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EC number: 200-819-5 | CAS number: 74-88-4
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Density
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- Vapour pressure
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- Ecotoxicological Summary
- Aquatic toxicity
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- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
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- Toxicological Summary
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- Acute Toxicity
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- Specific investigations
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- Additional toxicological data
Neurotoxicity
Administrative data
Description of key information
Under the conditions of this study, the NOEL (no-observed-effect level) for neurotoxicity was 27 ppm. The NOEL for systemic toxicity was 25 ppm. Clinical signs of systemic toxicity and decreases in body weight gain were noted at 93 and 401 ppm. In addition, one death occurred at 401 ppm.
Key value for chemical safety assessment
Effect on neurotoxicity: via inhalation route
Link to relevant study records
- Endpoint:
- neurotoxicity: acute inhalation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 April 2001 to 12 September 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 424 (Neurotoxicity Study in Rodents)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Remarks:
- Crl:CD®(SD)IGS BR
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: Young adult, 44-47 days old
- Weight at study initiation: Males, 111-146 g; Females, 90-121g
- Housing: Weanling animals were housed three per cage, by sex, in suspended, stainless steel wire-mesh cages for approximately three days following receipt. Thereafter, all animals were housed individually.
- Diet: ad libitum (except during the inhalation exposure period)
- Water: ad libitum (except during the inhalation exposure period)
- Acclimation period: at least 13 days
ENVIRONMENTAL CONDITIONS
- Temperature: 22.3 – 22.7 °C
- Humidity: 42.0 – 60.1 %
- Photoperiod: 12 hours light (6 a.m. - 6 p.m.)/12 hours dark - Route of administration:
- inhalation: vapour
- Vehicle:
- air
- Details on exposure:
- - Exposures were conducted in four 2.0 m^3 stainless steel and glass wholebody exposure chambers. Three chambers were dedicated for the test material exposure groups, while the fourth chamber was dedicated to the control group.
- The chambers were operated under dynamic conditions at a slight negative pressure with at least 12 to 15 air changes per hour. Exposure chambers were maintained at a minimum oxygen content of 19%. The oxygen content of the exposure atmosphere in each chamber was determined during the pre-study method development phase. Chamber temperature, relative humidity, ventilation rate, and negative pressure within the chambers were monitored and recorded approximately every 35 minutes through the use of Lab VIEW® for Windows Data Acquisition software and a personal computer. Mean temperatures ranged from 22-23 °C and mean relative humidity from 44-45 %.
- All animals were housed in normal animal colony rooms except during the single six-hour exposure. For the acute exposure, the animals were transferred to exposure caging, transported to the exposure chamber room, exposed for the requisite duration and returned to their home cages. Exposures occurred over four consecutive days to accommodate four study replicates.
- The daily exposure duration was six hours. Exposures were initiated when the generation of test material vapour was started and were terminated after six hours when the generation of the test material was stopped. The test material chambers were not to be opened until at least the T99 period (approximately 20 minutes) elapsed. The continued animal exposure during this chamber clearance period was considered to be the reciprocal equivalent of the “under exposure” experienced during the chamber equilibration (concentration build up) period. Therefore, the daily exposure duration was not adjusted for the calculated T99 for the exposure systems.
-Vapours of the test material were generated using an ambient temperature bubbler-type vaporisation system, in which the carrier gas (air) was dispersed (bubbled) through the liquid test material. In this process, the carrier gas picked up and vaporised the test material as the fine air bubbles passed through the liquid and into the vapour phase above the liquid. Glass gas washing bottles were used as bubblers. For gas dispersion, a 125-mL bottle with a 50-mm bottom fritted disc was used for Chamber 2, a 125-mL bottle with a 25-mm bottom fritted disc was used for chamber 3 and a 250-mL bottle with fritted cylinder was used for chamber 4. Liquid test article was added to the bubblers prior to daily exposures, as needed. Regulated compressed air controlled by ¼-inch Swagelok® S-series metering needle valves was dispersed through the fritted disc and carried the test article vapours to the chamber inlet through ¼” Teflon delivery tubing. The generator airflow rate was monitored by compact rotameters; 1-280 mL/min. rotameter for chambers 2 and 3, and a flowmeter for chamber 4. The rotameters had been calibrated prior to animal exposures using a mini Buck Flow Calibrator, and the Matheson flowmeter was calibrated using a dry test meter. The concentrated vapours were piped from the gas washing bottles, via ¼” Teflon tubing, to a 2” I.D. glass chamber inlet, where the concentration was diluted to the target level by the chamber ventilation air flow. Compressed air was also metered to the control chamber to maintain consistency between the control and exposure system. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- NOMINAL EXPOSURE CONCENTRATIONS
- A nominal exposure concentration was calculated for each daily exposure for each chamber from the total amount of test material used during the exposure and the total volume of air passed through the chamber during that day's exposure. The amount of test material used was obtained by weighing the gas-washing bottle containing the test material for each chamber prior to and after each daily exposure. The total volume of air passed through each chamber was calculated from the daily average chamber ventilation flow rate in litres per minute (LPM) and the exposure duration. The nominal concentration was calculated as follows:
ppm Iodomethane = (Wt. Iodomethane · Mol. Vol. · 10^6) / (MW · Ch. Flow · Exp. Dur.)
