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Toxicological information

Neurotoxicity

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Description of key information

OECD 424, (90 -day repeated exposure): NOAEL >= 1200ppm, no effects up to the highest dose tested (Gill, 2000, GLP)

single inhalation: NOAEL >= 3000ppm, no effects up to the highest dose tested (UCC, 2001, GLP)

Key value for chemical safety assessment

Effect on neurotoxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
neurotoxicity: sub-chronic inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 August 1996 - 29 November 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 424 (Neurotoxicity Study in Rodents)
Principles of method if other than guideline:
The test conducted was in compliance with guidelines set forth by the United States Department of Agriculture and the Canadian Council on Animal Care for the human care and use of laboratory animals, and was designed to meet or exceed US EPA Testing Guidelines requirements.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain as cited in the study report: Sprague-Dawley rats, Crl:CD (SD)BR
- Source: Charles River Canada Inc. (St. Constant, Quebec)
- Age at study initiation: 56-57 days
- Weight at study initiation: 291 to 350 g (males), 184 to 228 g (females)
- Housing: individually in stainless-steel mesh-bottomed cages equipped with an automatic watering valve
- Diet: standard certified commercial pelleted laboratory diet (PMI Certified Rodent Chow 5002), ad libitum, except during inhalation exposure and neurobehavioral observations
- Water: water treated by reverse osmosis and ultraviolet sterilization, ad libitum
- Acclimation period: approx. 3.5 weeks were allowed between receipt of the animals and the start of treatment to accustom the animals to the laboratory environment. Approximately 2 weeks were allowed between receipt of the animals and the conduct of the prestudy behavioral evaluations

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The test atmosphere was generated by a heated flow-through vapour generator.
Stainless-steel and glass whole-body inhalation chambers (400-l volume) were utilized in this experiment. While in the chamber, the rats were housed in stainless-steel wire-mesh-compartmentalized cages, with each compartment being ca. 7 inches x 3 inches x 3 inches. Each inhalation chamber had a minimum of 12 air changes per hour and was maintained at 20–24°C, 30–70% relative humidity and at least 19% O2.

TEST ATMOSPHERE
Chamber airflow was monitored continuously in the exhaust line by means of a precalibrated Magnehelic gauge. Inhalation chamber temperature and relative humidity were recorded hourly during each exposure. The oxygen concentration was recorded once during animal exposure on day 1 for
all groups.



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Each hour during daily exposure, inhalation chamber airflow was recorded and samples of the test atmosphere were collected from the animal breathing zone and analyzed for test substance concentration (Miran 1A infrared gas analysis). In addition, a sample of each test atmosphere was collected on at least one occasion (treatment day) each week during the 13 weeks of exposure and analyzed by high-resolution gas chromatography. Nominal chamber concentrations were calculated by dividing the weight of the test substance used during the vapor generation period by the total airflow through the chamber during the vapor generation period.
Duration of treatment / exposure:
6 hrs/day
Frequency of treatment:
5 days/week for 13 weeks, except during weeks 4, 8 and 13 when animals were exposed for 4 days to accommodate the schedule for behavioral evaluations
Remarks:
Doses / Concentrations:
300, 600 and 1200 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
15 rats/sex for control and high dose groups (5 animals each for a possibe post exposure recovery period)
10 rats/sex for low and intermediate dose groups
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: The maximum exposure concentration of 1200 ppm was selected based on the results of previous studies and was expected to produce clear acute effects on the nervous system during exposure, such as sedation or decreased activity. Additionally, the high exposure concentration exceeds the limit exposure concentration for EPA TSCA subchronic inhalation toxicity studies and is equivalent to a dose of 1.5 g/kg/day which exceeds the limit dose for the subchronic inhalation studies included in the EPA TSCA Multisubstance Test Rule for Neurotoxicity. The low and intermediate concentrations were expected to produce no effect or an intermediate degree of effect.

