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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Entry adopted from the OECD SIAR on sulfur dioxide without modification.Study meets generally accepted scientific principles, sufficiently documented, specific investigation of behavioural effects in parent generation and of somatic and neurobehavioral development in F1 generation; investigation of dose-dependency for 3 test concentrations; acceptable for assessment.

Data source

Reference
Reference Type:
publication
Title:
Behavioural disturbance in adult CD-1 mice and absence of effects of their offspring upon SO2 exposure.
Author:
Petruzzi, S. et al.
Year:
1996
Bibliographic source:
Arch. Toxicol. 70: 757-766

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Directive 86/509/EEC, specific investigation of fetotoxictiy and behavioral effects in parent generation and of somatic and neurobehavioural development in F1 generation
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
not specified

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Calco, Italy
- Weight at study initiation: (P) Males and Females: 25-27 g
- Housing: Upon arrival at the laboratory, the animals were housed in same sex pairs 33 x 13 x 14 cm Plexiglas boxes with a metal top and sawdust as bedding.
- Diet: pellet food, ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +/- 1°C
- Humidity: 60 +/- 10%
- Photoperiod: Lighting followed a 12L:12D reversed photoperiod.

No further details are given.

Administration / exposure

Route of administration:
inhalation
Vehicle:
other: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The apparatus (custom-made by A. & L. Industries) induced a control system and four steinless steel exposure chambers (52 x 135 x 113 cm) with a hatch glass in the front door. The chambers were equipped with racks which could hold up to 24 housing boxes.
- Method of holding animals in test chamber:
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols: SO2 was delivered from 40-l aluminium bottles. Different SO2 concentrations were obtained by varying the flow and gas pressure from the bottles.
- Temperature, humidity, pressure in air chamber:
- Air flow rate:
- Air change rate:
- Method of particle size determination:
- Treatment of exhaust air: The sulphurised air passed through appropriate filters before being discharged into the outdoor environment.


TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: yes/no


VEHICLE (if applicable)
- Justification for use and choice of vehicle:
- Composition of vehicle:
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle:
- Lot/batch no. of vehicle (if required):
- Purity of vehicle:
Details on mating procedure:
Ten breeding pairs were formed in each chamber on ED 9. Females were inspected daily for the presence of a vaginal plug and for delivery, the males being removed on pregnancy days 12. At birth, all litters were culled to four males and four females and fostered to untreated dams, which had given births to healthy litters within the previous 24 hours.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sulphur dioxide was measured using an ultraviolet SO2 analyser; periodic calibration of the analyser was performed using permeation tubes which produced known SO2 concentrations.
Duration of treatment / exposure:
24 days
Frequency of treatment:
nearly continuously (80 % of total time)
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
5 ppm (nominal)
Dose / conc.:
12 ppm (nominal)
Dose / conc.:
30 ppm (nominal)
No. of animals per sex per dose:
10 males and 10 females/test group
Control animals:
yes
Details on study design:
Exposure to SO2 was started on exposure day 0 (ED 0), 9 days before the formation of breeding pairs and went on until ED 24; i.e. until pregnancy day 12-14. Exposure was near-continuous, covering 80% of the indicated time.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
One week after arrival at the laboratory, 10 mice were assigned to each of eight combinations of sex and exposure conditions.
Video recording was performed on two pairs of females and two pairs of males chosen in each of the 12 ppm and 30 ppm groups. Animals were scored according to the following response end-points: bar-holding, feeding, self-grooming, digging, sniffing, social interaction and inactivity.
Behavioural observations in parent generation outside the exposure chambers: all exposed mice underwent 5-minute activity tests at 72-hour intervals, i.e. in the morning of exposure days 3, 6 and 9 when placed individually in a clean cage; end-points evaluated from video recording: crossing, rearing, digging, wall-rearing, self-grooming and inactivity

BODY WEIGHT: Yes
Body weight were monitored during exposure.

FOOD AND WATER CONSUMPTION AND COMPOUND INTAKE: Yes
Food intake were monitored during exposure. The former were determined by weighing food containers and water bottles before and after refilling. Measurements were taken at 72-hour intervals until male and female pairing on ED 9 and subsequently at 96-hour intervals until ED 21.

OTHER:
- Fertility index: number of females confirmed mated vs. number of females paired
- Gestational index: number of females with successful pregnancies vs. number of females pregnant
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
no data
Litter observations:
EXAMINATION OF THE FETUSES: litter size, birth weight, sex and sex ratio, viability index, postnatal growth, growth rate
PARAMETERS ASSESSED DURING STUDY in F1: At birth all litters culled to 4 males and 4 females and fostered to untreated dams, which had given birth to healthy litters within the previous 24 hours, in total 8 litters/test concentration.
- Clinical observations and frequency (N=64/concentration): daily from post-natal day 2 to 10 and on alternate days from post-natal day 10 to 16 or 18 body weight, body length and tail length measured in 2 males and 2 females/litter
- Somatic and neurobehavioral development (N=64/concentration): recording of day of eyelid and ear opening and of incisor eruption daily from post-natal day 2 to 10 and on alternate days from post-natal day 10 to 16 or 18 testing according to slightly modified Fox battery scoring the following reflexes and responses: righting reflex, cliff aversion, forelimb and hindlimb stick grasp reflexes, forelimb and hindlimb placing reflexes, vertical screen grasping response, weak tactile stimulation response
- Passive avoidance learning and retention: 2 male mice from each litter
Postmortem examinations (parental animals):
no data
Statistics:
Analysis of variance, Tkey's honestly significant difference (HSD) test, chi square test, Fisher's exact probability test with Bonferroni's correction.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
not specified