Where:
Wt. Iodomethane = weight of test material in grams
Mol. Vol. = Molar volume at 730 mmHg and 21 °C, 25.11 L/mole
10^6 = ppm conversion factor
MW = Iodomethane molecular weight, 141.93 g/mole
Ch. Flow = Daily average chamber flowrate for a given day, in LPM.
Exp. Dur. = Duration of a given day's exposure, in minutes
ACTUAL EXPOSURE CONCENTRATIONS
- Actual exposure concentrations were measured using a gas chromatograph (GC). Samples of the exposure atmospheres from each chamber were automatically collected at approximately 30-minute intervals using a sample loop and a multiposition valve. Samples were collected from the approximate animal-breathing zone of the inhalation exposure chambers.. The following summarises the GC conditions:
Instrument: Hewlett Packard 5890 Series II with a 3396 Series II integrator
Detector: Flame ionisation
Column: J & W DB-Wax, 30 m x 0.25 mm I.D., 0.25- micron film thickness
Gases: (Pressure (psig) Flow Rate (mL/min.)): Carrier - Helium 108 10.0, Fuel - Hydrogen 17 30, Air 37 300
Temperatures (°C): Injector 250, Column 45, isothermal and Detector 250
Injection volume (mL) 0.25
Retention time (min.) Approximately 0.985 min.
Integrator Run Parameters: Chart Zero Offset 0, Chart Attenuation 2, Chart Speed 2.5 cm/min, Peak Area Rejection Value 0, Peak Threshold 2 and Peak Width 0.04
- The chromatograph was standardised using 40-liter Tedlar® gas bags prepared to contain known concentrations of the test material. The standard bags were prepared by injecting known volumes of test material into a 500 mL glass vaporisation bulb. A continuous flow of air carried the vaporised test material into the bag. The total volume of air was measured by a dry test meter. Concentrations of the gas-phase standards were calculated as follows: Concentration = (VOL · R · T · D · 10^-3 · 10^6) / (L · GMW · P)
Where:
Conc. is in ppm
VOL = volume of test material vaporised into bag in μL
R = universal gas constant, 62.36 L mmHg/mole K
T = nominal laboratory temperature in K (273 + 21 °C = 294 K)
D = density of the test material, 2.280 g/mL
L = volume of air used to prepare bag, 30 L
GMW = gram molecular weight, 141.93 g/mole
P = nominal laboratory barometric pressure, 730 mmHg
10^-3 = μL to mL conversion factor
10^6 = conversion factor to ppm
- Two calibration curves were used to determine chamber concentrations. One curve (Low Prime Calibration) was used for the 25 ppm and control chambers and the second curve (High Prime Calibration) was used for the 100 and 400 ppm chambers.
- Each standard was prepared in triplicate prior to the exposure period and analysed with the GC. A least-squares line was fitted to the resulting peak areas. Concentrations were then calculated using the slope and intercept of this prime calibration curve. Prior to the initiation of each exposure, the integrity of the prime calibration curve was checked by analysing one freshly prepared standard. If the analysed concentration was within ± 10 % of the known concentration, the GC was considered within calibration specifications.
RESULTS OF CHARACTERISATION OF EXPOSURE ATMOSPHERES
- NOMINAL EXPOSURE CONCENTRATIONS: The overall mean nominal concentrations were 44, 140 and 423 for the 25, 100 and 400 ppm groups, respectively.
