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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
Based on the available data, conducting reproductive toxicity studies on ozone is not considered justified. This is also in line with animal welfare considerations. However, in a number of available scientific studies male and female animals have been exposed to ozone before and during mating. For further information please refer to the text field "Additional information".
Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
No studies, in which internationally accepted guidelines were followed for evaluation of the reproductive toxicity of pure ozone, were found in the public domain. However, the literature published during the last 50 years in the public domain has been reviewed for toxic effects on reproduction caused by ozone. A total of 5 studies (mice, rats) were found, in which either the stud or the dam or both were exposed for 6-30 days before mating. Although these studies are not in compliance with the design as recommended in the current OECD guidelines for fertility testing, these peer-reviewed and well-documented studies are considered accceptable for supporting information on the effects of ozone on fertility. The IUCLID entries for these studies are listed under IUCLID section 7.8.2 (Petruzzi et al., 1995 and 1999; Santucci et al., 2006; Dell´Omo et al., 1995 and Jedlinska-Krakowska et al., 2006).
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No studies, in which internationally accepted guidelines were followed for evaluation of the reproductive toxicity of pure ozone, were found in the public domain or in the archives of the applicants. In accordance with column 2 of Annex IX of Regulation (EC) No. 1907/2006, reproductive toxicity studies, required under section 8.7, can be omitted if it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure and that there is no significant human exposure. Based on available toxicokinetic data it is shown that ozone is not systemically available. After inhalation, 90% of the O3 metabolism takes place in the epithelial lining fluid, covering the respiratory airways and epithelial cellular membranes. The remaining 10% takes place at cell membranes. O3 reacts directly with polyunsaturated fatty acids, amino acids, electron donors such as vitamins or glutathione and proteins. By this mechanism O3 is entirely consumed and systemically not available (Pryor et al., 1996). With respect to systemic toxicity, in the NTP carcinogenicity study (20 months) conducted according to OECD 452, no systemic adverse effects were reported. Moreover, there is limited human epidemiological evidence to suggest an association between ozone and effects on fertility. Therefore, based on the available data,conducting reproductive toxicity studies on ozone is not considered justified. This is also in line with animal welfare considerations.

However, there are a number of studies, in which male and female animals were exposed to ozone before and during mating. These studies provide information on possible effects of ozone exposure on the male and female reproductive system and mating performance. In the study by Santucci et al. (2006) female mice received continuous exposure to 0, 0.3 and 0.6 ppm ozone 30 days prior to mating until gestational day 17. No effects on mating performance, delivery or pregnancy outcome were reported. Additional data to support that there are no ozone-mediated effects on reproduction performance can also be found in the studies by Petruzzi et al., (1995, 1999) and Dell´Omo et al. (1995). Jedlinska-Krakowska et al., (2006) showed that the successful matings and the survival of pups were equivalent between the control and the ozone-exposed treatment groups. The testes of the ozone-exposed and control rats also showed no differences with regard to morphology and sperm motility, but sperm concentration was 17% lower in the ozone-exposed rats.


