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Neurotoxicity

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Effect on neurotoxicity: via oral route

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Endpoint:
neurotoxicity, other
Remarks:
acute
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not specified
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: No guideline & No GLP, but peer reviewed. No purity reported.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The effects of a single acute exposure to the test material on spontaneous motor activity (SMA) in home cage were studied in male Wistar rats. The rats were given the test material intraperitoneally at a dosage of 0, 1.6 or 3.3 mg/kg, and the SMA was measured for five days after administration.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar Kyoto (WKY)
Sex:
male
Details on test animals and environmental conditions:
- Male Wistar rats (Std: Wistar-KY, Japan SLC, Inc., Hamamatsu) weighed 210-290 g were used throughout this study.
- Animals were housed in an air-conditioned room employing a timed light cycle of 12-hr of light per day (7:00 to 19:00 ) at 24 ± 1 °C, with a relative humidity of 55 ± 5 %.
- The rats were reared on a basal diet (F-2, Funabashi Farm Co., Inc., Funabashi) and tap water ad libitum.
Route of administration:
intraperitoneal
Vehicle:
olive oil
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Single dose.
Frequency of treatment:
Single dose.
Dose / conc.:
1.6 other: mg/kg
Dose / conc.:
3.3 other: mg/kg
No. of animals per sex per dose:
No data
Control animals:
yes
Details on study design:
The rats were distributed on a random basis to three groups consisting of eight animals, and were intraperitoneally giiven the test material dissolved in olive oil (Wako Pure Chemical Industries, LTD., Osaka) at a dosage of 0, 1.6 or 3.3 mg/kg. The volume of each dose was adjusted to 1 mL/kg of body weight. Administration of the test material was made at 16:00. Measurements of body weight and feeder and water bottle refills were made from 16:00 to 16:30 on every day. No other handling nor human activity occurred in the animal room at any other time.
The SMA of rats was measured with an MK-Animex-Auto (Muromachi Kikai Co., Ltd., Tokyo). A transparent plastic cage (31 x 36 x 17.5 cm, CL-0106, Clea Japan, Inc., Tokyo) in which a rat was housed individually was placed on the top of the MK-Animex-Auto. The MK-Animex-Auto and the plastic cage were housed inside a soundproof box (MC-50, Muromachi Kikai Co., Ltd., Tokyo) maintained at 24 ± 1 °C and timed light cycle of 12-hr of light per day (7:00 to 19:00) with a ventilating fan for attenuation of external light and sound. The SMA was continuously determined for five days after administration of the test material. The SMA of rats was determined for several days prior to the treatment of the test material, and the rats which showed stable magnitude and pattern of circadian cycle of SMA for at least two days were selected and used in the present study.
Statistics:
Statistical analysis of the data was carried out using analysis of variance with significant F values further analysed using Dunnett's multiple comparison test. The level of significance chosen was p < 0.05.
Clinical signs:
no effects observed
Description (incidence and severity):
Neither death nor clinical sign of toxicity was observed in the rats of any group.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
Neither death nor clinical sign of toxicity was observed in the rats of any group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain of rats administered the test material is presented in Figure 1. Body weight gain after administration of the test material in the 3.3 mg/kg group was lower than that in the control group and the difference was significant for rats on day 3 and day 4 after treatment. Body weight gain in the 1.6 mg/kg group was comparable to that in the control group.
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
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:
effects observed, treatment-related
Description (incidence and severity):
The circadian cycle of SMA of rats given the test material is shown in Figure 2. The SMA in the control group was almost constant and unchanged throughout the experimental period. The rats in all the groups were active during the last 3-hr epoch of the light phase (16:00-19;00). After injection of the test material, the SMA during 12-hr dark phase was decreased in a dose-dependent manner and returned to the control levels gradually in accordance with the progress of time after injection. The activity during the last 3-hr epoch of the dark phase (4:00-7:00) on day 0 and day 1 after administration of the test material in the 1.6 mg/kg group was significantly lower than that in the control group. In the 3.3 mg/kg group, the SMA during dark phase on day 0 and day 1 after the test material treatment was markedly reduced. A significant decrease in SMA was found in the second 3-hr epoch of the dark phase (22:00-1:00) by day 3 after treatment. During light phase, no significant change was detected in SMA in any 3-hr epoch except for the 16:00-19:00 epoch in the 1.6 mg/kg group and the 13:00-16:00 epoch in the 3.3 mg/kg group on day 4.
The circadian cycle of SMA is summarised in Figure 3. The histograms mean 24-hr total daily, 12-hr nocturnal and 12-hr diurnal activity. As can be seen in the total daily activity, the dose-related decrease in SMA was observed after administration in the test material-treated groups. This change was also evident in the nocturnal activity. The total daily and nocturnal activity in the test material-treated groups returned to the control levels until day 2 and day 3, respectively. No significant change was found in the diurnal activity for rats of the test material-treated groups throughout the experimental period.
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Conclusions:
The data of the present study indicate that the test material possesses behavioural toxicity and suggest that the decreased nocturnal SMA is a sensitive index for detecting toxicity of chemicals in rats.
Executive summary:

The effects of a single acute exposure to the test material on spontaneous motor activity (SMA) in home cage were studied in male Wistar rats. The rats were given the test material intraperitoneally at a dosage of 0, 1.6 or 3.3 mg/kg, and the SMA was measured for five days after administration.