- ACTUAL EXPOSURE CONCENTRATIONS: The overall mean concentrations were 27, 93 and 401 ppm for the 25, 100 and 400 ppm groups, respectively. - Duration of treatment / exposure:
- 6 hours
- Frequency of treatment:
- Once on study day 0.
- Dose / conc.:
- 27 ppm (analytical)
- Dose / conc.:
- 97 ppm (analytical)
- Dose / conc.:
- 401 ppm (analytical)
- No. of animals per sex per dose:
- 12 animals per sex per dose
- Control animals:
- yes
- Observations and clinical examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Animals were observed twice daily (morning and afternoon) for mortality and/or moribundity for at least 14 days
DETAILED CLINICAL OBSERVATIONS: Yes
- Detailed clinical observations were recorded daily (study days 0-14) for all animals. On study days 0, 7 and 14 when the FOB was conducted, no additional clinical signs were recorded.
BODY WEIGHT: Yes
- Individual body weights were recorded pre-study (study day -7) and on study days 0, 7 and 14. Body weights were also recorded during FOB procedures for animals undergoing these evaluations. - Neurobehavioural examinations performed and frequency:
- FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Same technicians used throughout testing: Yes
- Technicians were blind to treatment status of animals: Yes
- Time schedule for examinations: Observations on all animals were made prior to exposure, within three hours of exposure on study day 0 and on study days 7 and 14.
- Noise level: The FOB was performed in a sound-attenuated room equipped with a white noise generator set to operate at 70 ± 10db with one exception; home cage observations were performed in the animal room.
- Parameters:
- Home Cage Observations: Posture, Convulsions/Tremors, Faeces consistency, Biting and Palpebral (eyelid) closure.
- Handling Observations: Ease of removal from cage, Lacrimation/Chromodacryorrhoea, Piloerection, Palpebral closure, Eye prominence, Red/Crusty deposits, Ease of handling animal in hand, Salivation, Fur appearance, Respiratory rate/character, Mucous membranes/Eye/Skin colour and Muscle tone.
- Open Field Observations (evaluated over a 2-minute observation period): Mobility, Rearing, Convulsions/Tremors, Grooming, Bizarre/Stereotypic behaviour, Time to first step (seconds), Gait, Arousal, Urination/Defecation, Gait score and Backing.
- Sensory Observations: Approach response, Startle response, Pupil response, Forelimb response, Air righting reflex, Touch response, Tail pinch response, Eye-blink response, Hind-limb extension and Olfactory orientation.
- Neuromuscular Observations: Hind-limb extensor strength, Hind-limb foot splay, Grip strength-hind and forelimb and Rotarod performance.
- Physiological Observations: Catalepsy, Body temperature and Body weight.
LOCOMOTOR ACTIVITY: Yes
- Observations were made on all animals prior to exposure, within three hours of exposure on study day 0 and on study days 7 and 14.
- The testing of treatment groups was done according to replicate sequence. Each animal was tested separately.
- Type of equipment used: San Diego Instruments, Inc., Photobeam Activity System (San Diego Instruments, Inc., San Diego, CA). This personal computer controlled system utilises a series of infrared photobeams surrounding a clear plastic, rectangular cage to quantify each animal’s motor activity.
- Length of session, number and length of subsessions: Data were collected in five-minute epochs (print intervals) and the test session duration was 60 minutes. Data for ambulatory and total motor activity were tabulated.
- Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of one photobeam) and ambulatory motor activity (interruption of two or more consecutive photobeams). - Sacrifice and (histo)pathology:
- - Unscheduled Deaths: A complete necropsy was conducted on all animals found dead or euthanised in extremis. The necropsy included examination of the external surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities including viscera. No tissue samples were preserved.
- Neuropathology: After 15 days of observation, all surviving animals were anesthetised with an intraperitoneal injection of sodium pentobarbital and then perfused in situ with a buffered 4.0 % paraformaldehyde/1.4 % glutaraldehyde solution. The central and peripheral nervous system tissues were dissected and preserved. Fixed brain weight (excluding olfactory bulbs) and brain dimensions (length and width) were recorded. Any observable gross changes, abnormal coloration or lesions of the brain and spinal cord were recorded. The following nerve tissues were prepared for a qualitative histopathological examination (embedded in paraffin or plastic, sectioned and then stained with haematoxylin and eosin) from six animals per sex in the control and 400 ppm groups:
- Tissues Embedded in Paraffin
Brain - olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, tectum, cerebral peduncles, central grey matter, cerebellum, pons, medulla oblongata and tegmenta.
