A sampling rate of 4 minutes per port assured that all ports were sampled approximately twice per hour. Uniformity of ozone concentration in was measured quarterly during the 2-year and lifetime studies. While the majority of the determinations were within this range, some exceeded this value, and 10.1% was the maximum value found.
The vehicle was conditioned air (single HEPA filters and charcoal).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations were monitored using an ultraviolet spectrophotometric analyser (Dasibi Model 1003-AH or Dasibi Model 1003-PC systems) (Glendale, CA). Initially, the UV spectrophotometric analyser (Dasibi Model 1003-AH) was used to monitor the ozone concentration in the exposure chambers, control chamber, room, generator cabinet, and an on-line ozone standard. After approximately 14 months (2-year ozone study), or 16 months (2-year ozone/NNK study and lifetime studies) the Model 1003-AH ozone monitors were replaced with Dasibi Model 1003-PC ozone monitors/generators. This change reduced maintenance and repair costs and maintained an effective system for monitoring ozone.
For both monitoring systems, air sampled at each location was transported to the monitor by transfer lines of Teflon" tubing. Samples were directed to the ozone monitor through a set of eight computer controlled, multiplexed Teflon valves. A sampling rate of 4 minutes per port assured that all ports were sampled approximately twice per hour.
Duration of treatment / exposure:
2 Year ozone study: exposure 6h/d, 5d/w for 105 weeks;
Lifetime study: exposure 6h/d, 5d/w for 125 weeks;
2 Year ozone/NKK study: exposure 6h/d, 5d/w for 105 weeks.
Frequency of treatment:
daily for 5 days/week
Post exposure period:
no post exposure period
Dose / conc.:
0.12 ppm
Dose / conc.:
0.5 ppm
Dose / conc.:
1 ppm
No. of animals per sex per dose:
2 Year ozone study: 50 males and 50 females per dose;
Lifetime study: 50 males and 50 females per dose;
2 Year ozone/NKK study: 48 males
Control animals:
yes, concurrent vehicle
Details on study design:
Concentration levels were based on the National Ambient Air Quality standard (0.12 ppm) and the maximum concentration which the animals would tolerate (1.0 ppm).
Positive control:
n/a
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, twice daily for mortalities
DETAILED CLINICAL OBSERVATIONS (not in lifetime study): Yes, initially, monthly through week 92 then every 2 weeks until the end of the study.
BODY WEIGHT: Yes, initially, weekly through week 13, monthly through week 92 then every 2 weeks until the end of the study.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes
2 Year ozone study and lifetime study:
Complete histopathology was performed on all rats. In addition to gross lesions and tissue masses, the tissues examined included: adrenal gland, brain, clitoral gland, esophagus, femur, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lungs, lymph nodes (mandibular, mesenteric, bronchial, and mediastinal), mammary gland (with adjacent skin), nose, ovaries, pancreas, parathyroid gland, pituitary gland, preputial gland (rats), prostate gland, salivary gland, spleen, stomach, testes (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus
2 Year ozone/NKK study:
Histopathology was performed on all animals. In addition to gross lesions and tissue masses, the tissues examined included lymph nodes (bronchial and mediastinal), lungs, nose, larynx, and trachea.
Other examinations:
n/a
Statistics:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for possible dose-related effects on survival used Cox's method for testing two groups for equality and Tarone's life table test to identify dose-related trends. P values for the survival analyses are two sided.