Body weight gain in the 3.3 mg/kg group was significantly lowered, but that in the 1.6 mg/kg group was comparable to that in the control group. The SMA during light phase was not affected by the test material treatment, However, the SMA during dark phase was decreased in both of the test material treated groups. These decreases in SMA gradually returned to the control levels. The 24-hr total daily and 12-hr nocturnal activity in the test material treated groups were decreased in a dose-dependent manner. These data indicate that the test material possesses behavioral toxicity and suggest that the decreased nocturnal SMA is a sensitive index for detecting toxicity of chemicals in rats.

Endpoint:
neurotoxicity: acute oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not specified
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: No guideline & No GLP, but peer reviewed. No purity reported.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The behavioral effects of a single acute exposure to nonlethal doses of the test material were studied in male Wistar rats. The rats were given the test material by oral gavage at doses of 0, 6.3, 12.5, 25.0 or 50.0 mg/kg, and spontaneous motor activity (SMA) and acquisition of conditioned avoidance responses in a shuttle box were monitored.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar Kyoto (WKY)
Sex:
male
Details on test animals and environmental conditions:
- Male Wistar rats (Std: Wistar-KY, Japan SLC, Inc., Hamamatsu) weighing 197-281 g at the start of the experiment were used throughout this study.
- Animals were housed in groups in an air-conditioned room under a timed light cycle of 12 h of light per day (0700 to 1900) at 24 ± 1°C, with a relative humidity of 55 ± 5 %.
- The rats were given diet F-2 (Funabashi Farm Co., Inc., Funabashi) and tap water ad lib.
Route of administration:
oral: gavage
Vehicle:
olive oil
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Single oral dose.
Frequency of treatment:
Once
Dose / conc.:
6.3 mg/kg bw/day (actual dose received)
Dose / conc.:
12.5 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
No data
Control animals:
yes
Details on study design:
The rats were distributed on a random basis to five groups and given the test material dissolved in olive oil (Wako Pure Chemical Industries, Ltd., Osaka) by oral gavage at doses of 0, 6.3, 12.5, 25.0 or 50.0 mg/ kg. The volume of each dose was adjusted to 10 mL/kg of body weight.

Neurobehavioural examinations performed and frequency:
Spontaneous Motor Activity (SMA):
For determination of spontaneous motor activity (SMA), the rats were given the test material at 1600 to 1630. The SMA of rats was measured with an MK-Animex-Auto (Muromachi Kikai Co., Ltd., Tokyo). Rats were housed individually in transparent plastic cages (31 x 36 x 17.5 cm, CL-0106, Clea Japan, Inc., Tokyo), which were placed on the top of the MK-Animex-Auto. The MK-Animex-Auto and the plastic cage were housed inside a soundproof box (MC-50, Muromachi Kikai Co., Ltd., Tokyo) under a timed light cycle of 12 h of light per day (0700 to 1900) with a ventilating fan for attenuation of external sound. The SMA was continuously determined for five days after treatment with the test material. Rat SMA was monitored for several days prior to treatment with the test material, and only those rats that exhibited a stable circadian pattern and magnitude in SMA for at least two days were used in the present study (defined as 26000-51000 counts in 24 h, 24000-47000 counts in 12 h, and 4200 counts in each 3-h period during the dark phase). Measurements of body weight were made from 1600 to 1630 on each day; food and water were replaced at the same time. No human activity occurred in the animal room at any other time.