Spinal cord - at cervical swellings C3 - C7 and at lumbar swellings T13 - L4
Trigeminal ganglia/nerves Lumbar dorsal root ganglia at T13 – L4
Lumbar dorsal root fibres at T13 - L4
Lumbar ventral root fibres at T13 - L4
Cervical dorsal root ganglia at C3 - C7
Cervical dorsal root fibres at C3 - C7
Cervical ventral root fibres at C3 - C7
Optic Nerves
Eyes
Skeletal muscle - gastrocnemius
- Tissues Embedded in Plastic:
Sciatic nerves (mid-thigh region and at sciatic notch)
Sural nerves
Tibial nerves
Peroneal nerves - Statistics:
- - Statistical tests were performed using appropriate computing devices or programs. All analyses were two-tailed (except as noted) for significance levels of 5 and 1 %.
- Body weights, body weight changes, continuous Functional Observational Battery (FOB), Locomotor Activity Data, and brain weights/measurement data were analysed by a one-way analysis of variance (ANOVA). If significant differences were indicated by the ANOVA, Dunnett’s Test was used to compare the control and treated groups. Functional Observational Battery parameters that yielded scalar or descriptive data were analysed by Fisher’s Exact Test. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - Remarkable clinical signs observed in the 400 ppm group female that was found dead on study day 6 consisted of repetitive movement of the mouth and jaws and shallow respiration on study day 1 and decreased defecation on study days 1-5. Other findings observed for this animal consisted mainly of yellow or red material on various body surfaces and a pale left eye.
- For animals that survived to the scheduled necropsy, test material-related clinical findings consisted of red and/or yellow material on various body surfaces and decreased defecation in the 100 and 400 ppm groups. In addition, drooping eyelids were noted for a single 400 ppm group female.
- Limited occurrences of red and/or yellow material on various body surfaces were noted in a dose-related manner in the 100 and 400 ppm group males and/or females, typically during the first week following test material exposure. Decreased defecation was noted for a single 100 ppm group male and two 100 ppm group females on study day 1 and for most of the animals in the 400 ppm group. Decreased defecation was typically limited to the first week following acute exposure and was consistent with the lower body weight gains observed in these animals during this period. Drooping eyelids were noted for a single 400 ppm group female (no. 67421) on study day 1. Although the numbers and/or incidences of animals affected in the treated groups were not large, similar findings were not observed in the control group. Therefore, these findings were attributed to the test material.
- Other findings noted in the treated groups were common findings in laboratory rats of this strain and age, were observed at a similar incidence in the control group and/or were noted at a low incidence, typically in single animals. Therefore, these findings were not related to test material administration. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- - One female in the 400 ppm group was found dead on study day 6; this mortality was attributed to test material exposure since the rate of spontaneous mortality in young rats is low and other signs of toxicity were present. All other animals survived until the scheduled euthanasia (study day 15).
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- - Test material-related reductions in body weight gain were observed in the 100 and 400 ppm group males and females for study days 0 to 7.
- Lower mean body weight gains were noted in the 100 and 400 ppm group males and females for study days 0 to 7; the differences from the control
group were statistically significant (p<0.01) for the 100 ppm group males and females and the 400 ppm group males. A dose-related response was not
observed in the females. As a result of the reduced body weight gains, mean body weights in the 400 ppm group males at study days 7 and 14 were lower than the control group by 10 and 8 %, respectively (statistically significant at p<0.05 or p<0.01). Mean body weights in the 100 ppm group males and
females and the 400 ppm group females were generally similar to the control group values. No effects on mean body weights or body weight gains were apparent in the 25 ppm group males or females. - 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):
- effects observed, treatment-related
- Description (incidence and severity):
- FUNCTIONAL OBSERVATIONAL BATTERY (FOB):
- Home Cage Observations:
Test material-related effects on home cage observations were noted in the 100 ppm group males and the 400 ppm group males and females on study day 0 and included changes in posture, palpebral closure and/or repetitive movement of the mouth and jaw. Four males and eight females in the 400 ppm group were noted to have changes in posture (sitting with the head held low); the difference from the control group was statistically significant at p<0.05 for the females. There was also a reduction (statistically significant at p<0.05 for the females) in number of animals with eyelids wide open in the 400 ppm group (8/12 for males and 5/12 for females). The degree of palpebral closure in the 400 ppm group ranged from slightly drooping to completely shut. Repetitive movement of the mouth and jaws was noted for a single 100 ppm group male and 3/12 males and 4/12 females in the 400 ppm group; the differences from the control group were not statistically significant. These findings were not noted in the control or 25 ppm groups, nor did they persist to the study day 7 or 14 evaluations. No other remarkable differences were apparent between the 25, 100 and 400 ppm groups and the control group when home cage observations were evaluated during pretest and on study days 0, 7 and 14.
