2-methylpropyl-(R)-2-hydroxypropanoate

Neurotoxicity

Description of key information

Isobuty-R-lactate is not considered to be neurotoxic, based on evaluation of neurotixicity studies on the primary metatbolite isobutanol.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

neurotoxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Literature review concerning the neurotoxic effects of methanol, ethanol, 1-and 2-propanol, 1-, iso-and t-butanol, and ethylhexanol, which are considered to result from instantaneous hydrolysis of their respective lactate esters upon inhalation. In tissues and body fluids the breakdown (enzyme catalysed hydrolysis) of the target substance isobutyl-R-lactate is very fast, indicating that the compounds systemically available are (R)-lactic acid and isobutanol. Therefore, only data for isobutanol is in the following presented.

Principles of method if other than guideline:

Literature review by contract research organisation; proprietary data of Purac Biochem BV.

GLP compliance:

no

Neurotoxicity of isobutanol

Human data:

No data related to chronic toxic encephalopathy were found. From two studies among workers exposed to butanol isomers, it can be concluded that irritation of the eyes is the critical effect. In one of these reports, other effects such as headache, vertigo, and Ménière or Ménière-like vertigo were found. However, causal relationships with exposure to isobutanol per se or relationships with exposure levels could not be established (Health Council, 1994b, World Health Organization, 1987).

 

Animal data: Single exposure:

The neurotoxic effects of isobutanol have been examined by exposing male and female rats (Sprague-Dawley (CD); n= 10/sex/group) to concentrations of 0, 4500, 9000, and 24,000 mg/m³ for 6 hours. Besides observations of mortality and overt signs of toxicity (recorded by exception once every hour during the 6-hour exposure period) and body weight recordings, behavioural functioning (FOB, motor activity) was evaluated prior to exposure and immediately and 1, 7, and 14 days after ending exposure. Animals were sacrificed 15 days after ending exposure. From each group, 5 animals were perfused, and the female reproductive tract and selected central and peripheral nervous tissues were retained but not microscopically examined. The remaining animals from each group were completely necropsied while the reproductive tracts of the females were retained. During the 6-hour exposure period, a rapidly reversible general central nervous system depression (non-responsiveness to tapping on the inhalation chamber; hypo-activity) at the two higher concentrations was observed. At the lowest concentration of 4500 mg/m³, only minimal effects (hypo-activity) were seen during the exposure period. After ending exposure, treatment-related transient effects were seen in the animals of the 6000 ppm group only. They were considered to be residual effects of the anesthetic effects of the test compound and included, amongst others, decreased alertness, decreased motor activity, and incoordinated gait. At necropsy, there were no treatment-related findings in any tissues or organs. From this study, a lowest-observed-effect level of 4500 mg/m³ was established (Li et al, 1994). In another study, no signs of intoxication were observed in rats exposed to an average concentration of 6500 mg/m³ for 4 hours (BG Chemie, 1990). In a russian study in which rats and rabbits were exposed to 15700 mg/m³ for 4 hours, central nervous system depression and dystrophia of olfactory neurons in the brains were reported to occur three days after ending exposure. Exposure to 8000 mg/m³ caused similar but less severe symptoms while no such effects were reported at 1300 mg/m³ (WHO, 1987).

A single oral dose of 1200 mg/kg bw impaired the performance of rats on an inclined plane test, 2-methylpropanol-1 being 3.6 times as potent as ethanol on an equimolar basis (Wallgren, 1960). The ED50 for narcosis in rabbits was 1408 mg/kg bw (Munch, 1972). Following intraperitoneal injection, the threshold dose for ataxia in rats was 400 mg/kg bw (McCreery and Hunt, 1978), the ED50 for righting reflex in mice was 1104 mg/kg bw (Lyon et al, 1981). Following intravenous injections into the ear vein of rabbits, the minimal anaesthetizing dose (causing loss of corneal reflex) was 930 mg/animal (roughly 300 mg/kg) (BG Chemie, 1990).

