Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-guideline experimental study with clearly reported methods and results. Adequate for evaluation.

Data source

Reference
Reference Type:
publication
Title:
Gestational toluene exposure effects on spontaneous and amphetamine-induced locomotor behaviour in rats.
Author:
Bowen SE, Mohammadi MH, Batis JC and Hannigan JH
Year:
2007
Bibliographic source:
Neurotoxicol. Teratol. 29: 236-246

Materials and methods

GLP compliance:
not specified
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: technical grade
- Supplier: Fischer Scientific (T-324, Fischer Scientific Co., Fairlawn, NJ, USA)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, M1, USA
- Age at study initiation: dams - not reported - timed-pregnant
- Weight at study initiation: dams - 227g (mean, GD4)
- Housing: Individually in polypropylene cages with hardwood chip bedding
- Diet: Diet 5001 (PMI Nutrition International, Inc, Brentwood, MO) ad libitum except during exposure
- Water: drinking water ad libitum except during exposure
- Acclimation period: 4 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not reported
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod: 12hrs dark / 12hrs light (lights on at 0600hr)


IN-LIFE DATES: not reported

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Pregnant females exposed in one of 4 sealed 36 L cylindrical glass jars with Plexiglas lids and contained within a fume hood. Dams were placed individually into the chambers which had a vapour diffuser and fan in the lid. The chambers were sealed and toluene (0.0 ml for air controls, 1.36 mL for 8000 ppm, 2.05 mL for 12000 ppm) was injected into the diffuser. The injection port was sealed and toluene vapour dispersed around the chamber using the fan. Following a 15 min exposure, chambers were evacuated before continuing with the next group of dams. Within a given cohort, the air-only (0 ppm) exposures were restricted to one chamber.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Single wavelength monitoring infrared spectroscopy (Miran 1-A, Foxboro, Analytical, North Haven, CT, USA)
Duration of treatment / exposure:
Timed-pregnant Sprague Dawley rats were exposed to 0, 8000, or 12000 ppm for 15 min twice daily from gestation day 8 (GD8) through GD20
Frequency of treatment:
15 min twice daily from gestation day 8 (GD8) through GD20
Duration of test:
Experiment 1 pups tested for open field activity on PND22, PND42 or PND63
Experiment 2 pups tested on PND28
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 8000, 12000 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
Experiment 1: 0 ppm = 36 dams; 8000 ppm = 31 dams; 12000 ppm = 32 dams
Experiment 2: 0 ppm = 26 dams; 8000 ppm = 31 dams; 12000 ppm = 30 dams
Details on study design:
GENERAL
In the first experiment, separate groups of offspring were observed individually in an open-field on PND22, PND42 or PND63. In the second experiment, other offspring given identical prenatal toluene exposures were observed following a single i.p. injection of amphetamine (0, 0.56, 1.78 mg/kg) on PND28. Automated measurements of distance travelled and ambulatory time were recorded.

LOCOMOTOR ACTIVITY TESTING
Experiment 1: Open-field locomotor activity was assessed on PND22, PND42, or PND63 in one male and one female from each litter at each age. Animals were placed into automated activity monitors (Opto-Varimex-3, Columbus Instruments, Columbus, OH, USA) equipped with 3 15-by-15 infrared photo-beam arrays for 1 hr.

Experiment 2: On PND28 animals were injected i.p. with either D-amphetamine (0.56 mg/kg or 1.78 mg/kg) or vehicle (0.9% saline, 1 mL/kg) and then placed immediately into the activity monitors for 2 hr.

Testing sessions occurred between 1000 hr and 1600 hr in an isolated, test room dimly fit with a single red light and an ambient temperature of approximately 24 degrees C. No experimenters were present during testing and continuous background white noise was provided to reduce environmental disruptions during testing and while preparing the animals for testing.
Statistics:
Maternal body weight was analyzed using repeated measures ANOVA with prenatal treatment (0 ppm, 8000 ppm and 12000 ppm) and gestation day (GD4, GD8, GDI0, GD12, GD14, GD16, GD18 and GD20) as factors. Weight gain, litter size, gestation length, pup weight and litter sex-ratio were analysed by one-way ANOVAs . Tukey's B post hoc contrasts were used as appropriate.