- Handling Observations:
Test material-related effects on handling observations were noted in the 100 and 400 ppm group males and females on study day 0 and included salivation, drooping eyelids, gasping, red or crusty deposits around the eyes, nose and/or mouth, pale skin and/or slightly soiled fur. Salivation (slight to severe) was noted for 6/12 males and 7/12 females in the 400 ppm group (statistically significant at p<0.05). Other findings in the 400 ppm group males and females on study day 0 included gasping (3/12 males and 4/12 females), red deposits around the eyes, nose and/or mouth (4/12 males and 3/12 females), crusty deposits around the mouth for two females and pale skin for two males and two females. Findings in the 100 and 400 ppm groups consisted of slightly drooping eyelids for a single 100 ppm group female and one male and four females in the 400 ppm group and slightly soiled fur for one 100 ppm group female and one male and two females in the 400 ppm group. These findings were not noted in the control or 25 ppm groups, nor did they persist to the study day 7 or 14 evaluations. No other test material-related effects were observed on handling observations. The only other statistically significant (p<0.05) difference from the control group was an increased number of females in the 25 and 100 ppm groups that were easy to handle. However, these increases were not attributed to test material exposure since similar incidences were noted at the pretest evaluation. No other remarkable differences were apparent between the 25, 100 and 400 ppm groups and the control group when handling observations were evaluated during pretest and on study days 0, 7 and 14.
- Open Field Observations:
Test material-related effects on open field observations were noted in the 100 ppm group females and the 400 ppm group males and females on study day 0 and included repetitive movement of the mouth and jaws, abnormal gait, impaired gait score and/or low arousal level. Repetitive movement of the mouth and jaws was observed for one female in the 100 ppm group and three males and seven females in the 400 ppm group (statistically significant at p<0.05 in the 400 ppm group females). Abnormal gait (hunched body) was noted for four males and 11 females in the 400 ppm group, and slight, but definite impairment of the gait score was noted for one male and seven females in the 400 ppm group. The incidences of the gait findings were statistically significant (p<0.05) for the 400 ppm group females. Low, somewhat stuporous arousal levels were noted for three males and three females in the 400 ppm group. These findings were not noted in the control group, nor did they persist to the study day 7 or 14 evaluations. No exposure-related findings were noted in the 25 ppm group. No other remarkable differences were apparent between the 25, 100 and 400 ppm groups and the control group when open field observations were evaluated during pretest and on study days 0, 7 and 14.
- Sensory Observations:
Test material-related effects on sensorimotor observations consisted of slightly uncoordinated air-righting reflexes for 1/12 males and 4/12 females in the 400 ppm group and no reaction to the startle stimulus for 4/12 females in the 400 ppm group on study day 0. None of the differences from the control group were statistically significant. No other remarkable differences were apparent between the 25, 100 and 400 ppm groups and the control group when sensorimotor observations were evaluated during pretest and on study days 0, 7 and 14.
- Neuromuscular Observations:
Test material-related effects on neuromuscular observations consisted of marked reductions in rotarod performance for the 400 ppm group males and females on study day 0 (43-51% lower than the control group). The differences from the control group were statistically significant (p<0.05 or p<0.01). The study day 0 rotarod performance in the 400 ppm group males and females was also reduced when compared with the pretest values. No other test material-related effects were noted on neuromuscular observations. Mean rotarod performance in the 25 and 400 ppm group males was increased and statistically significant (p<0.05 or p<0.01) when compared with the control group at the study day 14 evaluation. However, these increases were not attributed to test material exposure since a dose-related response was not observed, similar changes were not observed in the female groups and similar changes were not observed at previous evaluations. No other remarkable differences were apparent between the control group and the 25, 100 and 400 ppm groups when neuromuscular observations were evaluated during pretest and on study days 0, 7 and 14.