Effects on developmental toxicity

Description of key information
Based on the available studies, no classification for developmental toxicity is warranted. In the study by Bignami et al. (1994) the NOAECs for maternal toxicity and developmental toxicity were set at 0.4 ppm (or 0.78 mg/m³) and 0.8 ppm (or 1.57 mg/m³), respectively. This finding has been supported by other studies, and since the observed effects of ozone appear to be a nonspecific consequence of maternal toxicity, classification for reproductive/developmental toxicity is not warranted.  
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
with respect to treatment
Deviations:
yes
Remarks:
readouts more in line with OECD 426
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
Ozone was produced with an electric arc discharge 03 generator (ozonator). Different O3 concentrations were obtained by varying the airflow directed to the Ozonator (Regulator 44-2200. Drager Tescom GM BH, Lubeck, Germany) and by subsequent regulation by flow regulators/meters (Model RMA-13-SSV. Dwyer Instruments. Inc.. Michigan City, IN) of the appropriate volume of ozonized mixture to be added to the clean air directed to each chamber.
Species:
mouse
Strain:
CD-1
Details on test animals or test system and environmental conditions:
Mice of an outbred Swiss-derived strain (CD-1) weighing 25-27 g were purchased from a commercial breeder (Charles River. Calco. Italy). Upon arrival at the laboratory the animals were housed in an air-conditioned room (21 ± 1°C. relative humidity 60 ± 10%) with lights on from 9.30 PM to 9.30 AM. Adult males and females were housed separatedly in groups of 8-10 in 42 x 27 x 15cm plexiglas boxes with metal tops and sawdust as bedding. Pellet food (enriched standard diet purchased from Piccioni, Brescia, Italy) and water were continuously available (ad libitum).
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
On gestational day 7 females started a 10-day exposure to 0, 0.4, 0.8, or 1.2 ppm of O3 (n= 11 per group).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Ozone levels were monitored in each chamber every 18 min for a 4.5 min period by an ultraviolet (uv) O3 analyzer (O3 41 M. Environnement SA. Poissy, France). Actual concentrations were recorded during the last 30 sec of each 4.5 min period. The analyzer was calibrated and checked with an absolute uv O3 photometer. Ozone concentration in the room was generally below 0.02 ppm and never exceeded 0.04 ppm.
Details on mating procedure:
After 1 week of acclimatization, breeding groups (two females and one male) were formed. Females were inspected daily for the presence of a vaginal plug (pregnancy Day 0) and for delivery (PND 1). The stud was removed 7 days after the finding of the plug, that is at the time when females were randomly assigned to the various exposure conditions.
Duration of treatment / exposure:
For 10 days 24 hours per day. Start on GD7 ending at GD17.
Frequency of treatment:
Continuous for 10 days
Duration of test:
Last testing of the offspring was done on post-natal day 60
Dose / conc.:
0 ppm
Dose / conc.:
0.4 ppm
Dose / conc.:
0.8 ppm
Dose / conc.:
1.2 ppm
No. of animals per sex per dose:
11 females
Details on study design:
At birth, all litters were inspected, reduced to five males and three females, and fostered to untreated dams which had given birth to healthy litters with in the previous 24 hr.
Due to failure of pregnancy in six cases (one in each of the 0 and 0.8 ppm groups and two in each of the 0.4 and 1.2 ppm groups) and the unavailability for a foster dam in one case (0 group), these and all subsequent experiments used a total of 37 litters (n= 9 in the 0, the 0.4. and the 1.2 ppm groups; n= 10 in the 0.8 ppm group).
Two males and two females from each litter were used for postnatal assessments of somatic and neurobehavioral development. On each day from PND 2 to 8 and on alternate days from PND 8 to 16 or 18 (depending on the time of completed maturation of all endpoints described below). Pups were weighed to the nearest 0.01 g on a Mettler PK-300 balance and their body and tail lengths were measured. Days of eyelid and ear opening and of incisor emption were also recorded. On the same days. The pups were tested according to a slightly modified Fox battery (reflex and response).

Ultrasonic vocalitations (Postnatal Days 3, 7, and 11). Recording of ultrasonic calls took place in a sound-attenuating chamber (Amplisilence, Robassomero. Italy), following a procedure similar to that of previous investigations (Cagiano e1 al.. 1986; see also Santucci et al., 1993). Pups (one male and one female from each litter not used in the tests indicated in the previous section) were removed from the Jitter and individually placed in a double-wall glass con1ainer (diameter 5 cm, height 5 cm}.Two openings in the walls allowed water to circulate between a warm bath and the interspace of the container to maintain the latter at a constant temperature of 27°C. A Bruel & Kjaer (B&K, Naerum, Denmark) Model 4135 micro­ phone (B&K 2633 preamplifier) was suspended 6 cm above the floor of the container. Vocalization signals were filtered (tunable band-pass Khroni­ Hite tilter 3500 set at 20 to 90 KHz), amplified (B&K 2610 measuring amplifier), and recorded for 1 min on a Racal Store 4DS tape recorder using a direct mode recording procedure (tape speed 76.8 cm/sec). The number of ultrasonic calls emitted during the 5-min test was assessed by listening to an audible output of the recorder upon reduction of the tape speed to 9.6 cm/sec. Characteristics of the acoustic signal (calls emitted during the first 15 sec of the recording session) were analyzed on a high resolution analyzer (B&K 2033) during replay of the tape at 76.8 cm/sec.