Analysis of Neoplasm incidences was by logistic regression analysis. Neoplasm prevalence was modelled as a logistic function of chemical exposure and time. Both linear and quadratic terms in time were incorporated initially, and the quadratic term was eliminated if the fit of the model was not significantly enhanced. The neoplasm incidences of exposed and control groups were compared on the basis of the likelihood score test for the regression coefficient of dose (prevalence analysis of Dinse and Lagakos). In addition to logistic regression, other methods of statistical analysis were used. These methods include the life table test (Cox; Tarone), appropriate for rapidly lethal neoplasms, and the Fisher exact test and the Cochran Armitage trend test, procedures based on the overall proportion of neoplasm-bearing animals. Tests of significance included pairwise comparisons of each exposed group with controls and a test for an overall dose-related trend. Continuity corrected tests were used in the analysis of neoplasm incidence, and reported P values are one sided. The procedures described above were also used to evaluate selected non-neoplastic lesions.
All non-neoplastic lesions in this study were considered to be incidental to the cause of death or not rapidly lethal. The primary statistical analysis used was logistic regression analysis. For lesions detected at the interim evaluation, the Fisher exact test was used.
Organ and body weight data were analysed using procedures test of Dunnett or William's, Jonckheere's test and the Mann­ Whitney U test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Hypoactivity was observed in male and female rats exposed to ozone. Rats, particularly those exposed to 1.0 ppm, were less active during and immediately after exposure.
Mortality:
mortality observed, treatment-related
Description (incidence):
Hypoactivity was observed in male and female rats exposed to ozone. Rats, particularly those exposed to 1.0 ppm, were less active during and immediately after exposure.
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
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:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Summary of the 2-Year and Lifetime Carcinogenesis Studies of Ozone

Doses 0, 0.12 (2-year study only), 0.5 and 1.0 ppm ozone by inhalation

2-Year males Lifetime males 2-Year females Lifetime females
Body weights 1.0 ppm group 1.0 ppm group 1.0 ppm group 1.0 ppm group
slightly lower than lower than controls slightly lower than slightly lower than
controls controls controls
Survival rates 8/49, 5/50, 7/50, 7/50 0/50, 0/50, 1/50 28/50, 24/50, 30/50, 6/50, 6/50, 7/50
27/50
Nonneoplastic effects
Nose: goblet cell Nose: goblet cell Nose: goblet cell Nose: goblet cell
hyperplasia (1/50, hyperplasia (1/50, hyperplasia (1/50, hyperplasia (0/50,
4/50, 41/50, 48/50); 46/49, 48/49); lateral 2/50, 45/50, 50/50); 47/49, 50/50); lateral
lateral wall wall hyperplasia lateral wall wall hyperplasia
hyperplasia (0/50, (10/50, 48/49, 47/49); hyperplasia (2/50, (4/50, 49/49, 50/50);
8/50, 50/50, 49/50) squamous metaplasia 8/50, 48/50, 50/50) squamous metaplasia
squamous metaplasia (10/50, 23/49, 40/49) squamous metaplasia (5/50, 25/49, 35/50)
(2/50, 6/50, 36/50, Larynx: squamous (2/50, 11/50, 21!50, Larynx: squamous
46/50) metaplasia (0/50, 45/50) metaplasia (2/49,
Larynx: squamous 20/48, 43/47) Larynx: squamous 16/47, 48/50)
metaplasia (0/50, Lung: metaplasia metaplasia (4/50, Lung: metaplasia
2/50, 16/50, 43/50) (0/50, 45/50, 50/50); 5!50, 9/50, 43/50) (0/50, 44/50, 50/50);
Lung: metaplasia histiocytic infiltration Lung: metaplasia histiocytic infiltration
(0/50, 9/50, 46/50, (0/50, 38/50, 49/50); (0/50, 6/50, 48/50, (0/50, 38/50, 49/50);
47/50); interstitial interstitial fibrosis 48/50); interstitial interstitial fibrosis
fibrosis (0/50, 2/50, (0/50, 44/50, 50/50) fibrosis (0/50, 0/50, (0/50, 41/50, 50/50)
40/50, 44/50) 42/50, 47/50)
Neoplastic effects
None None None None

Level of evidence of carcinogenic activity
No evidence No evidence No evidence No evidence

Ozone/NKK study

Lung: Alveolar epithelial metaplasia and interstitial fibrosis occurred in all groups of rats exposed to ozone (with or without NNK), but were not observed in vehicle controls or in animals exposed to NNK alone. The incidence of alveolar cellular infiltration was greater in males exposed to ozone than in the vehicle control males. There was a dose-related increased incidence of atypical alveolar hyperplasia in groups of rats receiving NNK, and the increase was significant. An increased incidence of alveolar/bronchiolar adenoma or carcinoma (combined) also occurred in rats administered 1.0 mg/kg NNK, with or without ozone. The administration of ozone did not affect the occurrence of pulmonary neoplasms or nonneoplastic lesions in rats administered NNK.