- Physiological Observations:
Test material-related effects on physiological observations consisted of dose-related reductions in body temperature for the 100 and 400 ppm group males and females on study day 0; the differences from the control group were statistically significant (p<0.01). The reductions in the 100 ppm group were mild (approximately 1 °C), while the reductions in the 400 ppm group were marked (approximately 4 °C). Mean body temperatures in the 100 and 400 ppm groups were similar to the control group at the study day 7 and 14 evaluations. Mean body weights in the 400 ppm group males were decreased at the study day 7 and 14 evaluations (statistically significant at p<0.05 or p<0.01). These decreases were attributed to the lower body weight gains noted in this group for study days 0 to 7 (see Section IV.C.). No other test material-related effects were noted on physiological observations. Mean body temperature in the 25 ppm group males was slightly increased (0.2°C) when compared with the control group at the study day 7 evaluation (statistically significant at p<0.05). This difference was not attributed to test material exposure since similar changes were not observed in the 100 or 400 ppm groups or at the study day 0 evaluation. No other remarkable differences were apparent between the control group and the 25, 100 and 400 ppm groups when physiological observations were evaluated during pretest and on study days 0, 7 and 14.
- Locomotor Activity:
Test material-related lower mean ambulatory and total motor activity counts (for the overall 60-minute test session) were noted for the 100 and 400 ppm group males and females on study day 0. The differences from the control group were statistically significant (p<0.01). Ambulatory and total motor activity in the 100 and 400 ppm groups was similar to the control group at the study day 7 and 14 evaluations. No other test material-related effects were noted on Locomotor Activity. The only other statistically significant (p<0.05) differences from the control group were slightly decreased ambulatory and total motor activity counts for the 25 ppm group females on study day 0. These differences from the control group were not attributed to test material exposure since the motor activity values in the 25 ppm group females at the study day 0 evaluation were similar to those noted at the pretest evaluation. No other remarkable differences were noted for motor activity data. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- No treatment-related effects were apparent on the group mean absolute fixed brain weights or brain length and width measurements in the treated groups when compared to the control group values. No statistically significant differences were observed
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- - One female in the 400 ppm group was found dead on study day 6. The only gross findings for this animal were red and yellow matting of the skin.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment-related microscopic lesions were observed in any of the central or peripheral nervous system tissues examined from six animals/sex in the 400 ppm group. Peripheral nerves of both the control and 400 ppm group animals had axonal degeneration typified by minimal numbers of digestion chambers containing cellular debris. Digestion chambers in peripheral nerves occur in spontaneous radiculoneuropathy, which has been detected in rats
as young as 100 days in age7. One 400 ppm group female also had minimal axonal degeneration (digestion chambers) in the lumbar dorsal root fibers. Axonal degeneration has been found in the spinal nerve roots as well as peripheral nerves in control animals in subchronic neurotoxicity studies. In any one nerve, although incidence of axonal degeneration typically was higher in the 400 ppm group when compared to the control group, the numbers were
similar. Additionally, in both the control and 400 ppm group animals, the severity of axonal degeneration was minimal and frequently only a single fiber was affected. Since it was not possible to differentiate a treatment effect based on the severity of axonal degeneration, since there were no test material-related effects at study days 7 or 14, and and since there was no particular pattern of nerve involvement in the 400 ppm group animals when compared to the control group, axonal degeneration was not considered test material-related. Other findings for animals in the 400 ppm group were not
attributed to test material administration since they were noted similarly in the control group, were findings commonly observed in laboratory rats and/or were noted at a low incidence, typically in single animals. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Key result
- Dose descriptor:
- NOEL
- Remarks:
- (systemic toxicity)
- Effect level:
- ca. 27 ppm (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical signs
- mortality
- Key result
- Dose descriptor:
- NOEL
- Remarks:
- (neutrotoxicity)
- Effect level:
- 27 ppm (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- behaviour (functional findings)
- Conclusions:
- Under the conditions of this study, the NOEL (no-observed-effect level) for neurotoxicity was 27 ppm. The NOEL for systemic toxicity was 25 ppm. Clinical signs of systemic toxicity and decreases in body weight gain were noted at 93 and 401 ppm. In addition, one death occurred at 401 ppm.