Conditioned Avoidance Responding (CAR):
For determination of learning ability, the rats received the test material 1 h prior to the initiation of shock avoidance responses. A shuttle box (RSC-001, Muromachi Kikai Co., Ltd., Tokyo), which consisted of transparent acrylic plastic panels, was used as the shock avoidance apparatus. The shuttle box was 46 cm in length, 20 cm in height and 19.5 cm in width: It was divided into two equal compartments by an aluminum plate partition with an arch-shaped opening (7.5 cm in width and 12 cm in height). The grid floor of each compartment consisted of 0.3-cm stainless steel rods spaced 1.1 cm center to center. An electric shock was delivered through the grid floor in the occupied compartment from a shock generator/scrambler (SGS-001, Muromachi Kikai Co., Ltd., Tokyo). Each end of the shuttle box wall contained a small 24-V DC cue lamp (1.27 cm in diameter) centered 14 cm from the grid floor. A Sonalert (3.5 cm in diameter) was placed outside and above the centre of the ceiling for delivery of a 2.8-KHz, 95-dB warning tone. The shuttle box was housed inside a soundproof box (MC-050, Muromachi Kikai Co., Ltd., Tokyo) with a ventilating fan for attenuation of external sound. On the first day of conditioned avoidance training, each subject was given five minutes for adaptation to the shuttle box after its introduction into one compartment. A trial began with the simultaneous presentation of the Sonalert tone and illumination of the cue lamp (conditioned stimuli, CS) in the compartment occupied by the rat. The CS preceded by five seconds the onset of electric shock (0.4 mA) to the floor as the unconditioned stimulus (UCS) and continued with the shock for five seconds. Thus the tone and light were presented for ten seconds (the last five seconds, together with the UCS). A conditioned response was recorded when the rat avoided the electric shock by crossing to the opposite side of the shuttle box within five seconds of CS onset, and the tone and light were immediately terminated. If a rat failed to avoid the electric shock, it could escape the shock by crossing to the other chamber during the five seconds of UCS. The tone, light and shock were then immediately terminated. Failure to either avoid or escape shock was designated as an error. A 30-second intertrial interval preceded the next presentation of the CS. Each rat was given 50 consecutive trials a day for four consecutive days.
Statistics:
Analysis of variance (ANOVA) was used to determine whether significant dose effects had occurred. When ANOVA revealed significant differences between groups, Dunnett's multiple comparison test was used to compare the test material treated groups to the control group. The level of significance chosen was p<0.05.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were found in any group.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain following administration of the test material is presented in Fig. 1. Body weight gain in the 50.0-mg/kg group was significantly lower than in the control group on days 2 through 7 after administration. By day 8, the body weight gain for this group returned to control levels. Body weight gains in the 6.3-, 12.5- and 25.0-mg/kg groups were comparable to that in the control group.
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
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
effects observed, treatment-related
Description (incidence and severity):
The circadian cycle of SMA of rats administered the test material is shown in Fig. 2. In all groups, the SMA during the 12-h dark phase was markedly higher than that during the 12-h light phase. After exposure to the test material, the SMA during the 12-h dark phase was decreased in a dose-dependent manner and returned to control levels within about four days after administration. The SMA during the dark phase on day 0 was strikingly lowered in all the test material-treated groups. The activity in the dark phase in the 50.0-mg/kg group was markedly reduced during the first three days after treatment and was nearly back to control levels on day 4. In the 25.0-mg/kg group, SMA during the dark phase was significantly decreased for the first two days following administration. A significant decrease in SMA was found in the last 3-h epoch of the dark phase (0400-0700) on day 0 for the 6.3-mg/kg group, on day 0 and day 1 for the 12.5- and 25.0-mg/kg groups, and throughout the experimental period for the 50.0-mg/kg group. Generally, rats in all groups were active during the last 3-h epoch of the light phase (1600-1900). Significantly higher levels of light-phase SMA occurred in several of the test material-treated groups (especially in the 50.0-mg/kg group), but these effects did not appear to be dose dependent.

SMA data are summarised in Fig. 3. The histograms represent 24-h total daily activity and 12-h nocturnal and 12-h diurnal activity. As can be seen in the total daily activity data, the test material-induced decrease in SMA was dose dependent. This effect was even more evident in the nocturnal activity data. Significant decreases in nocturnal activity were observed on day 0 for the 12.5-mg/kg group, on day 0 and day 1 for the 25.0 -mg/kg group, and on day 0, day 1 and day 2 for the 50.0-mg/kg group. Total daily and nocturnal activity in the test material-treated groups returned to control levels on day 2 and 3, respectively. Diurnal activity was higher for the 50.0-mg/kg group than that for the control group from day ;1 to day 4.