Nose: The incidence of hyperplasia in groups of rats exposed to ozone with and without NNK was greater than the incidence in males not exposed to ozone. Incidences of hyperplasia among groups of rats exposed only to NNK were low and similar to that of the controls. The nasal lesions were similar to those seen in rats exposed to ozone by inhalation for 2 years.





Conclusions:
The results of the large "NTP study" (2-year and lifetime inhalation studies) on rats showed that there was no evidence of carcinogenic activity of ozone in male or female F344/N rats exposed to 0.12, 0.5, or 1.0 ppm.
Executive summary:

The toxicity and carcinogenicity of ozone was evaluated in Fischer 344/N rats and B6C3Fl mice exposed 6 hr/day, 5 days/week, to 0.12 (2 years only), 0.5 or 1.0 ppm ozone by inhalation for 2-year and lifetime exposures. A 2-year cocarcinogenicity study (male rats only) included the subcutaneous administration of 0, 0.1 or 1.0 mg/kg body weight. of 4-(N-methyl-Nnitrosamino)- 1-(3-pyridyl)-1-butanone (NNK) for the first 20 weeks along with inhalation exposure to 0 or 0.5 ppm ozone followed by additional 84 weeks of ozone exposure alone.

Ozone exposure in rats did not cause an increased incidence of lung neoplasms. In the co-carcinogenicity study, ozone exposure did not have an additive carcinogenic effect. We conclude, that under the conditions of these studies: (a) ozone exposure is not carcinogenic to either male or female F-344/N rats, (b) ozone does not enhance the incidence of pulmonary neoplasms in F-344/N rats exposed to a known pulmonary carcinogen (NNK), and (c) mild site-specific toxic lesions characteristic of ozone exposure persist in the nasal passage and lung throughout the lifetime of the rat with continued ozone exposure