 

Animal data: Repeated exposure:

In a subchronic neurotoxicity study, groups of 10 (low/mid concentration) or 20 (control/high concentration) male and female Sprague-Dawley (CD) rats were exposed to 0, 750, 3000 and 7500 mg/m³ 6 h/d, 5 d/w, for approximately 3 months (rats received 70-73 exposures during a 102-day study period). Besides clinical observations (twice-daily checks for mortality, moribundity, noteworthy signs of toxicity; observations during the last hour of each exposure and upon daily removal from the cage, weekly detailed observations for toxicity signs), weekly body weight and food consumption recordings, and ophthalmic examinations (prior to the study, during week 14 in the control and high-concentration group), behavioural functioning (FOB, motor activity) was evaluated in 15 rats/sex from the control and high-concentration group and in 10 rats/sex from the two other exposure groups prior to exposure and within 24 hours of the end of the preceding exposure during exposure weeks 4, 8, and 13. At terminal sacrifice, groups of animals were, amongst others, selected for gross necropsy or for haematology and blood chemistry determinations, weighing and microscopic examination of selected tissues. In addition, the nervous tissues of 5 animals/sex from the control group and 5 males and 6 females from the high concentration group were examined microscopically. As to the end points relevant for the evaluation of the possible neurotoxicity of isobutanol, neither effects on FOB parameters or motor activity nor microscopic lesions in the nervous tissues were found in any of the groups at the post-exposure examinations. However, during (the last of hour of) exposure, none of the animals in any of the exposure groups responded to brushing over the inhalation chamber on any day while the control animals always did. Both exposed and control animals always responded to tapping on the chamber (Branch et al, 1996). Concurrently with the subchronic neurotoxicity study discussed above a scheduled operant behaviour study was performed in this study, groups of 10 male Sprague-Dawley rats were exposed to 0, 750, 3000, and 7500 mg/m³ 6 h/d, 5 d/w for approximately 3 months (65 exposures). Clinical observations and body weight recordings were made as well as ophthalmic examinations (prior to and 3 d after ending exposure). Scheduled-controlled operant behavioural parameters were analysed for 4 consecutive days before exposure and during exposure weeks 4, 8, and 13. At sacrifice, no necropsies were performed (except for 1 animal showing swollen genitalia during the final part of the study). As in the aforementioned neurotoxicity study, none of the exposed animals responded when brushing over the chamber during the last hour of exposure (Li and. Kaempfle, 1996). From these studies, it is concluded that exposure to concentrations up to 7500 mg/m³ for about 3 months does not cause severe persistent neurotoxic effects in rats. However, exposure to 750 mg/m³, the lowest level tested, induced very subtle effects on nervous system functioning. In an oral 13-week toxicity study, no clinical signs indicative of nervous system effects were found in male and female rats (SPF-Wistar, n= 10/sex/group) given up to 1450 mg isobutanol/kg bw/d in their drinking water (Schilling et al, 1997).

Conclusions:

Based on the data presented in the review report, isobutanol could not be identified as being neurotoxic to humans. Moreover, subchronic exposure did not produce evidence for chronic neurotoxic effects.

Executive summary:

In conclusion, based on the data presented in the review report isobutanol could not be classified as being neurotoxic to humans. Moreover, subchronic exposure in animals did not produce evidence for chronic neurotoxic effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 450 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on neurotoxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: sub-chronic inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Publication, which meets generally accepted scientific standards. Isobutanol used as read-across partner.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: Guideline 82-7; Subdivison F (Neurotoxicity Screening Battery)