Activity/ exploration and respons to d-amphetamine (at 60-61 days). These tests used the two remaining males in each litter when they were 60-61 days old. The tests were conducted in a quiet laboratory room, isolated from the animal colony and maintained under similar conditions, save for the use of dim red illumination . The experimental procedure took 2 days.
On Day 1 (First Test). animals were transferred to the experimental room prior to the stan of testing and then individually introduced for I hr in a freshly cleaned box identical t0 that used for housing, but without sawdust on the floor. The box was placed on a Varimex activity meter apparatus (Columbus Instrument s. Columbus, OH) with a sample rate of J O min to obtain six counts in each test. Immediately afterward, a novel stimulus object (SO. 35-m m film container) was gently introduced for 3-4 min in the box in the corner opposite that where the animal was standing. Videotape records were taken during SO exposure for subsequent scoring of mean latency to the first approach to the SO and number of contacts with it in the following 3 m in.
On Day 2 (Retest), one mouse in each litter received ip either 1 mg/kg d-amphetamine sulfate (in a volume of 0.01 mL/g body wt) or saline (0.9% NaCl) 15 min before being placed in the box for a 1-hr activity session. Since the previous day's data on responses to the SO failed to show significant O3 effects. The animals were not exposed to the SO in this session.
Maternal examinations:
Body weight, food and water intake. Reproductive performance parameters: pregnancy duration, number of pregnancy, litter size, sex ration, frequency of still birth, neonatal mortality
Ovaries and uterine content:
No expliciet information.
Fetal examinations:
Post natal body weight, appearance, ear opening, incisor eruption, hair growth, and body and tail lengths. Fox procedure for testing reflexes and responses. Ultrasonic vocalization and activity/exploration behavioral tests.
Statistics:
Body weight data from pregnant females was analyzed by a two-way analysis of variance (ANOVA) with O3 concentration as the grouping factor and the four measures indicated above (one before and three during exposure) as the within-subjects factors. The data on food and water intake were analyzed by separate randomized groups ANOVAs for each of the three periods indicated above since these periods were of unequal duration (72, 96, and 72 hr).
All parametric data obtained from the offspring of control and O3 exposed dams were analyzed by mixed-model ANOVAs considering the litter random factor and the exposure grouping factor. In addition, in the ANOVAs on postnatal body weight gain and on ultrasonic emissions sex was the within-litter grouping factor and the PNDs were the repeated measures factor: in the case of ultrasonic emissions. successive 1 min periods in the tests were also a second repeated measures factor. In the ANOVAs on Varimex activity scores at 60 and 61 days (males only) successive 10-min periods of the test were the repeated measures factor; the ANOVA on Retest had an additional grouping (with in-litter) factor (pretest saline vs d-amphetamine). Post hoc comparisons were performed by Tukey's HSD test, which can be used also in the absence of significant ANOVA results (Wilcox, 1987). The nonparametric data on somatic and neurobehavioral development were not amenable to analysis by an appropriate test respecting all main assumptions: in fact , there are no parametric tests which can consider litters (random factor) nested under prenatal treatments aswell asrepeated measures within subjects (Sachs and Thomas, 1985; Chiarotti et al., 1987). In addition. the scores had to be transformed to an "immature" (0 or 1)-"mature"' (2 or 3) dichotomy prior to being subjected to x² tests, so as to reduce the number of low expected frequencies to an acceptable level. Between-group comparisons after a sign. x² result were performed by Fisher's exact probability test.
Indices:
no
Historical control data:
no
Clinical signs:
not specified
Description (incidence and severity):
yes, see below
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see below
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
see below
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
see below
Pre- and post-implantation loss:
not examined
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Description (incidence and severity):
see below
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): see below
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Details on maternal toxic effects:
Table 1 shows that in the initial period of exposure (GD 7-10), O3 induced a concentration-dependent decrease of food and water intake. Food intake equal to control thereafter (GD 10-14), while water intake was still somewhat depressed in this period at 1.2 ppm. No significant effects were noted during GD 14-17.
However, maternal body weight during the exposure period is reduced for all exposure groups: 22.0, 20.6 and 15.5 g for 0.4, 0.8, 1.2 ppm, respectively as compared to controls 25.2 g.

Table 1 Body Weight and Intake of Food and Water" of Pregnant CD-1 Females during Ozone Exposure

Pregnancy Ozone exposure (ppm)

Days 0 0.4 0.8 1.2
Body weight (g) 7 (preexp.) 34.9± 0.5 34.7 ± 0.5 34.2± 0.6 34.8 ± 0.6
10 37.7± 0.9 36.4 ± 0.9 31.9± 1.0• 31.1 ± 0.6•
14 45.4± 1.1 42.7 ± 2.1 41.6± 1.3 39.2 ± 1.7
17 61. l± 4.0 56.7 ± 4. I 54.8± 2.8 50.3 ± 3.2

Food consumption (g) 7-10 8. 1± 1.2 5.8 ± 0.6• 5.2± 0.9•• 3.2 ± 0.1••
10-14 7.8± 0.3 7.2 ± 0.7 7.7± 0.6 6.8 ± 0.8
14-17 11.3± 0.7 10.2 ± 0.7 11.4± 1.1 10.8 ± 1.2

Water intake (ml) 7-10 12.8± 0.7 9.4 ± 1.2• 6.0± 0.4•• 5.0 ± 0.4 •
10-14 9.12 ± 0.3 8.8 ± 0.5 8.21 ± 0.5 6.9 ± 0.4••
14-17 14.7 ± 0.6 14.3 ±.1.2 12.9 ± 0.9 11.9 ± 0.7
• p <0.05.
•• p < 0.01 in comparisons with control (0).