Endpoint:
carcinogenicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.4200 (Carcinogenicity)
Principles of method if other than guideline:
The State of California and the Health Effects Institute (HEI) nominated ozone to the National Toxicology Program (NTP) for long-term studies in rats and mice to determine the potential toxicity and carcinogenicity of long-term ozone exposure. The studies reported here include exposures at the current EPA standard (0.12 ppm), the maximum concentration considered to be compatible with long-term survival (1.0 ppm) and an intermediate concentration (0.5 ppm). A second ozone inhalation study was included in which 0.5- and 1.0 ppm exposures were continued to 30 months based on lifetime studies in rats in which the majority of lung tumours developed after 24 months.
GLP compliance:
yes
Specific details on test material used for the study:
Ozone was generated by corona discharge using an OREC model 03V5-ozonator (Ozone Research and Equipment Corp., Phoenix, AZ) with 100% oxygen. The 03 concentration in each chamber was monitored by a multiplexed Dasibi model 1003-AH or 1003-PC ultraviolet spectrophotometric analyzer (Dasibi Environmental Corp., Glendale, CA). The monitor was calibrated by comparing it with a chemical­specific, calibrated monitor (neutral-buffered potassium iodide method) simultaneously sampling the exposure chambers.
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female mice were obtained from Simonsen Laboratories (Gilroy, CA). Mice were quarantined for 14 days (2-year study) or 21 days (lifetime study0 before the beginning of the study. Three male and two female mice were selected for bacterial culture and selected histopathology prior to the beginning of the study. Approximately 3 weeks after receipt, serology samples were collected for viral screening from two male and three female mice. Mice were approximately 6 weeks old at the beginning of the studies. The health of the animals was monitored during the studies according to the protocols of the NTP sentinel Animal Program.
All animals were housed individually. Water was available ad libitum, and feed (NIH-07 open formula meal diet, Zeigler Brothers, Inc., Gardners, PA; changed weekly) was available ad libitum except during exposure periods. Cage units were rotated vertically (2-year study) or horizontally (life time study) within each chamber weekly.
Environmental conditions:
Average temperature: 23.9 degrees centigrade;
Relative humidity: 40% to 70%;
Fluorescent light: 12 hours/day;
Room air: 12 to 18 changes/hour.
IN-LIFE DATES:
2 Year ozone study:
From: 9 November 1989 to 14 November 1991.
Lifetime study:
From: 16 November 1989 to 13 May 1992.
Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
Animals were exposed in H-2000 chambers. The animals were hold single in stainless steel, wire bottom cages.
The build-up of vapour concentration in the chamber at the beginning of exposure to 90% of its final stable concentration (T90) and the decay of concentration at the end of exposure to 10% (T10) were measured prior to the start of each study in chambers with a full complement of mature B6C3F1 mice. These tests were done in conjunction with the pre-start tests for the ozone study. A T90 value of 30 minutes was used based on the experimental data. The T10 value ranged from 5 to 11 minutes.
Chamber concentrations were monitored using an ultraviolet spectrophotometric analyser (Dasibi Model 1003-AH or Dasibi Model 1003-PC systems). Samples were directed to the ozone monitor through computer controlled, multiplexed Teflon valves. A sampling rate of 4 minutes per port assured that all ports were sampled approximately twice per hour. Uniformity of ozone concentration in was measured quarterly during the 2-year and lifetime studies. While the majority of the determinations were within this range, some exceeded this value, and 10.1% was the maximum value found. The vehicle was conditioned air (single HEPA filters and charcoal).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations were monitored using an ultraviolet spectrophotometric analyser (Dasibi Model 1003-AH or Dasibi Model 1003-PC systems) (Glendale, CA). Initially, the UV spectrophotometric analyser (Dasibi Model 1003-AH) was used to monitor the ozone concentration in the exposure chambers, control chamber, room, generator cabinet, and an on-line ozone standard. After approximately 14 months (2-year ozone study), or 16 months (2-year ozone/NNK study and lifetime studies) the Model 1003-AH ozone monitors were replaced with Dasibi Model 1003-PC ozone monitors/generators. This change reduced maintenance and repair costs and maintained an effective system for monitoring ozone.
For both monitoring systems, air sampled at each location was transported to the monitor by transfer lines of Teflon" tubing. Samples were directed to the ozone monitor through a set of eight computer controlled, multiplexed Teflon valves. A sampling rate of 4 minutes per port assured that all ports were sampled approximately twice per hour.

Duration of treatment / exposure:
2 Year ozone study: exposure 6h/d, 5d/w for 105 weeks;
Lifetime study: exposure 6h/d, 5d/w for 130 weeks;
Frequency of treatment:
daily for 5 days/weeks
Post exposure period:
no post exposure period
Dose / conc.:
0.12 ppm
Dose / conc.:
0.5 ppm
Dose / conc.:
1 ppm
No. of animals per sex per dose:
2 Year ozone study: 50 males and 50 females per dose;
Lifetime study: 50 males and 50 females per dose;
Control animals:
yes, concurrent vehicle
Details on study design:
Concentration levels were based on the National Ambient Air Quality standard (0.12 ppm) and the maximum concentration which the animals would tolerate (1.0 ppm).
Positive control:
n/a
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, twice daily for mortalities
DETAILED CLINICAL OBSERVATIONS (not in lifetime study): Yes, initially, monthly through week 92 then every 2 weeks until the end of the study.
BODY WEIGHT: Yes, initially, weekly through week 13, monthly through week 92 then every 2 weeks until the end of the study.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: No data