Observations and clinical examinations performed and frequency:
CLINICAL EXAMINATIONS
All animals were examined twice daily for mortality and clinical symptoms; they also were examined before, during and after exposure for reaction to treatment. In addition, a complete detailed examination was performed once weekly. During exposure, clinical observations were recorded on a
group basis, rather than on an individual animal basis, because all of the animals could not be seen clearly from the outside of the inhalation chamber.

BODY WEIGHT
The animals were weighed at least three times during the acclimation period, weekly during the study including days of behavioral testing, and at test ending, prior to sacrifice.

FOOD CONSUMPTION
Individual food consumption was measured weekly, starting two weeks prior to treatment initiation, and extending throughout the treatment period.
Neurobehavioural examinations performed and frequency:
FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
Behavioral function was evaluated prestudy and once each during the 4, 8 and 13 weeks of treatment using a FOB. The FOB testing was performed ca. 30 min after motor activity measurements.

- Observations in the home cage included body position, tremors, twitches, convulsions and bizarre or stereotypic behavior.
- When the animals were removed from the home cage, observations for ease of removal and vocalization were recorded.
- For open field observations, the open field arena consisted of a ca. 60 cm square of Plexiglas raised on a platform. In the arena, animals were observed for rearing, ataxic, hypotonic and impaired gait, overall gait incapacity, bizarre or stereotypic behavior, palpebral closure, tremors, twitches or convulsions, piloerection, respiratory rate/pattern, locomotor activity level, arousal, grooming, defecation, urinatio, and olfactory response.
- Handling observations included lacrimation, pupil size, salivation, urinary staining, diarrhea, body tone, extensor thrust, corneal reflex, pinna reflex, toe and tail pinch, visual placing, positional passivity, auricular startle and air righting reflex.
- Forelimb and hindlimb grip strength were measured using mechanical strain gauges. The mean of 2 trials was used as the forelimb and hindlimb grip strength value for each animal for statistics. Hindlimb splay was recorded twice for each animal and the mean of the two trials was used for statistical analyses.
- Body temperatures also were measured.

LOCOMOTOR ACTIVITY
Motor activity was evaluated for individual animals in figure-of-eight enclosures (San Diego Instruments, San Diego, CA). Motor activity testing was performed in a separate testing room prior to exposure and once during each of the 4, 8 and 13 weeks of the study. The testing room was maintained at a white noise level of 70 decibels during motor activity testing. The sessions were of 1-h duration and activity counts were recorded by a microcomputer in six successive 10-minutes intervals. The motor activity test sessions were performed at least 16 h after the previous inhalation exposure.
Sacrifice and (histo)pathology:
All animals were fasted overnight prior to scheduled sacrifice.

GROSS PATHOLOGY AND GENERAL HISTOPATHOLOGY
At study completion five animals per gender and group received a gross examination of the respiratory tract and the following tissues were collected and retained in neutral buffered 10% formalin for possible future histopathological examination: bronchi, larynx, two lobes of the lungs, bronchial lymph node, nasal cavity, pharynx and trachea.

NEUROPATHOLOGY
The tissues from 5 animals per gender from the control and high-dose groups were processed for in situ perfusion and neuropathological evaluation. Tissues were harvested and fixed with a mixture of 3% glutaraldehyde, 3% paraformaldehyde, 0.05% calcium chloride and 0.1% picric acid in 0.1 M cacodylate buffer (pH 7.3–7.5). The following adjacent sections of brain and spinal cord were stained with hematoxylin and eosin (H&E) and then examined by mean of light microscopy: forebrain; center of cerebrum; midbrain; cerebellum and pons; midcerebellum; medulla oblongata; cervical spinal cord; lumbar spinal cord; and gross lesions.

