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Description of key information

A study of acute neurotoxicity is available.  Neurotoxicity following repeated (sub-chronic) oral dosing was investigated as part of a 90-day rat study and is reported elsewhere.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records
Reference
Endpoint:
neurotoxicity: acute oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 January, 1992 - 8 April, 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was carried out under GLP and followed the appropriate guideline.
Qualifier:
according to
Guideline:
EPA OPP 81-8 (Neurotoxicity Screening Battery)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
- Source: The Charles River Breeding Laboratories, Inc.
- Age at study initiation:You adult, 43 days old
- Weight at study initiation: Males 197 - 276 g, Females 135 - 184 g
- Fasting period before study: None
- Housing: Individually, in general accordance with the NIH Guide for the Care and Use of Laboratory Animals
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002 adlibitum
- Water (e.g. ad libitum): municipal water ad libitum
- Acclimation period: 18 days
- Justification for selection: This species and strain is recognized to be appropriate for subchronic toxicity and neurotoxicity studies.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 23.9 °C
- Humidity (%): 48-78%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

IN-LIFE DATES: From: No data To:No data
Route of administration:
oral: gavage
Vehicle:
other: methyl cellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
An appropriate amount of the test material was weighed for each group and transferred to a mortar. The test material was triturated with a small amount of the vehicle until a slurry was formed. This was then transferred to a graduated cylinder and a sufficient amount of vehicle was added to attain an appropriate volume for mixing. The suspension was mixed on a mixer for approximately 5 minutes to reduce any large particles.
A magnetic stir bar was added and the mixture was stirred continuously throughout the sampling and dosing procedures. Dosing preparations were dispensed on a daily basis for dose administration. Dosing preparations were prepared twice during the study period and were stored at room temperature.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Concentration in vehicle: Each litre of vehicle was prepared by heating 1000 ml of deionised water to approximately 70 °C and gradually adding 5.0 g of the control material powder. The mixture was stirred until clear. The vehicle was prepared prior to study initiation and stored refrigerated between periods of use.
- Amount of vehicle (if gavage): Dosage volume 5 ml/kg
- Lot/batch no. (if required): 50H0209
- Purity: 0.5% aquous methyl cellulose
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical tests: Homogeneity , stability (14 day) and test article concentration analyses were conducted on Busan 11-M1 preparations
Strata sampled: the top, middle and bottom of the Busan 11-M1 preparations were sampled for homogeneity, the middle of a preparation was sample for stability and/or concentration.
Samples size: 10 ml
Sampling schedule: Samples were collected prior to study initiation (homogeneity and stability analyses) and from the preparation used for dosing (concentration analyses).
Performing laboratory: EPL BioAnalytical Services, Inc.
Duration of treatment / exposure:
- Duration of observation period following administration: 14 days
Frequency of treatment:
Once
Remarks:
Doses / Concentrations:
25, 50, 100, 200 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
12 rats/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a preliminary range-finding study
- Rationale for animal assignment (if not random): computer randomisation procedure
Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: recorded individually pre-study and on study days 0, 7 and 14

OPHTHALMOSCOPIC EXAMINATION: No
Specific biochemical examinations:
NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No

CHOLINESTERASE ACTIVITY: No
Neurobehavioural examinations performed and frequency:
FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters checked:
Home cage observations included posture, convulsions/tremors, faeces consistency, biting and palpebral (eyelid) closure.
Handling observations included ease of removal from cage, ease of handling animal in hand, lacrimation/chromodacryorrhea, salivation, piloerection, fur appearance, palpebral closure, respiratory rate/character, red/crusty deposits, mucous membranes/eye/skin colour, eye prominence and muscle tone.
Open field observations included mobility, gait, rearing, arousal, convulsions/tremors, urination/defecation, grooming, gait score, bizarre/stereotypic behaviour, backing, time to first step.
Sensory observations included approach response, touch response, startle response, tail pinch response, pupil response, eye blink response, forelimb extension, hind limb extension, air righting reflex and olfactory orientation.
Neuromuscular observations included hind limb extensor strength, grip strength-hind and forelimb, hand limb food splay and rotarod performance.
Physiological observations included catalepsy, bodyweight and body temperature.
- Description of procedures: Detailed description available in Appendix D to report
- Minimization of bias: No data
- Same technicians used throughout testing: No data
- Technicians were blind to treatment status of animals: No data
- Site of testing: sound-proofed room equipped with a white noise generator, except home cage observations which were performed in the animal room
- Time schedule for examinations: 4 hours after dosage administration and on study days 7 and 14
- Environmental conditions: sound-proofed room equipped with a white noise generator, except home cage observations which were performed in the animal room
- Noise level:sound-proofed room equipped with a white noise generator, except home cage observations which were performed in the animal room
- Other: No further data
- Scoring criteria (if any): available in Appendix D to report
- Duration of observation period for open field observations: 2 minutes
- Description of equipment where required: No further data

LOCOMOTOR ACTIVITY: Yes
- Replicates used: All animals
- Type of equipment used: Digiscan ‘Micro’ Animal Activity System. Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills including grooming; interruption of one or two adjacent photobeams and ambulatory motor activity the interruption of three or more consecutive photobeams.
- Length of session, number and length of subsessions: recorded after the completion of the FOB. Data were collected in one minute epochs and the test session duration was 41 minutes.
- Total activity: was defined as a combination of fine motor skills including grooming; interruption of one or two adjacent photobeams and ambulatory motor activity the interruption of three or more consecutive photobeams.
- Ambulatory activity: the interruption of three or more consecutive photobeams.