Pregnancy duration was slightly increased by O3 exposure at 0.8 and 1.2 ppm (18.70 ± 0.17 days and 18.60 ± 0.16 days, respectively) relative to control ( 18.2 ± 0.13 days). Concerning reproductive performance, O3 exposure at any concentration did not affect the proportion of successful pregnancies ( 1-2 failures per group of 11 mice), litter size, sex ratio, frequency of stillbirth, or neonatal mortality.
Dose descriptor:
NOAEC
Effect level:
0.4 ppm (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
see below
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
see below
External malformations:
not specified
Skeletal malformations:
not specified
Visceral malformations:
not examined
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Postnatal body weight gain was slightly but significantly depressed by prenatal O3 exposure at 1.2 ppm. Post hocs showed that the offspring of dams exposed to 1.2 ppm of O3 weighed significantly less than controls from PND 6 onward (p< 0.01).
Ear opening, incisor eruption, hair growth, and body and tail lengths were not affected by prenatal O3 exposure. Eyelid opening was delayed (by about 2 days). by prior O3 exposure. Fisher exact probability tests, however, showed a significant delay in only the 0.4 ppm group (p= 0.05). All other data, concerning various reflexes and responses showed no effect on sensorimotor development upon ozone exposure. Ultrasonic vocalization pattern of offspring form O3 exposed dams was not different from control pups. Maternal O3 exposure to 0.8 ppm did not affect offspring overall activity and habituation in either test session, nor did it affect latency of approach to stimulus object and number of approaches. Maternal O3 exposure (0.8 ppm) failed to affect response to d-amphetamine.
Key result
Dose descriptor:
NOAEC
Effect level:
0.8 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
not specified
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1.2 ppm (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
no
Conclusions:
Although this experimental study was not directly compliant with one of the OECD guidelines, it is an acceptable study which meets basic scientific principles and was conducted similar to the OECD 414. Thus, the study is considered to be reliable for risk assessment. Maternal toxicity was observed at 0.8 and 1.2 ppm as a body weight gain decrease and prolonged pregnancy. Thus, the NOAEC for maternal toxicity is considered to be 0.4 ppm. No effects on successful pregnancies, litter size, sex ratio, frequency of stillbirth or neonatal mortality was observed. But, a post natal decreased growth was observed in the 1.2 ppm treatment group. Thus, the reproductive/developmental NOAEC is considered to be 0.8 ppm.
Executive summary:

In a developmental toxicity study conducted equivalent to OECD 414, female CD-1 mice were exposed during pregnancy (Days 7-17) to different O3 concentrations (0, 0.4, 0.8, or 1.2 ppm) 24 hours/day. To avoid confounding by postnatal maternal effects, all litters were assigned shortly after birth to foster dams neither treated nor handled during pregnancy. Maternal toxicity was observed at 0.8 and 1.2 ppm as a body weight gain decrease and prolonged pregnancy. Thus, the NOAEC for maternal toxicity is considered to be 0.4 ppm. No effects on successful pregnancies, litter size, sex ratio, frequency of stillbirth or neonatal mortality was observed. But, a post natal decreased growth was observed in the 1.2 ppm treatment group. Ear opening, incisor eruption, hair growth, and body and tail lengths were not affected by prenatal O3 exposure. Thus, the reproductive/developmental NOAEC is considered to be 0.8 ppm.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
1.57 mg/m³
Study duration:
subacute
Species:
mouse
Quality of whole database:
Meets generally accepted scientific standards, well documented and acceptable
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A detailed discussion of the available studies is presented below.

Effects on the reproductive system and mating performance

In a number of studies, male and/or female animals have been exposed to ozone before and during mating. These studies provide information on possible effects of ozone exposure on the male and female reproductive system and mating performance. In the study by Santucci et al. (2006) female mice received continuous exposure to 0, 0.3 or 0.6 ppm ozone starting 30 days prior to pairing for mating and lasting until gestational day (GD) 17. At birth the litters were culled to eight pups. No ozone-mediated effects on mating performance, delivery or pregnancy outcome were reported.

An earlier study also showed that continuous (i.e. 24 h/day) exposure of females to 0, 0.2, 0.4 and 0.6 ppm ozone from 6 days prior to mating to GD 17 did not produce significant effects on reproduction performance (Petruzzi et al., 1995). These findings were confirmed in an independent study in female mice exposed to 0.6 ppm ozone also from 6 days prior to mating until weaning (Dell’Omo et al., 1995). In a later study by Petruzzi et al., the continuous exposure period of ozone (0, 0.3, 0.6 and 0.9 ppm) in female mice was extended to 6 days prior to mating until postnatal day 26, and here, no ozone-mediated effects were observed regarding the proportion of successful pregnancies, litter size, sex ratio and neonatal mortality (Petruzzi et al., 1999).

Male rats were exposed to ozone at 0.5 ppm or to control air for 5 h/day for 50 days, and it was reported that the number of successful matings and the survival of pups were similar between the two groups. The testes of the ozone-exposed and control rats showed no differences with regard to morphology and sperm motility, but sperm concentration was 17% lower in the ozone-exposed rats (Jedlinska-Krakowska et al., 2006).