The tissues listed below, from animals in the control and high-dose groups, were rinsed in 0.1 M sodium cacodylate buffer and placed in 2% osmium tetroxide for 2 h. Tissues were rinsed in buffer, stained in a 1% aqueous solution of uranyl acetate for 2 h, rinsed in distilled water, dehydrated in ascending concentrations of ethyl alcohol and embedded in a mixture of Jembed and Araldite. Epoxy sections were stained with borate-buffered 1% toluidine blue and examined by means of light microscopy. From the peripheral nervous system (PNS), cross-sections were taken of the sciatic (mid-thigh and sciatic notch), sural and tibial nerves. From the central nervous system (CNS), cross-sections were taken of the left Gasserian ganglion, lumbar dorsal root and ganglion, lumbar ventral root, cervical dorsal root and ganglion and cervical ventral root. Microscopic examination of the prepared nervous system tissues was performed for all animals in the control and high-dose groups.
Statistics:
Group variances for body weight, food consumption, FOB (including count data, which were transformed before analysis) and brain measurement data were compared using Bartlett’s test. When the differences between group variances were not significant, a one-way analysis of variance (ANOVA) was performed. If significant (P<0.05) differences were indicated by the ANOVA, Dunnett’s test was used to compare the control and treated groups for significant differences. When the differences between group variances were significant (P< 0.001) by Bartlett’s test, the Kruskal–Wallis test was then performed.
Where significant (P<0.05) differences between groups were indicated by the Kruskal–Wallis test, the control and treated groups were compared using Dunn’s
or Wilcoxon’s test.
Qualitative FOB data were statistically evaluated by comparing the control group to the treated groups using a Fisher’s exact probability test. If a significant (P<0.05) difference was noticed, then comparisons were made using all scores within a category between the control group and each of the treated groups.
Motor activity data were evaluated using a repeated measures analysis. The repeated-measures values were collapsed into two parameters: total counts, and a linear constructed variable (LCV) that evaluated the rate of linear change within the test session. Group variances for total counts and the LCV were then compared using Bartlett’s test. The differences between group variances were not significant using Bartlett’s tests, therefore an ANOVA for the pre-study data and an analysis of covariance (ANCOVA) with the pre-study data as the covariate for weeks 4, 8 and 13 were performed using the GLM procedure of the SAS statistical package (version 6.08). If significant (P<0.05) differences were indicated by the ANOVA/ANCOVA, a t-test was used to compare the control and treated groups.
Details on results:
CLINICAL SIGNS AND MORTALITY
No animals died on study and no animals were sacrificed prior to the end of the study. No signs of mucous membrane irritation during exposure and no clinical signs of toxicity were observed during weekly detailed clinical examinations. During the first 2 weeks of the treatment period, animals in the 600 and 1200 ppm treatment groups showed a general reduction in activity while they were in the inhalation chambers during exposure, compared to the activity levels of animals in the control and 300 ppm groups. This tendency for decreased activity was not observed immediately after exposure.

BODY WEIGHT AND FOOD CONSUMPTION
There were no changes in body weight or food consumption.

NEUROBEHAVIOUR
There were no treatment-related changes in the automated measure of motor activity. Motor activity tended to be decreased for females in the 600 ppm group throughout the study, including during the pretreatment assessment, and the differences between groups were not attributed to exposure with amyl acetate. The only statistically significant change in motor activity noted during the treatment phase of the study was an increase in total counts for 300 ppm males during the week 8 assessment. This statistical difference was not attributed to treatment with the test material because the change in motor activity was not dose related and no differences were noted between the control and 300 ppm males on the previous or subsequent testing occasions.
There were no clear treatment-related differences observed for qualitative or quantitative FOB evaluations. At week 4, there was an increase in the incidence of males in the 1200 ppm group with decreased locomotor activity (subjective measure) during the 2-min open field observation period. The toxicological significance of this finding and the relationship of this finding to treatment with the test material are questionable because of the lack of corroborative effects on arousal, the lack of a similar finding at the week 8 and week 13 assessments, the subjective nature of the measure and the lack of a similar effect on the automated measure of motor activity performed immediately prior to the FOB examination.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of the central and peripheral nervous system did not reveal any treatment-related alterations or pathology. There were no changes in either brain weight or length for male or female rats following 13 weeks of exposure to amyl acetate. There was a small, albeit statistically significant (P<0.05), decrease in brain width for female rats in the 600 ppm group (2.9% decrease compared to control) that was not attributed to treatment or considered toxicologically significant based on the small magnitude of the change, the lack of a dose response and the lack of corroborative histopathological findings. No changes in male brain width were observed in the study.
Dose descriptor:
NOEL
Effect level:
1 200 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment related adverse effects were observed at the highest concentration tested (1200ppm corresponding to 6.4 mg/L air)

