Ozone

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key 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:

Medium and Long-Term Behavioral Effects in Mice of Extended Gestational Exposure to Ozone

Author:

Petruzzi et al.

Year:

1995

Bibliographic source:

Neurotoxicology and Teratology 17(4), 463-470

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Design close to OECD 421 screening study with post natal assessments closely compliant with OECD 426.

GLP compliance:

no

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 produced with an electric arc discharge O3 generator (ozonator).

Test animals

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

The animals were housed in an air conditioned room (temperature 21 ± 1 °C, relative humidity 60 ± 10%) with lights on from 9.30 p.m. to 9.30 a.m.. Adult males and females were housed in same sex pairs in 33 x 13 x 14 cm plexiglas boxes with a metal top and sawdust as bedding. Pellet food (Enriched standard diet purchased from Mucedola, Settimo Milanese, Italy) and water were continuously available.

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

Ozone was produced with an electric arc discharge O3 generator (ozonator). Different O3 concentrations were obtained by varying the airflow directed to the ozonator (Regulator 44-2200, Drager Tescom GMBH, Lübeck, Germany) and by subsequent regulation with flow regulators-meters (model RMA-13-SSV, Dwyer Instruments, Inc., Michigan City, USA) of the appropriate volume of ozonized mixture to be added to the clean air directed into each chamber.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Ozone levels were monitored in samples drawn at the bottom of the chamber (close to exhaust) every 18 min for a 4.5 min period, after extensive preliminary verification that 0, levels in these samples were representative of those in different parts of the chamber. Ozone was measured by an ultraviolet (UV) 0, analyzer (0,41 M, Environnement SA, Poissy, France) equipped with a control system for the repeated check of the calibration between successive measure cycles, based on the measurement of known 0, concentrations. Actual concentrations were recorded during the last 30 s of each 4.5-min period.

Details on mating procedure:

After 1 week of acclimatization, 16 males and 16 females were randomly assigned to each of the three O3 exposure groups (see below), while 20 males and 20 females were assigned to the control condition. Breeding pairs were formed after a 6-day exposure period. Females were inspected daily for the presence of a vaginal plug (pregnancy day 0) and for delivery (PND 1); because the interval between pairing and plug varied from 1 to 4 days, the time range between start of exposure and start of pregnancy was 7-10 days. The stud was removed ten days after discovery of the plug.

Duration of treatment / exposure:

Exposure to O3 was continuously (0, 0.2, 0.4, or 0.6 ppm) lasted from 6 days prior to the formation of breeding pairs (7-10 days prior to the start of pregnancy to the morning of pregnancy day 17)

Frequency of treatment:

6 days prior formation breeding pairs until pregnancy day 17.

Duration of test:

Test start: at first exposure day at 6 days before formation breeding pairs. Test end: at the last observation day PND 98.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.2 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

0.4 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

0.6 ppm (nominal)

No. of animals per sex per dose:

Control air : n=20 M and 20 F
0.2 ppm ozone: n= 16 M and 16 F
0.4 ppm ozone: n= 16 M and 16 F
0.6 ppm ozone: n= 16 M and 16 F

Control animals:

yes, concurrent vehicle

Details on study design:

At birth, all litters were reduced to four males and four females and fostered to untreated dams which had given birth to healthy litters within 24 h.
Assessment of Somatic and Neurobehavioral Development: post-natal day 2-18
Social Interaction Tests: post-natal days 23-25 and 43-45
Locomotor Activity: at post-natal days 57-60
Eight-Arm Radial Maze Learning: at post-natal days 84-98

Examinations

Maternal examinations:

Body weight and food and water intake were measured to the nearest 0.01 g (Mettler PK-300 balance set to automatic compensation of movements). For this purpose, food containers and water bottles were weighed and refilled every 3 days until the day after exposure discontinuation (pregnancy day 18).

Proportion of pregnancies carried to term, litter size, sex ratio, and neonatal mortality were also noted to assess any effect on reproductive performance.

Ovaries and uterine content:

No

Fetal examinations:

Reproductive performance:
At birth, all litters were reduced to four males and four females and fostered to untreated dams which had given birth to healthy litters within 24 hours. Proportion of pregnancies carried to term, litter size, sex ration and neonatal mortality were assessed.

Two males and two females from each of 10 litters in each treatment group were used for postnatal assessment of somatic and neurobehavioral development. On each day from PND 2 to 10 and on alternate days from PND 10 to 16 or 18 (depending on the time of completed maturation of all end-points of interest), pups were weighed to the nearest 0.01 g (see above) and their body and tail lengths were measured. Day of eyelid and ear opening and of incisor eruption was also recorded.