Maternal toxicity

Effects of prenatal ozone exposure on the pregnant females have been published in a number of studies in both rats and mice. In a study with pregnant female CD-1 mice exposed to 0, 0.4, 0.8 and 1.2 ppm ozone during GD 7-10, the maternal food and water intake as well as body weight gain were decreased in a concentration-dependent manner from 0.8 ppm onwards. However, tolerance to these effects developed as exposure continued until GD 17 (Bignami et al., 1994). In three different studies, female CD-1 mice were continuously exposed to ozone in the range of 0.2-0.9 ppm. Exposure started at 6 days prior to pairing for mating and continued during breeding until GD 17 (Petruzzi et al., 1995), or postnatal day 26 (Petruzzi et al., 1999, Dell’Omo et al., 1995). Only in one study, transient decreases in food consumption, water intake and body weight gain were observed at 0.4 and 0.6 ppm ozone during the initial period of exposure (days 1-3) and lasted slightly longer with the 0.6 ppm ozone exposure (days 3-6) . Recovery was nearly complete at the start of the mating period (Petruzzi et al., 1995).

In several experiments, Kavlock et al. (1979, 1980) exposed pregnant Long-Evans rats at different periods of gestation to ozone concentrations in the range of 0.44-1.97 ppm. Continuous exposure of pregnant rats to 0, 1.0, 1.26 and 1.49 ppm ozone from GD 9 to 12 revealed lower maternal weight at 1.26 and 1.49 ppm with dose-related reduction in food and water intake. In a second experiment with similar experimental design, dose-related decrease in maternal body weight gain and reduction in food and water intake were recorded with exposures to 0.64, 0.93 and 1.97 ppm ozone. Exposure to 0.44 ppm ozone for 8 h/day during organogenesis (GD 6-15) reduced maternal weight gain (24%) as compared to control animals (Kavlock et al., 1979). Custodio et al. (2010) reported that the body weight gain of pregnant rats exposed to 1 ppm ozone for 12 h/day during the first 20 days of the gestation period was comparable with the growth in the control group. Rats at several stages of pregnancy and lactation, as well as age-matched virgin females, received a single exposure to 1 ppm ozone for 6 h to determine whether pregnant rats are more sensitive to the inflammatory effects of ozone than age-matched virgin females. Components of bronchoalveolar lavage fluid were used to assess the severity of pulmonary inflammation. The results showed that, compared to virgin female rats, ozone exposure during pregnancy significantly enhanced the sensitivity to ozone-induced pulmonary inflammation. The pulmonary inflammation, which developed during pregnancy, persisted during lactation but was no longer observed following lactation (Gunnison et al., 1992). Later, uterine contractile responsiveness to acetylcholine and oxytocin was studied in virgin and pregnant rats. Virgin and pregnant (at GD 5, 10 and 18) rats were exposed for 1 h to air or 3 ppm ozone. Uterine strips were isolated from these animals and were tested 16-18 h later on contractile responsiveness to acetylcholine and oxytocin. The authors suggested that ozone inhalation can produce abnormal contractility in the pregnant uterus (Campos-Bedolla et al., 2002).

The study by Gunnison et al. (1999) provides supporting information that exposure to 0.5, 0.8 or 1.1 ppm ozone enhanced polymorphonuclear leukocyte (PMN) inflammation in the lung, and the severity of PMN inflammation is directly related to the relative dose of ozone, or its metabolites, to the lower lung, regardless of physiological status (i.e. pregnancy).

Embryo-foetal toxicity and reproduction performance

The number of published studies concerning the effect of ozone exposure during gestation on embryo-foetal development and delivery is very limited. In addition, none of these is compliant with the current standards of the OECD Test Guidelines. The international standards for reproduction toxicology clearly define the different stages of the reproduction cycle and the period of treatment for each cycle in order to distinguish among adverse effects occurring during fertility, mating performance, early-/mid-/late gestation, delivery or postnatal care and development. Because the available published studies which were reviewed for this dossier were mainly driven by different objectives, their study designs, such as exposure periods, are mostly not in line with the reproduction segments as defined in the international test guidelines for evaluation of reproductive toxicity. Despite this fact, the majority of the studies published during the last twenty years are considered sufficiently good quality studies. The experimental design of these studies with respect to animal species used, animal housing conditions, group size, exposure regime, exposure control, observation methodology and statistics are in compliance with international scientific standards. Although study design and methodology of some studies are only described in few details, e.g. due to the space limitation of the journals, the overall quality of the studies is considered acceptable and sufficient to allow a risk assessment of health effects on offspring as a consequence of maternal exposure to ozone during pregnancy. The effects of ozone on embryo-foetal development and delivery were investigated in mice and rats. Six studies in mice reveal information on embryo-foetal development and/or reproduction performance upon ozone exposure. For the rat, only one study, by Kavlock et al. (1979) revealed information on the effect of ozone on embryo-foetal development and reproduction performance.