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
- Source: Charles River Laboratories, Raleigh, NC, USA
- Age at study initiation: 8 weeks
- Weight at study initiation: Males: 285-365 g, females: 170-242 g
- Housing: Individually in stainless steel wire-mesh cages
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Three weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70 %
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE
The test atmosphere was generated by metering liquid isobutanol using an adjustable-flow valveless pump (Fluid Metering Inc, Oyster Bay, NY) to a Laskin-type nebulizer mounted in the supply air inlet at the top of the chamber. Animals were exposed to isobutanol vapour in 2000 liter stainless steel and glass Hazelton H-2000 chambers. The chambers operated at an airflow of approximately 500 liters/minute (15 air changes/ hour). Chamber environmental conditions were recorded every 30 minutes during exposures. The study mean chamber temperature and humidity ranged from 23-24 °C and 48-53 %, respectively.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The chamber concentrations of isobutanol vapour were determined by drawing test atmosphere samples from the animals breathing zone at evenly spaced intervals (18 samples per exposure) through a Fourier Transform infrared analyser (FVB/Analect, Irvine, CA) calibrated for isobutanol.

Duration of treatment / exposure:

3 months

Frequency of treatment:

6 hours/day, 5 days/week (at least 70 exposures)

Remarks:

Doses / Concentrations:
0, 250, 1000, 2500 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

Control and 2500 ppm exposure group: 20/sex
250 and 1000 ppm exposure groups: 10/sex

Control animals:

yes

Details on study design:

- Dose selection rationale:
Based on the results of a pilot study, particularly the laboured respiration and unresponsiveness of the rats to a fairly strong external stimulus during exposure, the highest exposure concentration used for the subchronic study was 2500 ppm. The intermediate and low concentrations, 1000 and 250 ppm, respectively, were selected as fractions of the high target concentration.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS
Animals were observed twice daily for mortality, morbundity and signs of toxicity. They were observed as they were removed from exposure chambers and returned to their home cages. Additionally, rats were observed for signs of toxicity during the last hour of every exposure period. Observations during exposure included subjective assessments of reaction to brushing and tapping the exterior walls of the exposure chambers. Detailed clinical observations for signs of toxicity were made once weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly

FOOD CONSUMPTION
-Time schedule: Once weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Twice, prior to assignment on study and during the 14th week of exposure
- Dose groups that were examined: All rats were examined prior to assignment. The control and 2500 ppm group animals were examined during the 14th week of exposure

Specific biochemical examinations:

N.A.

Neurobehavioural examinations performed and frequency:

The behavioral tests consisted of a functional observational battery (FOB) and an automated test of motor activity (MA). The behavioural tests were conducted prior to initiation of exposure and during the 4th, 8th and 13th weeks of exposure. For each time period, behavioral tests were conducted over a 4 day period with 25 animals tested each day. The time of testing was balanced across the different exposure concentrations. Animals were not exposed to isobutanol on the days that they were given these behavioral tests.

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
The FOB was performed to detect functional and behavioural effects using explicit, operationally defined scales for each measure. Animals were observed in their home cage and prior to any handling for general posture/activity level and any clonic or tonic movements. As the animals were removed from the home cage, ease of removal, reactivity towards the investigator, degree of lacrimation, salivation, presence or absence of piloerection and any general descriptive clinical effects were noted. The animal was placed in an open field for a two minute interval and observed for evidence of clonic or tonic movements, abnormal gait, spontaneous vocalisation, stereotypic or unusual behaviour, level of activity. Impairment of gait or mobility and any other unusual observations. Additionally, the number of animal rears, faecal boli and urinary pools were counted and recorded. Following the open field observation period, each animals response to an approaching object, tail pinch response, startle response, pupillary reflex, righting reflex, forelimb and hind limb grip strength, landing foot splay and body weight were determined.

LOCOMOTOR ACTIVITY: Yes
Motor activity was measured using an automated photocell recording apparatus designed to measure activity in a novel environment (Cage Rack Photobeam Activity System, San Diego Instruments, USA). Each monitoring session was one hour long determined previously to be long enough for the activity of untreated rats to approach asymptotic levels for the last 20 % of the session. Total motor activity is defined as the total number of Photobeam breaks that occurred during the one hour session. Intercession motor activity level is defined as the number of Photobeam breaks at each of the six 10 minute’s intervals.