Neurobehavior development:
On each of the days indicated above, the pups were tested on neurobehavior development (reflexes and responses) according to a slightly modified Fox battery. The tests were conducted during the dark period between 10 a.m. and 3 p.m., each subject being tested at approximately the same time of day.

Social Interaction Test:
Upon weaning (PND 21), the subjects not used in the assessments described in the previous section were housed in pairs of the same sex and litter in 33 x 13 x 14 cm cages. On one of PNDs 23-25 (prejuvenile stage) and again on one of PNDs 43-45 (juvenile stage) each pair underwent between 10 a.m. and 3 p.m. a 15-min social encounter with another pair of the same sex and treatment.

Locomotor Activity:
Out of the subjects used for the social interaction test, 48 males (12 per treatment) were subsequently tested at 57-60 days for locomotor activity. Animals were transferred to the experimental room and then individually introduced for 20 min into a freshly cleaned box identical to the home box but without bedding. The box was placed on a Varimex Activity Meter apparatus (Columbus) with a sample rate of 4 min, so as to obtain five counts in each test.

Eight- Arm Radial Maze Learning:
Thirty-two out of the 48 males used in the activity test (8 per treatment) were tested at adulthood (84-98 days) in an eight-arm radial maze

Statistics:

Analyses of variance (ANOVAs) considering prenatal exposure as grouping (between-subjects) factor and exposure days as repeated measures (within-subject) factor were performed on dams’ food and water intake and body weight. Postnatal body weight data were subjected to a mixed model ANOVA considering litters as random factor, prenatal exposure, and sex as grouping factors (between- and withinlitter, respectively), and PNDs as repeated-measure factor. Two separate ANOVAs on frequency and duration data were performed for each of the social test items with treatment and sex as grouping factors and the two ages as repeated measures. For the reason given in the Section on Method (Social behavior) the statistical unit was composed by the group of four subjects. In the ANOVAs on Varimex activity scores at 57-60 days (males only) and those on eight-arm radial maze performances, prenatal treatment was the only grouping factor while repeated measures were successive 4-min periods of the activity test and successive daily maze sessions, respectively. Post hoc comparisons were performed by Tukey’s HSD test, which can also be used in the absence of significant ANOVA results (46). Nonparametric data on somatic and neurobehavioral development (Fox scores) were simplified prior to analysis by x2 test, using an “immature” (O-l) “mature” (2 or 3) dichotomy which reduced the number of low expected frequencies to an acceptable level. Between-group comparisons after a significant x2 result were performed by Fisher’s exact probability test with Bonferroni’s correction. This type of analysis cannot avoid the bias created by the non-availability of nonparametric tests which can simultaneously take into account the nesting of litters under treatments (a random variable), the use of litters as blocks, and repeated measures within subjects.

Indices:

no

Historical control data:

no

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

During the initial period of exposure (Days 1-6) O3 induced the expected concentration dependent decreases of food and water intake and of body weight, followed by a rapid attenuation of these effects. In other words, tolerance to the initial depressant effects of O3 on consummatory responses and body weight was complete or near-complete by the time the mice were enabled to mate after the initial period of exposure.

Effect levels (maternal animals)

Dose descriptor:

NOAEC

Effect level:

0.6 ppm (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxicity / teratogenic effects:
As concerns reproductive performance, O3 exposure at any concentration did not affect either the proportion of successful pregnancies (14/20, 16/16, 14/16, and 13/16 in the 0, 0.2, 0.4, and 0.6 ppm groups, respectively), litter size, sex ratio, and neonatal mortality.

Somatic and Neurobehavioral Development (PNDs 2-18):
Prenatal O3 exposure did not exert significant effects on weight at birth and postnatal body weight gain in the presence of a significantly higher weight of males than females. The data on postnatal somatic and neurobehavioral development also failed to show any significant effects of O3 exposure. These data need not to be reported in detail, being indistinguishable from those of the previous experiment with shorter exposure from day 7 to 17 of pregnancy (Bignami 1994).