Mouse

Bignami et al. (1994) exposed CD-1 mice continuously to ozone concentrations of 0, 0.4, 0.8 or 1.2 ppm from GD 7-17. The study was conducted similar to OECD 414. They found that duration of pregnancy slightly increased 0.8 and 1.2 ppm ozone. Exposure at any ozone concentration did not affect the proportion of successful pregnancies, litter size, sex ratio, frequency of still birth or neonatal mortality. The pups were separated from their mothers immediately after birth. The only ozone-related finding was a slight reduction in birth weights at the highest ozone concentration tested (1.2 ppm) (Bignami et al., 1994). As mentioned earlier, prenatal exposure to 0.2, 0.4 and 0.6 ppm ozone did not exert a significant effect on birth weight or reproductive performance. Additionally, the exposure did not affect the proportion of successful pregnancies, litter size, sex ratio or neonatal mortality (Petruzzi et al., 1995). Similarly, there were no reported effects by ozone on reproduction performance and birth weight of mice continuously exposed to 0.6 ppm ozone from 6 days prior to pairing for mating to postnatal day 22 (Dell’Omo et al., 1995), and continuous exposure to 0, 0.3, 0.6 or 0.9 ppm ozone from 6 days prior to pairing for mating until postnatal day 26 was found to have no effect on the proportion of successful pregnancies, litter size, sex ratio and neonatal mortality. There was also no significant difference in pup weight at birth between the pups from ozone-exposed mothers and those from control air-exposed mothers (Petruzzi et al., 1999). However, in one study, a reduction of succesful pregancies was reported in female mice exposed to 0.6 ppm from 6 days prior to mating until GD 17 (Sorace et al, 2001).

Sharkhuu et al. (2011) reported the findings of three separate experiments (in order to generate sufficient offspring for postnatal testing) on BALB/C mice. Exposure of pregnant mice to 0, 0.4, 0.8, or 1.2 ppm ozone took place for 4 h/day for 10 days during GD 9-18. The average percentage of successful pregnancies of the three experiments was 58% for the control air exposure group, 45% for both 0.4 and 0.8 ppm ozone groups and 33% for the 1.2 ppm ozone group. The litter size and sex ratios of the ozone-exposed groups were not different from the controls. The offspring from the 1.2 ppm-exposed dams weighed less than the pups from the air-exposed mothers. Strain-related factors are likely to be responsible for the difference in response to 1.2 ppm ozone between this study and the study of Bignami et al. (1994). It is well known that reproductive processes are generally much less robust in BALB/C mice than in outbred strains such as CD-1 mice as used in the Bignami study. It is general knowledge that BALB/C mice have 44.4% unsuccessful mating rate.

Brinkman et al. (1964) did suggest that ozone exposure may exert postnatal adverse effects. Paired male and female mice of 2 different inbred strains were exposed to 0.1 or 0.2 ppm ozone for 7 h/day, 5 days/week for 3 weeks, and the mice were allowed to give birth. Average litter size was not significantly reduced in ozone-exposed groups in comparison with air-exposed controls. However, there was a significant increase in postnatal mortality in the first 3 weeks after birth. It is worth mentioning though that animal studies from the 1950s and 60s are more difficult to evaluate and assess since limited methodological details are available in the public domain.

Rat

Female rats exposed to ozone during pregnancy and the consequent effects on prenatal development of offspring have been studied. Long-Evans rats were exposed either for 24 h/day during early (GD 6-9), mid- (GD 9-12) or late (GD 17-20) gestation to concentrations of ozone varying from 0 to 1.97 ppm or for 8 h/day throughout the period of organogenesis (GD 6-15) to 0.44 ppm ozone. Exposure to 1.04 ppm ozone during early gestation caused significantly fewer implantation sites and an increase in resorptions. Surviving foetuses were heavier and more advanced in skeletal development, which could potentially be related to the reduction in the number of viable foetuses in the litters. However, exposure to a lower dose of ozone (0.44 ppm) during organogenesis had no effect on implantation or resorption rates, and also, no ozone-mediated effects were observed on foetal weight, skeletal ossification and visceral development. The only increase in anomalies noted in the group exposed to 1.04 ppm ozone was enlarged renal pelvis affecting 5.8% of the foetuses among 20% of the litters compared with none in the concurrent controls. However, in the group exposed during mid-gestation, 6.1% of the foetuses from the concurrent control groups had enlarged renal pelvis, whereas 2.2%, 0% and 0% of foetuses exhibited this anomaly in the 1.00, 1.26 and 1.49 ppm ozone-exposed groups, respectively (Kavlock et al., 1979). Thus, the frequency of the anomaly was apparently random with no suggestion of a dose-response relationship.