Sacrifice and (histo)pathology:

Animals were sacrificed during the 14th week of exposure with the exception of one female that was sacrificed in a moribund condition after approximately two months of exposure. Five rats/sex/exposure level were perfused for neuropathology examination. The one female sacrificed in moribund condition was also perfused for neuropathology examination. A separate set of five rats/sex/exposure level were euthanized and given a full histopathology and clinical pathology evaluation. The remaining 10 rats/sex in the control group and 9 females at the 2500 ppm level were given a full gross necropsy. These remaining animals had been added to study reversibility or persistence of toxic effects in the event that a specific effect on the nervous system was observed. Since no specific effects were observed, these animals were euthanized after 3 months of exposure along with the other animals in this study.

Neuropathology evaluation:
At study termination, 5 animals/sex/group of the animals that underwent behavioural testing, were anaesthetised by intraperitoneal injection of sodium pentobarbital and tissues were fixed by intracardial infusion of sodium nitrite followed by 4 % formaldehyde/1.5 % glutaraldehyde. The brain (7 coronal sections), spinal cord (cervical, thoracic and lumbar segments), dorsal and ventral spinal nerve roots with dorsal root ganglia (cervical and lumbar swelling), Gasserian ganglia, sciatic, tibial and sural nerves were removed and stored in perfusion fixative. Peripheral nerves and ganglia were embedded in glycol methacrylate and stained with toluidine blue. The brain and spinal cord were embedded in paraffin and stained with haematoxylin and eosin. Tissues from the control and 2500 ppm exposure groups were examined by light microscopy.

Necropsy:
A complete necropsy was performed on each animal, selected organs weighed and fixes by immersion in 10 % neutral buffered formalin with exception of eyes which were fixed in 5 % neutral buffered formalin/0.5 % glutaraldehyde. The fixed tissues were washed, dehydrated, embedded in paraffin, sectioned and stained with hematoxylin and eosin. The tissues examined microscopically included the adrenals, brain (5 coronal sections), eyes, heart, kidneys, liver, lungs, nose (3 sections), ovaries, testes, epididymides, skin, spleen, uterus and vagina

Other examinations:

Haematology and blood chemistry:
At termination of the study, 5 rats/sex/exposure group were fasted overnight and anaesthised with CO2. Blood was drawn from the posterior vena cava for haematology and blood chemistry determinations.
Serum clinical chemistries included blood urea nitrogen, creatinine, glucose, total protein, albumin, globulin, glutamic pyruvic transaminase, alkaline phosphatase, gamma glutamyl transpeptidase, glutamic oxaloacetic transaminase, creatinine phosphokinase, total and direct bilirubin, cholesterol, sodium, potassium, calcium, chloride and phosphorous.
Haematological evaluations included total erythrocyte count (RBC), total leukocyte count, haematocrit (HCT), haemoglobin level (HGB), platelet count, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, activated partial thromboplastin time, leukocyte differential and reticulocyte count.

Statistics:

Haematology data, clinical chemistry data, terminal body weights, absolute organ weights and organ/body weight, and organ/brain weight ratios were evaluated by decision-tree statistical analyses. The decision-tree methodology performs both control-treatment comparisons and a test for trend. The specific statistical tests used for these analyses is determined by a set screening procedures for normality (i.e., skewness and kurtosis) and equality of variance (Bartlett-Box test). Data consistent with normality and homogeneity of variance were analyzed using Dunnett's 2-sided comparison of treatments to a control. Differences were considered significant if p < 0.05. The trend test in this case is a simple linear contrast from an analysis of variance model (but compared against pure, rather than residual, error). Data that were strikingly inconsistent with either normality or constant variance were analyzed using non-parametric procedures.
Treatments are compared with control using a protected Mann-Whitney test. (Protection is given in this case by performing the Mann-Whitney tests only if a Kruskal-Wallis test for overall group differences is significant at the 5 % level.) Jonckheere' s test is the non-parametric trend test used.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Behaviour (functional findings):

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no treatment-related abnormal clinical signs seen immediately after exposure at any concentration or during the weekly, detailed clinical observations.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant differences between treated and control groups for body weights and cumulative body weight changes.