AUDITORY STARTLE REFLEX HABITUATION: No

LEARNING AND MEMORY TESTING: No
Sacrifice and (histo)pathology:
- Time point of sacrifice: Day 14
- Number of animals sacrificed: All
- Parameters measured:
- Brain weight: yes excluding olfactory bulbs
- Length and width of brain: yes
- Other: Any observable gross changes, abnormal coloration or lesions of the brain and spinal cord were recorded
- Procedures for perfusion: in situ
- Number of animals perfused: All
- Tissues evaluated: The central and peripheral tissues. Central nervous system tissues: brain (forebrain, centre of cerebrum, midbrain, cerebellum and pons, and the medulla oblongata), spinal cord (at cervical swellings C3-C8 and at lumbar swellings T13-L4), gasserian ganglion/trigeminal nerves, lumbar dorsal root ganglion at T13-L4, lumbar dorsal root fibres at T13-L4, lumbar ventral root fibres at T13-L4, lumbar dorsal root ganglion at C3-C8, lumbar dorsal root fibres at C3-C8, lumbar ventral root fibres at C3-C8, optic nerves, eyes.
Peripheral nervous system tissues: Sciatic nerve (mid-thigh region and at sciatic notch), sural nerve, tibial nerve, peroneal nerve, forelimbs.
- Type of staining:hematoxylin and eosin
- Methodology of preparation of sections: embedded, sectioned and the stained
- Thickness: No data
- Embedding media: plastic or paraffin
- Number of sections: No data
- Number of animals evaulated from each sex and treatment group: 5 animal/sex in the control and 200 mg/kg group. The Sciatic nerve (mid-thigh region and at sciatic notch) and the lumbar dorsal root fibres at T13-L4 were also examined from 5 females each in the 25, 50 and 100 mg/kg groups.
Other examinations:
No further data
Positive control:
No further data
Statistics:
Statistical tests for body weight, histopathological and brain data were performed by a Digital MicroVAX 3400 computer with appropriate programming. All analyses were two-tailed for significance levels of 5% and 1%. Each mean was presented with the standard deviation and the number of animals used to calculate the mean. Body weights, body weight changes, brain weights and brain dimensions were analyzed by a one-way ANOVA. If significant differences were indicate, Dunnett’s test was used to compare the control and treat groups. Histopathological findings in the treated groups were compared to the control group data by the one-tailed Kolmogorov-Smirnov test.
All statistical tests for the FOB and locomotor activity data were performed using SAS/STAT statistical software. Each mean was presented with the standard deviation and the number of animals used to calculate the mean. Contiguous FOB data were analyzed using a two-way repeated measures ANOVA. If significant treatment or treatment-time interactions occurred, a one-way ANOVA was conducted at each point. If significant treatment effects were observed at a time point, Dunnett’s multiple T-test was conducted to determine significant treatment differences from the control group (p<0.05). FOB parameters yielding scalar or descriptive data were analyzed using the repeated measures SAS CATMOD procedure. If significant treatment or treatment-time interactions occurred, Fisher’s Exact test or Dunnett’s test were conducted to determine significant treatment differences from the control group. These tests were performed by a Digital MicroVAX 3400 computer with appropriate programming.
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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Clinical biochemistry findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Other effects:
not examined
Details on results:
NEUROBEHAVIOUR

No remarkable differences between animals receiving the test substance and the control animals in locomotor activity.
Differences were found between control animals and males receiving 100 and 200 mg/kg and females receiving 200 mg/kg test substance when the FOB was performed. Specifically, a slight impairment of gait was noted on study day 0, four hours after treatment during the open field testing. The only other possible treatment-related effect was the impairment of hind limb extension for two males in the 200 mg/kg group during the sensory observations on day 0. These effects were transient in nature. No signs of toxicity were apparent on study days 7 and 14. No remarkable differences between the treated and control animals were apparent during the home cage, handling, neuromuscular and physiological observations during pre-test evaluations or on study days 0, 7 and 14.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Changes in gait at higher dose levels
Remarks on result:
other:
Conclusions:
Based on the results of this study, the NOAEL for acute neurotoxicity was considered to be 50 mg/kg.
Executive summary:

In an acute neurotoxicity study performed by Lamb (1993) groups of 12 rats/sex/dose were dosed with 25, 50, 100, 200 mg/kg Busan 11-M1 by oral gavage. The animals were then observed for mortality and clinical effects, body weight and neurobehavioral parameters, until day 14 at which point they were sacrificed and gross necropsy and neuropathology examinations was performed. There were no mortalities. No effects attributable to treatment were observed in regards to clinical signs, body weight, gross necropsy and neuropathology examinations. There were some neurobehavioral differences that were attributable to treatment found in the 100 and 200 mg/kg dose groups. Based on the results of this study, the NOAEL for acute neurotoxicity was considered to be 50 mg/kg.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Species:
rat
Quality of whole database:
A guideline-complinat acute neurotoxicity study is avaialble

Effect on neurotoxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In an acute neurotoxicity study performed by Lamb (1993) groups of 12 rats/sex/dose were dosed with 25, 50, 100, 200 mg/kg Busan 11-M1 by oral gavage. The animals were then observed for mortality and clinical effects, body weight and neurobehavioral parameters, until day 14 at which point they were sacrificed and gross necropsy and neuropathology examinations was performed. There were no mortalities. No effects attributable to treatment were observed in regards to clinical signs, body weight, gross necropsy and neuropathology examinations. There were some neurobehavioral differences that were attributable to treatment found in the 100 and 200 mg/kg dose groups. Based on the results of this study, the NOAEL for acute neurotoxicity was considered to be 50 mg/kg.


Justification for selection of effect on neurotoxicity via oral route endpoint:
Only one study available

Justification for classification or non-classification

The findings of this study do not trigger classification according to the CLP Regulation