Table1: Subjective assessment of activity level during FOB for male rats (activity assessed for individual animals during a 2 -min period)

 Week  Level of locomotor activity  Incidence of animals         
     control  300 ppm 600 ppm  1200 ppm
 Prestudy  Moderate activity  15/15  10/10  10/10  15/15
 Week4  Low activity  0/15  2/10  0/10  5/15*
   Moderate activity  15/15  8/10  10/10  10/15*
 Week8  Low activity  3/15  3/10  1/10  4/15
   Moderate activity  12/15  7/10  9/10  11/15
 Week13  No activity  0/15  1/10  0/10  0/15
   Very low activity  0/15  1/10  0/10  1/15
   Low activity  8/15  4/10  3/10  5/15
   Moderate activity  7/15  4/10  7/10  9/15

* p<0.05 (Fisher's Test) compared with the control group

Table2: Brain weight, length and width measurements in rats

   Amyl Acetate concentration         
   Control  300 ppm  600 ppm  1200 ppm
 Males        
 Body weight (g)  518.0 ± 37.6  519.3 ± 30.5  541.5 ± 49.7  540.2 ± 39.6
 Brain weight (g)  1.99 ± 0.25  1.95 ± 0.13  1.93 ± 0.17  1.94 ± 0.08
 Brain/body weight (%)  0.38 ± 0.05  0.38 ± 0.04  0.36 ± 0.07  0.36 ± 0.03
 Brain length (mm)  25.43 ± 1.19  24.99 ± 0.48  24.74 ± 0.60  24.90 ± 0.76
 Brain width (mm)  15.17 ± 0.33  15.68 ± 0.39  15.60 ± 0.32  15.64 ± 0.32
 Females        
 Body weight (g)  292.4 ± 28.2  276.8 ± 10.4  289.5 ± 19.3  276.9 ± 12.6
 Brain weight (g)  1.85 ± 0.11  1.86 ± 0.10  1.76 ± 0.10  1.75 ± 0.10
 Brain/body weight (%)  0.63 ± 0.06  0.67 ± 0.04  0.59 ± 0.06  0.63 ± 0.02
 Brain length (mm)  23.91 ± 0.64  23.98 ± 0.67  23.77 ± 0.60  23.25 ± 0.64
 Brain width (mm)  15.32 ± 0.24  15.22 ± 0.21  14.87 ± 0.27*  15.40 ± 0.24

Data expressed as mean ± SD for groups of 5 rats per gender selected for neuropathological evaluation.

* p<0.05 (Dunnett's test) compared with the control group

Conclusions:
The no-observed-effect level (NOEL) for subchronic neurotoxicity in this study was at least 1200 ppm, based on the lack of evidence of persistent or delayed effects on nervous system function or structure following repeated exposure.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
6 380 mg/m³
Study duration:
subchronic
Species:
rat