HISTOPATHOLOGY: Yes
2 Year ozone study and Lifetime study:
Complete histopathology was performed on all mice. In addition to gross lesions and tissue masses, the tissues examined included: adrenal gland, brain, esophagus, femur, gallbladder, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lungs, lymph nodes (mandibular, mesenteric, bronchial, and mediastinal), mammary gland (with adjacent skin), nose, ovaries, pancreas, parathyroid gland, pituitary gland, preputial gland (rats), prostate gland, salivary gland, spleen, stomach, testes (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus

2 Year ozone/NKK study:
Histopathology was performed on all animals. In addition to gross lesions and tissue masses, the tissues examined included lymph nodes (bronchial and mediastinal), lungs, nose, larynx, and trachea.
Other examinations:
n/a
Statistics:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for possible dose-related effects on survival used Cox's method for testing two groups for equality and Tarone's life table test to identify dose-related trends. P values for the survival analyses are two sided. Analysis of neoplasm incidences was by logistic regression analysis. Neoplasm prevalence was modelled as a logistic function of chemical exposure and time. Both linear and quadratic terms in time were incorporated initially, and the quadratic term was eliminated if the fit of the model was not significantly enhanced. The neoplasm incidences of exposed and control groups were compared on the basis of the likelihood score test for the regression coefficient of dose (prevalence analysis of Dinse and Lagakos). In addition to logistic regression, other methods of statistical analysis were used. These methods include the life table test (Cox; Tarone), appropriate for rapidly lethal neoplasms, and the Fisher exact test and the Cochran Armitage trend test, procedures based on the overall proportion of neoplasm-bearing animals. Tests of significance included pairwise comparisons of each exposed group with controls and a test for an overall dose-related trend. Continuity corrected tests were used in the analysis of neoplasm incidence, and reported P values are one sided. The procedures described above were also used to evaluate selected non-neoplastic lesions. All non-neoplastic lesions in this study were considered to be incidental to the cause of death or not rapidly lethal. The primary statistical analysis used was logistic regression analysis. For lesions detected at the interim evaluation, the Fisher exact test was used. Organ and body weight data were analysed using procedures test of Dunnett or William's, Jonckheere's test and the Mann­ Whitney U test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Hypoactivity was observed in male and female rats exposed to ozone. Mice, particularly those exposed to 1.0 ppm, were less active during and immediately after exposure.
Mortality:
mortality observed, treatment-related
Description (incidence):
Hypoactivity was observed in male and female rats exposed to ozone. Mice, particularly those exposed to 1.0 ppm, were less active during and immediately after exposure.
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
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:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Summary of the 2-Year and Lifetime Carcinogenesis Studies of Ozone

Doses 0, 0.12 (2-year study only), 0.5 and 1.0 ppm by inhalation

2-Year males Life time males 2-Year females Life time females

Body weights 1.0 ppm group 1.0 ppm group lower All exposed groups 1.0 ppm group lower
slightly lower than controls than controls lower than controls than controls

Survival rates 30/50, 34/50, 25/50, 14/50, 11/50, 12/50 29/50, 37/50, 33/48, 9/50, 12/50, 10/50
27/50 40/50