Results and discussion

Observed effects

Comment: all study results (except maternal and litter data) presented graphically.

MATERNAL AND LITTER CHARACTERISTICS
Dams showed initial qualitative increases in activity in the chambers (locomotion and rearing) followed by concentration-dependent decreases in activity. As the number of toluene exposures increased, the dams appeared to develop a tolerance to the activating effects of toluene. Following the first day of exposure to 12000 ppm toluene, the dams were essentially immobile and appeared sedated, with post-exposure recovery taking approx. 20 min. By the final day, all dams showed rapid recovery (within around 10 min of removal from the chamber).
In experiments 1 and 2, both toluene-exposed groups gained significantly less weight than controls during gestation. There were significantly more pups born in the 12000 ppm group than the 8000 ppm group in experiment 1. Pups in the 12000 ppm group weighed significantly less than pups in the control and 8000 ppm groups on post natal day 1 in both experiment 1 and 2, with female pups weighing less than the males.

LOCOMOTOR ACTIVITY
Experiment 1 - spontaneous locomotor activity:
Distance travelled: There were no significant effects on distance travelled following prenatal toluene treatment. Significant effects were observed for sex and age with females and older animals travelling further than males or younger animals.
Ambulatory time: There were no significant effects on ambulatory time involving prenatal toluene treatment and the pattern of time spent moving generally paralleled those for distance travelled. A significant effect was observed for age with older animals being more active than younger animals.
Rearing: Rats exposed prenatally to 8000 ppm toluene had significantly fewer rears than the controls and the 12000 ppm groups.

Experiment 2 - Amphetamine challenge:
Distance travelled: A significant increase was observed for amphetamine dose at PND28. There were no significant main effects or interactions involving prenatal toluene treatment.
Ambulatory time: Animals receiving amphetamine were more active than the saline-treated group. Animals exposed prenatally to toluene were more active than controls overall. A significant amphetamine dose x pre-natal toluene interaction was also observed, which post hoc analyses indicated was due to toluene- exposed animals (8000 ppm) being more sensitive to the locomotor-stimulatory effects of the highest dose of amphetamine than other groups.
Rearing: There were no significant main effects or interactions involving pre-natal toluene treatment. A significant main effect was observed for amphetamine dose with animals given amphetamine showing more rearing activity than controls. A significant sex x amphetamine dose interaction was also observed with males displaying more rears than females at the higher dose of amphetamine.

Applicant's summary and conclusion

Conclusions:
Prenatal toluene exposure resulted in small increases in spontaneous activity compared to non-exposed rats on PND22, but not on PND42 or PND63. Prenatal exposure to 12000 ppm toluene resulted in significant hyposensitivity to the locomotor stimulatory effects of the amphetamine challenge in male but not female rats on postnatal day 28.
Executive summary:

Timed-pregnant Sprague-Dawley rats were exposed to 0, 8000 or 12000 ppm toluene vapour for 15 min twice daily from GD8 through GD20. In the first experiment, separate groups of offspring were observed individually in an "open field" on postnatal days 22, 42 or 63. In the second experiment, other offspring given identical prenatal toluene exposures were observed in an "open field" following an acute i.p. injection of amphetamine (0, 0.56, 1.78 mg/kg) on post natal day 28. Automated measurements of distance travelled and ambulatory time were recorded.

Prenatal toluene exposure resulted in small alterations in spontaneous activity compared to non-exposed rats. Prenatal exposure to 12000 ppm toluene resulted in significant hyposensitivity to the locomotor stimulatory effects of the amphetamine challenge in male but not female rats on postnatal day 28. The results demonstrate that prenatal exposure to high concentrations of toluene vapour alters spontaneous and amphetamine-induced locomotor behaviour in rats. The expression of these effects also appears to depend upon the postnatal age of the testing.