Additional information

Repeated exposure

To evaluate the neurotoxicity potential of the test substance (Gill et al., 2000), 4 groups of male and female Sprague-Dawley rats were exposed to test substance vapor by inhalation (whole-body) at target concentrations of 0 (air control), 300, 600 or 1200 ppm (15 rats per sex in the control and 1200 ppm groups, 10 rats per sex in the 300 and 600, and 5 rats per sex in the control and 1200 ppm groups to assess recovery) for at least 13 consecutive weeks, 6 hours per day, 5 days per week (with the exception of weeks 4, 8 and 13 when animals were exposed for 4 days to accommodate the schedule for behavioral evaluations; the animals received at least 65 exposures), in a test protocol equivalent or similar to the OECD Guideline 424 (Neurotoxicity Study in Rodents). During each exposure, the animals were observed for overt signs of reaction to treatment. In addition, animals were examined individually before and after each exposure and a complete detailed clinical examination was performed on each animal once weekly. Body weights and food consumption were measured weekly. Individual animals were examined for possible changes in behavior using automated motor activity measurements and a functional observational battery (FOB) pre-study and once during each of weeks 4, 8 and 13. Following completion of treatment, 5 rats per sex per group were perfused in situ with fixative and those animals in the control and 1200 ppm groups underwent a microscopic examination of nervous system tissues. A gross examination of respiratory tract tissues was performed for an additional 5 rats per sex per group and these tissues were retained for possible future microscopic examination. The remaining animals, including those animals designated for a possible post-exposure recovery period, were euthanized and discarded.

No overt clinical signs of toxicity or changes in body weight and food consumption were observed in this study. Transient subtle decreases in activity during exposure were noted for the 600 and 1200 ppm groups. Functional observational battery evaluations, automated motor activity measurements and the neuropathological examination did not reveal a treatment-related effect. The no observable effect level (NOEL) for subchronic neurotoxicity for this study was at least 1200 ppm based on a lack of cumulative neurotoxicity following repeated exposure.

 

Single exposure

A Union Carbide Corporation (2001) study was also conducted with the objective to evaluate the potential for test substance vapours to produce evidence of neurotoxicity in Sprague-Dawley CD® rats when treated (male and female animals treated only once) by whole-body inhalation exposure for 6 hours (exposure levels: 0, 500, 1500 or 3000 ppm; corresponding to approx. 2.66, 7.97 and 15.94 mg/l, calculated assuming test substance molecular weight of 130.19 g/mol). Animals were examined in detail for clinical signs of toxicity following exposure and weekly thereafter during a 2-week post-exposure period. Body weights were measured weekly. Individual animals were examined for possible changes in behaviour using automated motor activity measurements and a functional observational battery (FOB). Animals were examined pre-study and as soon as possible after exposure (motor activity commencing within 30 minutes; FOB within approximately 2.5 hours) and 7 and 14 days after exposure. Motor activity was also examined approximately 22-23 hours after exposure.

Single treatment of male and female rats with test substance vapours at concentrations as high as 3000 ppm did not result in overt clinical signs of toxicity or changes in body weight, FOB evaluations, or automated motor activity measurements. Under the conditions of this study, the no observable effect level for neurotoxicity was at least 3000 ppm.

CAS No. 123-92-2

In this neurotoxicity study 4 - 6 male Sprague-Dawley rats inhaled isoamyl acetate (no data about the concentrations) for 2 hours (Stengard, 1994). An increased motor activity (sniffing, rearing, and motility) was observed during the first 15 min of exposure to isoamyl acetate, likely due to the strong odour of the test substance. The greatest activity was seen during the onset of exposure. After approximately 30 min exposure the rats (also in the control group) were essentially immobile. No signs of increased motor activity after termination of exposure were seen in the exposed group compared to the control. There was no sign of increased eating behaviour in the exposed or control group. No difference in the mean basal concentration of dopamin and homovanillic acid concentration in the striatum between the exposed and control group was found. Exposure to isoamyl acetate had no demonstrable effect on extracellular dopamin or homovanillic acid levels within the striatum.

Justification for classification or non-classification

Based on the above results on the evaluation of the neurotoxicity potential of the test substance or its surrogates after acute or repeated inhalation treatment, there is no need for classification according to the current regulations.

  • EU classification according to Annex I and VI of Directive 67/548/EEC: no classification required
  • GHS classification (REGULATION (EC) No 1272/2008 (CLP)):no classification required