Nonneoplastic effects
Nose: hyperplasia Nose: hyperplasia Nose: hyperplasia Nose: hyperplasia
(0/50, 0/50, 42/50, (2/49, 33/48, 45/49); (0/50, 0/50, 42/48, (1/50, 42/49, 47/50);
50/50); squamous squamous metaplasia 50/50); squamous squamous metaplasia
metaplasia (0/50, (1/49, 2/48, 20/49) metaplasia (1/50, (2/50, 3/49, 28/50)
3/50, 3/50, 36/50) Larynx: hyperplasia 1/50, 11/48, 36/50) Larynx: hyperplasia
Larynx: hyperplasia (4/49, 7/49, 15/50); Larynx: hyperplasia (13/50, 11/49, 24/50);
(1/50, 0/50, 0/50, squamous cell metaplasia (0/50, 0/50, 0/49, squamous cell
6/50) (2/49, 1/49, 10/50) 7/50) metaplasia (2/50,
Lung: histiocytic Lung: histiocytic Lung: histiocytic 2/49, 19/50)
infiltration (0/50, infiltration (3/49, infiltration (0/50, Lung: histiocytic
0/50, 18/50, 31/50); 40/49, 41/50); 0/50, 11/49, 42/50); infiltration (5/50,
metaplasia (0/50, metaplasia (0/49, metaplasia (0/50, 39/49, 45/50);
0/50, 48/50, 50/50) 48/49, 47/50) 0/50, 43/49, 49/50) metaplasia (0/50,
43/49, 50/50)

Neoplastic effects
None None Lung: alveolar/ Lung: alveolar/
bronchiolar adenoma bronchiolar adenoma
or carcinoma or carcinoma
(combined) (6/50, (combined) (6/50,
7/50, 9/49, 16/50) 8/49, 12/50)
Uncertain effects
Lung: alveolar/ Lung: alveolar/ Decreased incidence of None
bronchiolar adenoma bronchiolar adenoma hepatocellular adenoma
or carcinoma or carcinoma (combined) or carcinoma (combined)
(combined) (14/50, (16/49, 22/49, 21/50) (27/50; 22/50; 20/50,
13/50, 18/50, 19/50) 11/50)

Level of evidence of carcinogenic activity
Equivocal evidence Some evidence
Dose descriptor:
NOAEC
Effect level:
1 mg/m³ air
Based on:
test mat.
Sex:
female
Basis for effect level:
other: 1 mg/m³ = 0.5 ppm mild, site-specific, non-neoplastic lesions in the nasal cavity and centriacinar lung of male and female mice exposed to 0.5 or 1.0 ppm of ozone for 2 yrs or lifetime, but no neoplasms at 0.5 ppm were observed.
Conclusions:
The results of the large "NTP study" on mice showed that long-term exposure to relatively high concentrations of ozone (1.0 ppm) can increase the incidence of lung neoplasms in B6C3F1 mice. Neoplasms were only observed in female mice after at least 52 weeks of exposure to 1.0 ppm of ozone. Results also indicate that non-neoplastic toxic effects, with potential for adverse effects on human health, occur and persist in the nasal cavity and lung with prolonged exposure. The ‘NTP’ study in mice is the only study which was performed in close accordance with current test guidelines for carcinogenicity testing. The neoplasms were however only observed in female mice after at least 52 weeks of exposure to 1.0 ppm of ozone, a concentration which is 20 fold higher than the normal ambient ozone concentration (ca 0.05 ppm background level) and 10 times higher than most 8 h occupational exposure limits for ozone (0.1 ppm). The A/J mouse is frequently used as a model for testing of chemicals by inhalation for lung tumour multiplicities. It has a high incidence of spontaneous lung adenomas, and lung tumours readily develop in response to carcinogens. Concluding, only limited evidence (only one animal species, one sex and one dose level; no dose response) was obtained for a carcinogenic potential of ozone in animals. An NOAEC (carcinogenicity) of 0.5 ppm (1 g/m³) could be derived from the results on female mice.
Executive summary:

To evaluate the toxicity and carcinogenic potential of long-term exposure to ozone, B6C3F1 mice were exposed by whole-body inhalation to 0, 0.12, 0.5, or 1.0 ppm and 0, 0.5, or 1.0 ppm ozone for 24 or 30 months (lifetime), respectively. The incidence of alveolar/bronchiolar adenomas and carcinomas (combined) increased (p < 0.05) in female mice exposed to 1.0 ppm for 24 or 30 months and marginally increased (p> 0.05) in male mice exposed to concentrations of 0.5 or 1.0 ppm. An increased incidence of non-neoplastic lesions were observed in the nasal cavities and in the centriacinar region of the lung of mice exposed to 0.5 or 1.0 ppm for 24 and 30 mo. Nasal cavity lesions were mild and included hyaline degeneration, hyperplasia, squamous metaplasia, fibrosis and suppurative inflammation of the transitional and respiratory epithelium of the lateral wall, and atrophy of the olfactory epithelium. Lung lesions included replacement of the epithelium of the alveolar ducts and adjacent alveolar septa with epithelium similar to that normally found in terminal bronchioles (metaplasia) and associated alveolar histiocytosis. Based on the results of these studies, we conclude that inhalation exposure of B6C3F1 mice to ozone for 24 or 30 months (a) is carcinogenic in female B6C3F1 mice exposed to 1.0 ppm of ozone based on an increased incidence of alveolar/bronchiolar adenoma or carcinoma and (b) results in mild, site-specific, non-neoplastic lesions in the nasal cavity and centriacinar lung of male and female mice exposed to 0.5 or 1.0 ppm of ozone for 2 years, which persist with continued exposure to 30 months. It is uncertain whether or not the marginal increase (p> 0.05) of alveolar/bronchiolar neoplasms in male B6C3F1 mice resulted from exposure to ozone.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
The US NTP's two-year and lifetime studies on ozone using F344/N rats and B6C3F1 mice were performed in close accordance to internationally accepted guidelines (Boorman et al. 1994, Boorman et al. 1995).

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the existing available data that was assessed in a weight-of-evidence approach, it has been concluded that there is insufficient evidence from animal studies (as well as from human epidemiological studies) to determine the carcinogenicity of ozone, and thus, classification for carcinogenicity for ozone is considered not appropriate.

Additional information

Two 2-year and lifetime studies (30 months) from the US National Toxicology Program (NTP) of ozone using F344/N rats and B6C3F1 mice are published and available in the public domain (Boorman et al., 1994, 1995). In these NTP studies, which were performed according to internationally accepted guidelines, the animals were exposed to one of the three tested levels of ozone: (1) the US EPA standard at the time of the study (0.12 ppm), (2) the maximum concentration considered to be compatible with long-term survival (1.0 ppm) and (3) an intermediate level (0.5 ppm). In the lifetime studies, the tested ozone levels were 0.5 and 1.0 ppm. The animals were exposed to ozone for 6 hours per day, 5 days per week. Ozone had no effect on survival rates or body weights in both species as well as in both the 2-year and lifetime studies. Under the conditions of these two studies, there was no evidence of carcinogenic activity of ozone in male or female rats exposed to 0.12, 0.5 and 1.0 ppm. However, it should be mentioned that the mortality in the male rats ranged from 82 to 90% at the end of the 2-year study, and this exceeds the acceptable limit according to international test guidelines such as OECD 451 and EPA OPPTS 870.4200. In the 2-year mouse study increased incidences of alveolar/bronchiolar adenoma or carcinoma (combined) in female B6C3F1 mice exposed to 1.0 ppm ozone were observed. No effects were seen at 0.5 ppm and 0.12 ppm. In male mice no effect was observed. In the lifetime study an increased incidence of adenomas (but not carcinomas or total neoplasm) were seen at 1.0 ppm in female mice. In male mice the number of total neoplasms were not significantly increased, but a marginally increased incidence of carcinomas was observed at 0.5 and 1.0 ppm. Based on the results, there was equivocal evidence of carcinogenic activity of ozone in male mice and some suggestive evidence of carcinogenic activity of ozone in female mice.