- Executive summary:
The acute neurotoxic potential of the test material when administered by inhalation during a single six-hour exposure to BR rats was evaluated using a neurotoxicity screening battery. The study was performed in accordance with the standardised guidelines OECD 424 and OPPTS 870.6200, under GLP conditions.
The test material was administered via wholebody inhalation for a single six-hour session exposure. Target dosage levels were 25, 100 and 400 ppm. A concurrent control group received filtered air on a comparable regimen. Each group consisted of 12 males and 12 females. Experimental parameters recorded for all animals included viability, clinical signs and body weights. Functional Observational Battery and Locomotor Activity data were recorded prior to exposure, within three hours of exposure and on study days 7 and 14. All surviving animals were euthanized on study day 15 and perfused in situ. Brain weights (excluding olfactory bulbs) and brain dimensions were recorded for each of these animals. In addition, a neuropathological evaluation was performed on six animals/sex in the control and 400 ppm groups. Analytical verification of dose concentrations was performed and the overall mean concentrations were 27, 93 and 401 ppm for the 25, 100 and 400 ppm groups, respectively.
The results of a single inhalation exposure of the test material in rats at exposure levels of 25, 100 and 400 ppm can be summarized as follows: One female in the 400 ppm group was found dead on study day 6; the mortality was attributed to exposure. All other animals survived to the scheduled necropsy. Test material-related clinical findings consisted of limited occurrences of red and/or yellow material on various body surfaces and decreased defecation in the 100 and/or 400 ppm groups and drooping eyelids for a single 400 ppm group female. These findings were typically observed during the first week following test material exposure. Test material-related reductions in body weight gain were observed in the 100 and 400 ppm group males and females for study days 0 to 7. Mean body weights in the 400 ppm group males were 10 and 8 % lower than the control group values at study days 7 and 14, respectively. Mean body weights and body weight gains in the 25 ppm group were unaffected by treatment. Test material-related effects on Functional Observational Battery evaluations (collectively involving home cage, handling, open field, sensorimotor, neuromuscular and physiological observations) were noted in the 100 and 400 ppm groups on study day 0. None of the findings in the 100 and 400 ppm groups persisted to the study day 7 or 14 evaluations. A dose-related response was noted in the number of animals affected and the severity of the effect. Functional Observational Battery evaluations were unaffected in the 25 ppm group. Effects on home cage observations consisted of repetitive movement of the mouth and jaws in the 100 and 400 ppm groups and changes in posture (sitting with the head held low) and changes in palpebral closure (fewer animals with eyelids wide open) in the 400 ppm group. Effects on handling observations consisted of slightly drooping eyelids and slightly soiled fur in the 100 and 400 ppm groups and salivation (slight to severe), gasping, pale skin, red deposits around the eyes, nose and/or mouth and crusty deposits around the mouth in the 400 ppm group. Effects on open field observations consisted of repetitive movement of the mouth and jaws in the 100 and 400 ppm groups and abnormal gait (hunched body), slight (but definite) impairment of the gait and low arousal levels in the 400 ppm group. Effects on sensorimotor observations consisted of slightly uncoordinated airrighting reflexes and no response to the startle stimulus in the 400 ppm group. Effects on neuromuscular observations consisted of marked reductions in rotarod performance for the 400 ppm group (43-51% lower than the control group). Effects on physiological observations consisted of reductions in body temperature for the 100 and 400 ppm groups (approximately 1 and 4 °C lower, respectively, than the control group). Lower mean ambulatory and total motor activity counts were noted for the 100 and 400 ppm group males and females on study day 0. These lower motor activity counts did not persist to the study day 7 and 14 evaluations. No treatment-related effects were apparent in fixed brain weights or brain dimensions for perfused animals. No treatment-related neuropathological lesions were observed upon microscopic examination of six animals/sex in the 400 ppm group.
Based on the results of the Functional Observational Battery evaluations, all of the six domains described by Moser were affected by a single inhalation exposure of the test material at 400 ppm and four domains (autonomic, CNS excitability, CNS activity and physiological) were affected at 100 ppm. No effects were noted following a single exposure at 25 ppm.
Under the conditions of this study, the NOEL (no-observed-effect level) for neurotoxicity was 27 ppm. The NOEL for systemic toxicity was 25 ppm. Clinical signs of systemic toxicity and decreases in body weight gain were noted at 93 and 401 ppm. In addition, one death occurred at 401 ppm.