The effects of the test material on avoidance responses are shown in Fig. 4. A general increase in the number of avoidance responses was observed as training proceeded in all groups. However, the increase in the number of avoidance responses made by all the test material-treated groups was smaller than for the control group. The acquisition of avoidance responses in the test material-treated groups was inhibited in a dose-dependent manner. The increase in the number of avoidance responses in the 25.0- and 50.0 -mg/kg groups was significantly smaller than that for the control group.
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Conclusions:
This report demonstrates that a single acute exposure to non-lethal doses of the test material can cause significant changes in rat behavior for at least four days, as evidenced by decreased SMA and avoidance responding, and also demonstrates that a comparison of three indexes indicates the relative order of sensitivity to the test material as SMA, CAR, and body weight gain. With respect to effects on SMA, oral adminstration of the test material caused a decrease in SMA in a dose-dependent manner.
SMA was more sensitive to the test material than body weight gain. Even a dose of 6.3 mg/kg caused a significant decrease in SMA during the last 3-h epoch of the dark phase on day 0. Thus the circadian pattern of SMA in rats is sensitive to single oral test material exposures at doses as low as 6.3 mg/kg.
The continuous measurement of SMA in rats appears useful for the evaluation of the toxic potential of some chemicals, because the disruptive effects of handling and testing procedures can be avoided or decreased. Although efforts were made to minimize the effects of handling in the present experiment, a handling effect was observed. The rats were more active during the last 3-h epoch of the light phase, because daily handling occurred during this time.
CAR was more sensitive to the test material than body weight gain. The test material-induced deficits noted in CAR are not thought to be due to any test material-induced hypoactivity, because the behavioural deficits noted on day 0 in the two high-dose groups were accompanied by hyperactivity as shown in SMA. Thus it is possible that the test material exposure, at doses above 25.0 mg/kg, affects the acquisition of CAR in rats.
Decreases in body weight gain are one of the most easily detectable indicators of toxicity. Behavioral changes that occur concurrently with decreases in body weight gain may be of questionable neurotoxic significance since they may occur secondary to physiological dysfunction. In the present study, certain doses of the test material caused significant changes in SMA and impaired CAR at doses that did not affect body weight gain. These findings suggest that the test material may be producing neurological dysfunction via relatively direct mechanisms.
It is concluded that the test material possesses behavioral toxicity and that SMA can serve as a very sensitive index for detecting test material effects.
Executive summary:

The behavioral effects of a single acute exposure to nonlethal doses of the test material were studied in male Wistar rats. The rats were given the test material by oral gavage at doses of 0, 6.3, 12.5, 25.0 or 50.0 mg/kg, and spontaneous motor activity (SMA) and acquisition of conditioned avoidance responses in a shuttle box were monitored. Body weight gain in the 50.0-mg/kg group was significantly lowered, but weight gain in the 6.3-, 12.5- and 25.0-mg/kg groups was comparable to that in the control group. The test material caused a dose-related decrease in SMA during the dark phase. The 24 h total daily and 12 h nocturnal activity was decreased at doses of 12.5 mg/kg and above. The acquisition of shock avoidance responses was inhibited in all test material-treated groups in a dose-dependent manner, and the difference was significant for rats given the test material at doses of 25.0 mg/kg and above. The data indicate that an acute exposure to the test material can cause significant changes in rat behaviour and suggest that SMA can serve as a sensitive index for detecting its toxicity.

Effect on neurotoxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Both studies were performed by the same author and published in the same year. It should be noted that a dose-related decrease in nocturnal activity was noted in both studies, despite the difference in route of exposure, and that both of the studies noted that the nocturnal activity of the animals was found to recover to the level of the controls after a period of time.

In the Ema et al, 1991 (001) paper, the effects of a single, intraperitoneal acute exposure of the test material on spontaneous motor activity (SMA) in home cage was studied in male Wistar rats.

Body weight gain in the 3.3 mg/kg group was significantly lowered, but that in the 1.6 mg/kg group was comparable to that in the control group. The SMA during light phase was not affected by the test material treatment, However, the SMA during dark phase was decreased in both of the the test material-treated groups. These decreases in SMA gradually returned to the control levels. The 24-hr total daily and 12-hr nocturnal activity in the the test material-treated groups were decreased in a dose-dependent manner. These data indicate that the test material possesses behavioural toxicity and suggest that the decreased nocturnal SMA is a sensitive index for detecting toxicity of chemicals in rats.

A reliability rating of 4 was assigned to this study, according to the criteria of Klimisch, 1997 as the method does not follow recognised guidelines and there is no GLP or purity information presented.

 

In the Ema et al, 1991 (002) paper, male Wistar rats dosed with a single non-lethal dose of

the test material by oral gavage were studied for behavioural effects (spontaneous motor activity (SMA) and acquisition of conditioned avoidance responses in a shuttle box). Body weight gain in the 50.0-mg/kg group was significantly lowered, but weight gain in the 6.3-, 12.5- and 25.0-mg/kg groups was comparable to that in the control group.The test material caused a dose-related decrease in SMA during the dark phase. The 24-h total daily and 12-h nocturnal activity was decreased at doses of 12.5 mg/kg and above. The acquisition of shock avoidance responses was inhibited in all the test material-treated groups in a dose-dependent manner, and the difference was significant for rats given the test material at doses of 25.0 mg/kg and above. The data indicate that an acute exposure to the test material can cause significant changes in rat behaviour and suggest that SMA can serve as a sensitive index for detecting its toxicity.

A reliability rating of 4 was assigned to this study, according to the criteria of Klimisch, 1997 as the method does not follow recognised guidelines and there is no GLP or purity information presented.

Justification for classification or non-classification