Reaction products of 3-methylbutanal and 3-methylbut-3-en-1-ol, distillation overheads, acid-treated

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted in accordance with TSCA guidelines under GLP and foccused on neurotoxicity/respiratory tract (no evaluation of systemic toxicity effects (clinical chemistry, haematology, urinalysis, effects on other organs besides neurological system).

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2000

Materials and methods

Principles of method if other than guideline:

USEPA (1985). Toxic Substances Control Act (TSCA) Test Guidelines, Neurotoxicity. 40 CFR 798.6050, 798.6200 and 798.6400. Fed. Reg. 1985; 50: 39458–39470.
USEPA. Revocation of Final Multi-substance Rule for the Testing of Neurotoxicity. 40 CFR Part 799. Fed. Reg 1995; 60: 4514–4516.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Amyl acetate

IUPAC Name:

Amyl acetate

Details on test material:

- Name of test material (as cited in study report): Amyl acetate
- Analytical purity: 99.8%
- Composition of test material, percentage of components: 65% amyl acetate (CAS 628-63-7), 35% 2-methylbutyl acetate (CAS 624-41-9)
- Stability under test conditions: stable for the duration of the study

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Canada Inc.
- Age at study initiation: 56 to 57 days
- Weight at study initiation: males - 291 g to 350 g, females - 184 g to 228 g
- Housing: individually in stainless-mesh bottomed cages (during non-exposure periods)
- Diet: pelleted diet (PMI certified Rodent Chow 5002), ad libitum
- Water: ad libitum
- Acclimation period: 3.5 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: Not applicable

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless-steel and glass whole-body inhalation chambers
- Method of holding animals in test chamber: rats were housed in stainless steel wire-mesh-compartmentalized cages inside the chamber
- System of generating particulates/aerosols: The test atmospheres were generated with a heated flow-through vapor generator.
- Temperature, humidity: 20-24°C, 30-70%
- Air change rate: minimum of 12 air changes per hour

TEST ATMOSPHERE
- Brief description of analytical method used: infrared gas analysis and weekly analysis by high-resolution gas chromatography
- Samples taken from breathing zone: yes

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 by 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 using n-amyl acetate as an analytical standard. 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:

13 weeks

Frequency of treatment:

6 h/day, 5 days/week (4 days/week during weeks 4, 8 and 13 to acommodate behavioral testing), at least 65 exposures

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
300, 600, 1200 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1.6, 3.2, 6.4 mg/L
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
299, 600, 1199 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

15 (control and 1200 ppm)
10 (300 and 600 ppm)

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: based on the results of previous studies, 1200 ppm was expected to produce clear acute effects on the nervous system, such as sedation and decreased activity
- Post-exposure recovery period in satellite groups: no post-exposure period due to ne effects during exposure period

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before, during and after exposure (observations during exposure were recorded on a group basis, as all of the animals could not be seen clearly from outside the chamber); a complete physical examination was conducted weekly

BODY WEIGHT: Yes
- Time schedule for examinations: pre-study and weekly during the study, including on days of behavioral testing and pre-sacrifice

FOOD CONSUMPTION:
- Food consumption: Yes, weekly (beginning pre-study and continuing throughout the study)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to exposure and once during week 4, 8 and 13 (at least 16 hours after exposure)
- Dose groups that were examined: all dose groups
- Battery of functions tested: grip strength / motor activity / other: behavioral function, body temperature

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
1) 5/gender/group: gross examination of respiratory tract and collection of tissues for histopathology
2) 5/gender from control and high dose group: in situ perfusion and neuropathological examination

HISTOPATHOLOGY: Yes
1) 5/gender/group: bronchi, larynx, two lobes of the lungs, bronchial lymph node, nasal cavity, pharynx and trachea
2) in situ perfusion animals: the following adjacent sections of brain and spinal cord were stained with hematoxylin and eosin (H&E) and then examined by light microscopy: forebrain; center of cerebrum; midbrain; cerebellum and pons; midcerebellum; medulla oblongata; cervical spinal cord; lumbar spinal cord and gross lesions

- all animals of control and high dose groups: preparation of nervous system tissues (from the peripheral nervous system (PNS), cross-sections were taken of the sciatic (midthigh 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

Statistics:

Group variances for body weight, food consumption, FOB and brain measurement data were compared using Bartlett’s test. When the differences between group variances were not significant (P>0.001), 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 analyzed by comparing the control group to the treated groups using a Fisher’s exact probability test. Initially, the comparison included all scores within a category, where appropriate, for all groups together. If a significant (P<0.05) difference was detected, then comparisons were made using all scores within a category between the control group and each of the treated groups. Motor activity data were analyzed 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 (P>0.001) 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.

Results and discussion

Results of examinations

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.

Effect levels

Dose descriptor:

NOEL

Remarks:

neurotoxicity

Effect level:

>= 6 400 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: lack of evidence of persistent or delayed effects on nervous system function or structure (corresponds to 1200 ppm, highest dose tested)

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Transient, subtle decreases in general activity level during the 6-h exposure period were noted for animals in the 600 and 1200 ppm groups during the first 2 weeks of the study. The no-observed-effect level (NOEL) for subchronic neurotoxicity in this study was at least 1200 ppm amyl acetate vapor, based on the lack of evidence of persistent or delayed effects on nervous system function or structure following repeated exposure.

Executive summary:

An inhalation toxicity study was conducted with rats to examine the potential for amyl acetate vapor to produce alterations in nervous system structure and function following repeated exposure. The study was performed as part of a USEPA Toxic Substances Control Act (TSCA) Section 4e Enforceable Consent Agreement for amyl acetate. Rats were exposed to amyl acetate vapor at concentrations of 0, 300, 600 or 1200 ppm, 6 h per day, 4 or 5 days per week, for at least 65 exposures. Treatment groups consisted of 15 rats per gender for the control and 1200 ppm groups and 10 rats per gender for the 300 and 600 ppm groups. Evaluations included clinical observations before, during and after each exposure, weekly detailed clinical examinations, weekly body weight and food consumption measurements and monthly functional observational battery and motor activity measurements conducted on non-exposure days. Neuropathology examination of the central and peripheral nervous system was performed at the end of the study. Exposure to amyl acetate did not result in overt clinical signs of toxicity or changes in body weight or food consumption. Transient, subtle decreases in general activity level during the 6-h exposure period were noted for animals in the 600 and 1200 ppm groups during the first 2 weeks of the study. Functional observational battery evaluations, automated motor activity measurements and neuroanatomy were unaffected by exposure. The no observed-effect level (NOEL) for subchronic neurotoxicity for this study was at least 1200 ppm, which is greater than an order of magnitude above the occupational threshold limit value (8-h, time-weighted average) for amyl acetate (100 ppm).