Details on results (P0)

PARENTAL TOXIC EFFECTS BY DOSE LEVEL:
- Body weight: no effect until mating, from exposure day 13 (ca. GD 4) significant decrease in females of 30 ppm group, p<0.05
- Food consumption: no effect until mating, from exposure day 13 (ca. GD 4) significant concentration-dependent depression for 12-ppm and 30-ppm groups, p<0.05, less marked effect on exposure days 13-17
- Water consumption: significantly depressed on exposure days 9-13 for 12-ppm and 30-ppm groups, p<0.05
- Fertility index: 100 % mated in all groups
- Gestational index: 100 % successful pregnancies in all groups
- Behavioural observations - Activity tests inside the exposure chambers (only 12 and 30 ppm groups, single testing due to continuous exposure):
significant concentration-dependent increase in the frequency of rearing, sniffing and social interactions shortly after the start of sulphur dioxde exposure, followed by a return to pre-exposure baseline values within 30-60 min; significantly higher effects in males than in females
- Behavioural observations - Activity test outside the exposure chambers (all animals, on exposure days 3, 6 and 9): more marked effects in females than in males; dose-dependent decrease of grooming and increase of digging, changes significant on exposure day 9 at 30 ppm in comparison to control, p<0.05; changes in chamber crossing and wall-rearing in females at 30 ppm without dose-dependency as there was a decreasing tendency for the lower test concentrations in comparison to the control

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
30 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Fetotoxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified

Details on results (F1)

EXAMINATION OF FETUSES AND POSTNATAL DEVELOPMENT:
No changes of fetotoxicity endpoints, of postnatal somatic and neurobehavioral development as well as of passive avoidance learning and retention.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: for somatic and neurobehavioral development in F1 generation

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

LOAEL (LOEL): maternal toxicity based on reduced body weight gain and food intake
PARENTAL TOXIC EFFECTS BY DOSE LEVEL:
- Body weight: no effect until mating, from exposure day 13 (ca. GD 4) significant decrease in females of 30 ppm group, p<0.05
- Food consumption: no effect until mating, from exposure day 13 (ca. GD 4) significant concentration-dependent depression for 12-ppm and 30-ppm groups, p<0.05, less marked effect on exposure days 13-17
- Water consumption: significantly depressed on exposure days 9-13 for 12-ppm and 30-ppm groups, p<0.05
- Fertility index: 100 % mated in all groups
- Gestational index: 100 % successful pregnancies in all groups

- Behavioral observations - Activity tests inside the exposure chambers (only 12 and 30 ppm groups, single testing due to continuous exposure): significant concentration-dependent increase in the frequency of rearing, sniffing and social interactions shortly after the start of sulfur dioxde exposure, followed by a return to pre-exposure baseline values within 30-60 min; significantly higher effects in males than in females
- Behavioral observations - Activity test outside the exposure chambers (all animals, on exposure days 3, 6 and 9): more marked effects in females than in males; dose-dependent decrease of grooming and increase of digging, changes significant on exposure day 9 at 30 ppm in comparison to control, p<0.05; changes in chamber crossing and wall-rearing in females at 30 ppm without dose-dependency as there was a decreasing tendency for the lower test concentrations in comparison to the control
EXAMINATION OF FETUSES AND POSTNATAL DEVELOPMENT:
No changes of fetotoxicity endpoints, of postnatal somatic and neurobehavioral development as well as of passive avoidance learning and retention

Applicant's summary and conclusion

Conclusions:
The absence of effects of sulfur dioxide exposure (5-30 ppm prior to mating and during pregnancy) on reproductive performance of mice as well as the absence of changes of somatic and neurobehavioral development of the offspring suggested that the risk to the developing organism from gestational sulfur dioxide exposure is low. In the parent mice marked, acute transient behavioral effects were reported when the continuous exposure started being more pronounced in males than in females. Subsequent acitvity tests on exposure days 3, 6 and 9 showed subacute response changes which were either stable over successive exposure days or showed a tendency to increase with time of exposure. These subacute effects were more pronounced in females than in males. The behavioral effects in adult mice were considered to be due, at least partly, to a functional interference with olfactory modulation of mouse behavior. Interferences of the higher sulfur dioxide concentrations with the olfactory system were also suggested to play a role for reduced food intake and decreased body weight gain. Due to the probability that olfactory mechanisms are involved in the changes in the adult mice the relevance to humans of the mouse data is questionable.