Social interaction tests:
The effects of sex and age on various social and nonsocial responses cannot be described here in detail. Briefly, the former effects were limited to only one of the responses in the nonsocial repertoire, namely digging, which was more frequent at both ages in males than in females, F(1, 32) = 4.24, p< 0.05. On the other hand, three responses in the social repertoire (follow, squire, and mutual circle) were more frequent at both ages in females than in males, F(1, 32)= 6.74, 10.92, and 20.43, respectively, p< 0.01.
Both these effects of sex, which did not interact with other factors, and the response changes with increasing age often produced different control baselines within the same response category, making it possible to verify whether the presence or absence of O3 induced changes was related to response baseline. For example O3 was without significant effects on several social and nonsocial responses with widely differing baselines which depended either mainly on response characteristics (sniffing vs. mutual circle) or on age higher frequency of digging at 43-45 than at 23-25 days, F(1, 36) = 26.97, p < 0.01, and vice versa in the case of jumping, F(1, 36)= 18.98,~ < 0.011.
On the other hand, prenatal O3 exposure produced selective effects in opposite directions on frequency of self-grooming and duration of exploration, increasing the former, F(3, 36) = 3.64, p < 0.05, and reducing the latter, F(3, 36)= 5.00, p< 0.01, respectively in the presence and in the absence of a significant age effect [self-grooming F(1, 36) = 63.50, p< 0.01)]. In the case of self-grooming, the results of posthots suggested that the O3 effects observed at the younger age became attenuated or disappeared at the older age, although the interaction between the two factors failed to reach statistical significance. In the case of exploration, younger and older mice were similarly affected, although O3 effects were significant only at the highest 0.6.ppm concentration.

Locomotor Activity at 57-60 Days:
The data on locomotor activity and within-session activity decrement [habituation; repeated measures, F(3,22) = 63.56, p< 0.011] need not to be reported in detail, since no significant effects of prenatal O3 exposure were found.

Eight-Arm Radial Maze Learning
The data concerning the number of rewarded entries in the first eight trials of each session (blocks of four sessions each), show different trends in control and O3 animals; namely, a higher initial level followed by minor fluctuations in the former and a lower initial level followed by a marked increase in the latter, treatment x repeated measures, F(12, 112)= 2.06, p< 0.05. Because the posthocs failed to locate the between-group differences responsible for the interaction just mentioned, a subsequent ANOVA was devoted to a session-by-session analysis of the first four sessions. This ANOVA confirmed the treatment induced reduction in the number of rewarded trials in the initial phase of training, F(3, 28)= 3.18, p< 0.05, but only the 0.2 ppm group was significantly different from controls (p< 0.05). The data concerning the total time taken to make the first eight visits to maze arms suggest that two of the O3 groups (0.2 and 0.4 ppm) were slower than the controls. The ANOVA yielded a significant effect of repeated measures, F(4, 112)= 4.34, p< 0.01, whereas the effect of treatment just missed statistical significance, F(3, 28)= 2.84, p= 0.056. Posthocs failed to show significant differences between controls and O3 animals but yielded significant results in comparisons between the 0.6 ppm group on the one side and the 0.2 and 0.4 ppm groups on the other (p< 0.05).

Effect levels (fetuses)

Dose descriptor:

NOAEC

Effect level:

0.6 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, in this study CD-1 mice were continuously exposed to ozone from 6 days before the formation of breeding pairs to Day 17 of pregnancy. The concentrations used were 0, 0.2, 0.4, and 0.6 ppm. Ozone failed to produce significant effects on either reproductive performance, postnatal somatic and neurobehavioral development (as assessed by a Fox test battery) or adult motor activity (including within-session habituation). In social interaction tests performed in the pre-juvenile period (23-25
days) and the juvenile period (43-45 days), social response endpoints were not modified in O3 mice, but exploration and self-grooming showed concentration dependent effects (decrease and increase, respectively). Performance at 84-98 days in an eight-arm radial maze with water reinforcement was initially impaired in mice, but the results were not entirely consistent; e.g., the data failed to show a concentration dependence of the effects.

Executive summary:

CD-1 mice were continuously exposed to ozone from 6 days before the formation of breeding pairs to day 17 of pregnancy. The concentrations used were 0, 0.2, 0.4, and 0.6 ppm. Ozone failed to produce significant effects on either reproductive performance (proportion of pregnancies, litter size, sex ratio and neonatal mortality), postnatal somatic and neurobehavioral development (as assessed by a Fox test battery) or adult motor activity (including within-session habituation). In social interaction tests performed in the pre-juvenile period (23-25 days) and the juvenile period (43-45 days), social response endpoints were not modified in O3 mice, but exploration and self-grooming showed concentration dependent effects (decrease and increase, respectively). Performance at 84-98 days in an eight-arm radial maze with water reinforcement was initially impaired in O3 mice, but the results were not entirely consistent; e.g., the data failed to show a concentration dependence of the effects. Based on the results, the NOAEC for maternal and reproductive/developmental toxicity is considered to be 0.6 ppm.