Reference
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEC
- 540 mg/m³
Additional information
The acute neurotoxic potential of the test material when administered by inhalation during a single six-hour exposure to BR rats was evaluated using a neurotoxicity screening battery. The study was performed in accordance with the standardised guidelines OECD 424 and OPPTS 870.6200, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).
The test material was administered via wholebody inhalation for a single six-hour session exposure. Target dosage levels were 25, 100 and 400 ppm. A concurrent control group received filtered air on a comparable regimen. Each group consisted of 12 males and 12 females. Experimental parameters recorded for all animals included viability, clinical signs and body weights. Functional Observational Battery and Locomotor Activity data were recorded prior to exposure, within three hours of exposure and on study days 7 and 14. All surviving animals were euthanized on study day 15 and perfused in situ. Brain weights (excluding olfactory bulbs) and brain dimensions were recorded for each of these animals. In addition, a neuropathological evaluation was performed on six animals/sex in the control and 400 ppm groups. Analytical verification of dose concentrations was performed and the overall mean concentrations were 27, 93 and 401 ppm for the 25, 100 and 400 ppm groups, respectively.
The results of a single inhalation exposure of the test material in rats at exposure levels of 25, 100 and 400 ppm can be summarized as follows: One female in the 400 ppm group was found dead on study day 6; the mortality was attributed to exposure. All other animals survived to the scheduled necropsy. Test material-related clinical findings consisted of limited occurrences of red and/or yellow material on various body surfaces and decreased defecation in the 100 and/or 400 ppm groups and drooping eyelids for a single 400 ppm group female. These findings were typically observed during the first week following test material exposure. Test material-related reductions in body weight gain were observed in the 100 and 400 ppm group males and females for study days 0 to 7. Mean body weights in the 400 ppm group males were 10 and 8 % lower than the control group values at study days 7 and 14, respectively. Mean body weights and body weight gains in the 25 ppm group were unaffected by treatment. Test material-related effects on Functional Observational Battery evaluations (collectively involving home cage, handling, open field, sensorimotor, neuromuscular and physiological observations) were noted in the 100 and 400 ppm groups on study day 0. None of the findings in the 100 and 400 ppm groups persisted to the study day 7 or 14 evaluations. A dose-related response was noted in the number of animals affected and the severity of the effect. Functional Observational Battery evaluations were unaffected in the 25 ppm group. Effects on home cage observations consisted of repetitive movement of the mouth and jaws in the 100 and 400 ppm groups and changes in posture (sitting with the head held low) and changes in palpebral closure (fewer animals with eyelids wide open) in the 400 ppm group. Effects on handling observations consisted of slightly drooping eyelids and slightly soiled fur in the 100 and 400 ppm groups and salivation (slight to severe), gasping, pale skin, red deposits around the eyes, nose and/or mouth and crusty deposits around the mouth in the 400 ppm group. Effects on open field observations consisted of repetitive movement of the mouth and jaws in the 100 and 400 ppm groups and abnormal gait (hunched body), slight (but definite) impairment of the gait and low arousal levels in the 400 ppm group. Effects on sensorimotor observations consisted of slightly uncoordinated airrighting reflexes and no response to the startle stimulus in the 400 ppm group. Effects on neuromuscular observations consisted of marked reductions in rotarod performance for the 400 ppm group (43-51% lower than the control group). Effects on physiological observations consisted of reductions in body temperature for the 100 and 400 ppm groups (approximately 1 and 4 °C lower, respectively, than the control group). Lower mean ambulatory and total motor activity counts were noted for the 100 and 400 ppm group males and females on study day 0. These lower motor activity counts did not persist to the study day 7 and 14 evaluations. No treatment-related effects were apparent in fixed brain weights or brain dimensions for perfused animals. No treatment-related neuropathological lesions were observed upon microscopic examination of six animals/sex in the 400 ppm group.
Based on the results of the Functional Observational Battery evaluations, all of the six domains described by Moser were affected by a single inhalation exposure of the test material at 400 ppm and four domains (autonomic, CNS excitability, CNS activity and physiological) were affected at 100 ppm. No effects were noted following a single exposure at 25 ppm.
Under the conditions of this study, the NOEL (no-observed-effect level) for neurotoxicity was 27 ppm. The NOEL for systemic toxicity was 25 ppm. Clinical signs of systemic toxicity and decreases in body weight gain were noted at 93 and 401 ppm. In addition, one death occurred at 401 ppm.
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