In two separate experiments from the same authors, female rats were exposed during GD 9-12 to either 0, 1.0, 1.26 or 1.49 ppm ozone (experiment 1) or 0, 0.64, 0.93 or 1.97 ppm ozone (experiment 2). There was a significant increase in resorption rates observed at 50.4% and 58.8% in the 1.49 and 1.97 ppm ozone-exposed groups, respectively, compared to 8-13% observed resorption rates in the other groups including controls. This was largely due to complete resorption of a number of litters in these two treatment groups. Effects on foetal weight and ossification were variable: significant reductions in foetal weight and delayed ossification seen in the 1.49 ppm ozone group from experiment 1 were not seen in the 1.97 ppm ozone group from experiment 2. The absence of any statistically significant effect at 1.97 ppm ozone-exposed group is probably due to the high numbers of resorptions, and the effects among the surviving foetuses were likely to be very variable. There were no significant major malformations or anomalies observed in these two experiments. Although exposure to ozone from 0.44-1.97 ppm caused reductions in maternal body weight gain, it is unlikely that the significant embryo lethality observed with 1.49 and 1.97 ppm ozone exposure during mid-gestation was secondary to these maternal effects since the average maternal weight loss was not greater than that that from groups exposed to lower ozone concentrations (Kavlock et al., 1979). Thus, it seems likely that levels above 1.04 ppm can trigger early embryo lethality but are not teratogenic.

In a study by Rivas-Manzano and Paz (1999), dams exposed to 1 ppm ozone for 12 h/day during the entire pregnancy had no offspring and complete stillbirth. Lopez et al. (2008) studied the ultrastructural alterations at the bronchiolar level during the intra-uterine lung development. Pregnant rats were exposed to 1 ppm ozone for 12 hours on GD 18, 20 or 21. The ultrastructural analysis revealed swollen mitochondria, cytoplasmic vacuolisation of the epithelial cells and structural disorder caused by the oxidative stress. At GD 20, flake-off epithelial cells and laminar bodies in the bronchiolar lumen were observed. In the GD 21-exposed group, the mitochondria were edematous, and their cristae were disrupted by the damage caused in mitochondrial membranes.

Postnatal developmental response

Several studies on the postnatal development of offspring of mothers exposed to ozone during pregnancy have been reported for mice and rats.

Mice

A study of somatic and neurobehavioural development in female CD-1 mice exposed during GD 7-17 to 0, 0.4, 0.8 or 1.2 ppm ozone showed slight but significant decrease in postnatal body weight gain in the pups from the 1.2 ppm ozone-exposed dams. These pups had delayed eye opening, but there were no negative effects on their behavioural performance (Bignami et al., 1994). A second study on CD-1 mouse offspring exposed to 0, 0.2, 0.4, or 0.6 ppm ozone in utero (prior to conception to GD 17) exhibited no significant deficits in postnatal somatic and neurobehavioural development or in adult motor activity (Petruzzi et al., 1995). In a third study by the same group, CD-1 mouse offspring postnatally exposed to 0.6 ppm ozone from birth through weaning demonstrated no impairment of navigational performance during acquisition and only subtle changes during reversal learning. Offspring showed a significant reduction in body weight. Effects on neurobehavioural development from this ozone exposure were minor with some attenuation of activity responses and impairment of passive avoidance acquisition (Dell’Omo et al., 1995). A fourth study by the same group, in which exposure of the females to 0.3, 0.6, or 0.9 ppm ozone started from before conception and continued after birth of the pups until weaning, showed subtle changes in handedness and morphine reactivity. Exposures to 0.6 ppm ozone caused a reduced preference for the right paw in adulthood. Exposures to 0.9 ppm ozone altered hot plate avoidance after i.p. injection of morphine in adulthood (Petruzzi et al., 1999). In a fifth study, the offspring of CD-1 mice, which had been continuously exposed to 0, 0.3, or 0.6 ppm ozone from 30 days prior to pairing for mating until GD 17 showed small and selective effects on somatic and sensorimotor development up to an age of 100 days. The effects were more pronounced in the 0.3 ppm-exposed group than in the 0.6 ppm-exposed group (Sorace et al., 2001). The offspring of pregnant BALB/c mice exposed to 0, 0.4, 0.8 or 1.2 ppm ozone for 4 hours/day for 10 days during GD 9-18 had decreased weight gain in the 1.2 ppm-exposed group. The pulmonary inflammation and immune responses were assessed in the offspring at 6 weeks of age. At 0.8 and 1.2 ppm, delayed-type hypersensitivity responses to bovine serum albumin were suppressed in the female offspring, whereas humoral immune responses to sheep red blood cells were not altered in either sex. Maternal exposure to 1.2 ppm ozone increased the activity of lactate dehydrogenase (LDH) but had no effect on the number of inflammatory cells, the level of total protein or the level of cytokines in bronchoalveolar lavage fluid (BALF) of the offspring. The offspring from air-exposed dams that were sensitised early in life (postnatal day 3) to ovalbumin antigen (OVA) and then challenged as adults, developed eosinophilia, elevated levels of LDH activity and total protein in BALF as well as increased pulmonary responsiveness to methacholine compared to offspring sensitised later in life, e.g. at postnatal day 42. Maternal exposure to 1.2 ppm ozone decreased BALF eosinophilia and serum OVA-specific IgE in the female offspring sensitised early in life but did not affect the development of allergic airway inflammation by offspring sensitised later in life. In summary, maternal exposure to ozone affected reproductive outcome and produced modest decreases in immune function and indicators of allergic lung disease in surviving offspring (Sharkhuu et al., 2011).