The A/J mouse is frequently used as a model for testing of chemicals by inhalation for lung tumour multiplicities and has a high incidence of spontaneous lung adenomas. In the study by Witschi et al. (1999) female A/J mice were exposed to 0.12, 0.5 and 1.0 ppm of ozone. After 5 months of treatment, one-third of the animals were killed. The remaining animals were then split into two groups: one group with continued exposure to ozone for 4 additional months (for a total of 9 months) and the other group transferred into filtered air to allow a recovery period of 4 months. In animals exposed for 5 months to ozone a doubling of lung tumour multiplicity in the highest dose group was observed, but there was no sign of significantly increased tumour response after the full 9 months. A significant increase in tumour incidence was found after 9-month exposure to 0.5 ppm. In animals that were allowed to recover for 4 months, there was only a significant increase in tumour multiplicity and tumour incidence in the low dose group. Altogether, no clear dose-response relationship was observed, and the authors concluded that ozone is not a lung carcinogen in A/J mice.

Other studies also showed significant increase in lung tumour incidence in A/J mouse strain but not in the Swiss Webster mouse strain after exposure to 0.3-0.8 ppm ozone (Hassett et al., 1985; Last et al., 1987). Hassett et al. (1985) exposed two groups of female A/J mice to ozone. The first group was exposed to 0.31 ppm ozone for a total of 103 hours/week, every second week, for 6 months. The second group was exposed to 0.50 ppm ozone for a total of 102 hours/week, the first week of every month, for 6 months. Exposure to both concentrations was performed in two separate experiments with its own control group. With both concentrations, the animals were necropsied and examined for lung tumors 5 months after the last ozone exposure. Increased numbers of lung tumors relative to clean air controls were observed in the ozone-exposed animals. However, the number of lung tumour-bearing animals differed by 2-fold between the two control groups, and the percentage of mice with tumours was lower in the 0.50 ppm-exposed animals compared to the 0.31 ppm. It is likely that the finding was confounded due to the extremely low control values in one of the two experiments. Last et al. (1987) exposed male A/J mice and Swiss Webster mice to 0.4 and 0.8 ppm ozone for 8 hours/day (namely during nighttime), 7 days/week for 18 weeks. Although the exposure period was shorter than the aforementioned studies here, this might have been partly compensated by the timing of exposure to ozone during nighttime, when mice are known to be more active. An increased incidence of lung tumours was observed at 0.8 ppm in the A/J mice, a strain which is prone to develop lung tumours. However, the tumour incidence in the controls was abnormally low, especially given the higher sensitivity of this inbred mice strain for lung tumours compared to other strains. Thus, these results appear questionable such that the positive response might be a consequence of statisical analysis using the abnormally low control values rather than a true carcinogenic effect of ozone. On the other hand, no increase of lung tumours was observed in the ozone-exposed Swiss Webster mice. Lastly, Kim et al. (2001) and Kim and Cho (2009a, b) exposed male and female B6C3F1 mice to 0.5 ppm ozone for 6 hours/day, 5 days/week for 12, 16, 32 or 52 weeks. Oviductal carcinomas were observed after 12 and 16 weeks of exposure, in 3 out of 10 and 2 out of 20 animals respectively, but not after 32 or 52 weeks of exposure. It is considered very unlikely that the carcinomas were induced by the ozone treatment since the carcinomas were absent in the groups of animals with longer ozone treatments. Witschi et al. (1993) exposed hamsters to 0.8 ppm ozone for 23 hours/day, 7 days/week for 16 or 24 weeks. No evidence of carcinogenic action of ozone was seen in this study.

Altogether, there is limited scientific evidence to indicate the carcinogenic potential of ozone in animals. On the other hand, carcinogenicity of ozone in humans has been assessed by the US EPA as part of the "Integrated Science Assessment for Ozone and Related Photochemical Oxidants" (EPA 2013, pages 80-85). In this assessment, the authors carefully reviewed the epidemiological studies on the effects of ozone on human health and concluded that, albeit that ozone exposure may contribute to DNA damage, the evidence is inadequate to determine if a causal relationship exists between ambient ozone exposure and cancer. Since there was insufficient evidence for carcinogenic effects of ozone obtained from animal or human epidemiological studies, classification for carcinogenicity is considered not appropriate.