FOOD CONSUMPTION
There were no statistically significant differences between treated and control groups for food consumption.

OPHTHALMOSCOPIC EXAMINATION
There were no ocular abnormalities noted at the 2500 ppm and control exposure levels.

NEUROBEHAVIOUR
There were no statistically significant differences between treated and control groups for any parameter evaluated by the functional observational battery at any time point in the study. Motor activity was unaffected by exposure to isobutanol at any concentration.

GROSS PATHOLOGY/HISTOPATHOLOGY
There were no statistically significant differences in terminal body weights, absolute organ weights, organ to body weight ratios, or organ to brain weight ratios in any group or for any tissue. There were no treatment-related gross or microscopic abnormalities noted in any of the tissues.
The female rat at the 2500 ppm exposure level that was sacrificed in mid-study was diagnosed to have lymphoblastic leukaemia.

NEUROPATHOLOGY
No morphological signs of neurotoxicity were noted.

HAEMATOLOGY and BLOOD CHEMISTRY
There were statistically significant increases in total erythrocyte count, haematocrit and haemoglobin parameters in female rats of the 2500 ppm group as compared to the control females. These changes may have been related to treatment, but their toxicological significance was not determined. There was also a significant increase in serum calcium level in the males from the 1000 ppm exposure group but since the change did not occur
in a dose-related fashion, it is not considered to be related to isobutanol exposure. There were no other significant differences in the serum chemistry or haematological parameters.

Dose descriptor:

NOAEC

Remarks:

Neurotoxicity

Effect level:

> 2 500 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

other:

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

> 2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Slight increases in RBC, HGB and HCT in the 2500 ppm female rats were not taken into account due to unknown biological relevance.

Remarks on result:

other:

Conclusions:

Subchronic exposure to atmospheres containing isobutanol vapour at concentrations up to 2500 ppm did not result in any functional or morphological signs of neurotoxicity.

Executive summary:

In a subchronic inhalation study, male and female Sprague-Dawley CD rats were exposed to atmospheres containing isobutanol (99 % purity) vapour at concentrations of 0, 250, 1000 and 2500 ppm for 6 hours/day, 5 days/week for 3 months. The treatment did not result in any functional or morphological signs of neurotoxicity as measured by functional observational battery, motor activity and neuropathology. Therefore the NOAEL for neurotoxicity can be considered to be greater than 2500 ppm.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No data is available for isobutyl-R-lactate. Due to the rapid, enzymatically catalysed, hydrolysis of isobutyl-R-lactate into isobutanol and lactic acid, the toxicology of isobutyl-R-lactate can be understood in terms of the toxicology of isobutanol and lactic acid. Lactic acid is an ubiquitous and integral element of mammalian metabolism and therefore of minor toxicological relevance in comparison to isobutanol which is, as an alcohol, more important for the toxicological assessment. Thus, available data from isobutanol (source substance) was used in a read-across approach to assess the neurotoxic potential of isobutyl-R-lactate. In an oral 13-week toxicity study, no clinical signs indicative of nervous system effects were found in male and female rats (SPF-Wistar, n= 10/sex/group) given up to 1450 mg isobutanol/kg bw/d in their drinking water (Schilling et al, 1997).

In a subchronic inhalation study, male and female Sprague-Dawley CD rats were exposed to atmospheres containing isobutanol (99 % purity) vapour at concentrations of 0, 250, 1000 and 2500 ppm for 6 hours/day, 5 days/week for 3 months. The treatment did not result in any functional or morphological signs of neurotoxicity as measured by functional observational battery, motor activity, scheduled-controlled operant behaviour and neuropathology.

Justification for classification or non-classification

Based on the available data, isobutyl-R-lactate does not warrant classification for neurotoxicity.