Rat

Pregnant rats were exposed to 0, 1.0, or 1.5 ppm ozone during either mid-gestation (GD 9-12) or late gestation (GD 17-20). The dams were allowed to deliver, and the early morphological and behavioural development of their pups was monitored. Both ozone exposure regimes transiently reduced neonatal growth rates, except for male offspring who had lower weight at 60 days postnatal. The late gestation exposure regimen with 1 ppm ozone produced retardations in early reflex development and open field behaviour at 15 days of age. Several males from this exposure regimen remained permanently stunted in growth (Kavlock et al., 1980).

Five studies regarding the effects of ozone on postnatal development of pups from mothers exposed to 1 ppm ozone for 12 h/day during the entire pregnancy were reviewed. Offspring exposed in utero to 1 ppm ozone (i.e. via dams exposed to ozone for 12 h/day during pregnancy) showed significantly reduced noradrenaline concentration in the cerebellum at postnatal days 10, 20 and 30, and in the cerebral cortex and the pons noradrenaline was found to be significantly reduced at postnatal days 10 and 30, respectively, as compared to controls. The authors concluded that prenatal exposure to 1.0 ppm ozone causes embryonic-foetal changes as manifested by the modulation of postnatal levels of noradrenaline concentrations in the brains of rats (Custodio et al., 2010). Another study, with similar exposure regime, showed alterations in catecholamine neurotransmitter levels in the cerebellum during early postnatal life. This suggests that prenatal ozone exposure disrupts the cerebellar catecholamine system rather than the indoleamine system (Gonzalez-Pina et al., 2008). Rivas-Manzano and Paz (1999) reported morphological changes in the cerebellar lobe of rat offspring from dams exposed to 1 ppm during the entire gestation. Offspring showed cerebellar necrotic signs at birth, diminished area of the molecular layer with Purkinje cells with pale nucleoli and perinucleolar bodies at 12 days of age as well as Purkinje cells showing nuclei with unusual clumps of peripheral chromatin at 60 days of age. Romero-Velazquez et al. (2002) further studied the morphology of the rat cerebellum in offspring from dams exposed to 1 ppm ozone for 12 h/day during the entire gestation. They observed circular structures with a fibrillar composition meshed in an amorphous matrix with scattered cells in the cerebellum of 90-day-old rat offspring from the ozone-exposed mothers. Nearby these circular bodies, zones with noticeable depopulation of Purkinje cell were seen. Also, some lobules frequently showed big zones of degenerating Purkinje cells, and debris. A major quantity of capillaries and incomplete folding pattern of some lobes were found in this group as well. Haro and Paz (1993) studied the sleep organisation in rat offspring from mothers exposed to 1 ppm ozone for 12 h/day during pregnancy. Severe sleep disturbances such as a decrease in paradoxical sleep duration as well as inversion of the light-dark cycle or a circadian phase-shift of vigilance states were reported. The authors suggested that, in rats, ozone exposure during pregnancy may affect the generating mechanisms of paradoxical sleep and the regulation of circadian rhythms. The effect of immobilisation stress on neuronal response in the nucleus tractus solitarius (NTS) in rat offspring from mothers exposed to 0.5 ppm ozone for 12 h/day on GD 5-20 was studied by Boussouar et al. (2009). The adult NTS regulates respiratory control. The results indicated that prenatal exposure to ozone led to the loss of adult NTS reactivity to stress in the offspring and that long-lasting sequelae were detected in the offspring beyond the prenatal ozone exposure.

Altogether, 21 studies on rats and mice were evaluated, and based on these data, the NOAEC for maternal toxicity is considered to be around 0.4 ppm (or 0.78 mg/m³), the NOAEC for reproduction and embryo-fetal toxicity is considered to be around 0.8 ppm (or 1.57 mg/m³).

Justification for classification or non-classification

Based on the available data, the observed effects of ozone are considered to be a non-specific consequence of maternal toxicty. Thus, classification for reproductive/developmental toxicity is not warranted.

Additional information