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Administrative data

Description of key information

A 90-day oral toxicity study in the rat (with neurotoxicity assessment) is available for the submission substance.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 June, 1992 - 14 April, 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Carried out under GLP and conducted according to US EPA guideline 82-1 and 82-7 but follows OECD guideline 408 with the exception of the following organ weights for epididymes, uterus, thymus, spleen.
Qualifier:
according to
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
Qualifier:
according to
Guideline:
other: EPA OPP 82-7 (Neurotoxicity)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: The Charles River Breeding Laboratories, Inc.
- Age at study initiation:You adult, 43 days old
- Weight at study initiation: Males 155 - 227 g, Females 123 - 175 g
- Fasting period before study: prior to blood collection for clinical pathology evaluations
- 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: 19 days
- Justification for selection: This species and strain is recognized to be appropriate for subchronic toxicity and neurotoxicity studies.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 - 23.3 °C
- Humidity (%): 38-63%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

IN-LIFE DATES: From: 04/08/1993 To:23/02/1993
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Purina Cerhtified Rodent Chow #5002
- Storage temperature of food: room temperature
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 diet preparations
Strata sampled: the top, middle and bottom of the Busan 11-M1 p[reparations were sampled for homogeneity, the middle of a preparation was sample for stability and/or concentration.
Samples size: 100 g
Sampling schedule: Samples were collected prior to study initiation (homogeneity and stability analyses) and weekly during the study (concentration analyses conducted on diet samples collected weeks 0, 1, 2, 3, 7 and 11 which correspond to the 1st, 2nd, 3rd, 4th, 8th and 12th weeks of dose administration).
Performing laboratory: EPL BioAnalytical Services, Inc.
Duration of treatment / exposure:
91, 92, 93 or 94 consecutive days prior to necropsy
Frequency of treatment:
Daily in feed
Remarks:
Doses / Concentrations:
1000, 5000, 10000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10 animals/sex/dose for the subchronic toxicity evaluation
5 animals/sex/dose for the subchronic neurotoxicity evaluation
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results obtained from a preliminary range-finding study with Busan 11-M1
- Rationale for animal assignment (if not random): computer-based randomisation ensuring homogeneity of group means and variances for body weight
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, begningy week one prior to treatment and also prior to scheduled terminal eithanization.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake was calculated from the mean food consumption and the appropriate concnetration of Busan 11-M1 in the food (ppm): Yes

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to study initiation and near termination
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination, from the inferior vena cava at the time of necropsy
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight
- How many animals: All surviving animals
- Parameters examined: Total leukocyte count, erythrocyte count, hemaglobin, hematocrit mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, RBC morphology, platelet estimate, Differential WBC count – percent and absolute (segmented neutrophil, lymphocyte, monocyte, basophil, eosinophil, unsegmented neutrophil), prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination, from the inferior vena cava at the time of necropsy
- Animals fasted: Yes, overnight
- How many animals: All surviving animals
- Parameters examined: Glucose, urea nitrogen, creatinine sodium, potassium, serum aspartate aminotranferase, chloride, calcium, globulin, A/G ratio, lactate dehydrogenase, serum alanine aminotransferase, serum alkaline phosphatase, total bilirubin, total cholesterol, total protein, phosphorus, albumin, gamma glutamyltransferase.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: pre-study and then during week 3, 7 and 12 of the exposure
- Dose groups that were examined: All dose groups, observations were made on 10 animals/sex/group
- Battery of functions tested:
Home cage observations included posture, convulsions/tremors, faeces consistence, 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, re/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.

OTHER:
Locomotor activity, recorded after the completion of the FOB was measured automatically using the 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.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals euthanized in extremis and all surviving animals allocated to subchronic toxicity evaluation.
Animals were euthanized by carbon dioxide inhalation and exsanguinated. External surfaces, all orifices and the cranial, thoracic, abdominal and pelvic cavities including the viscera were examined. The following tissues and organs were collected and placed in 10% neutral buffered formalin: Adrenals, aorta, bone with marrow, bone marrow smear, brain, exorbital lacrimal gland, eyes with optic nerve, femur, gastrointestinal tract, (oesophagus, stomach, duodenum, jejunum, Ileum, cecum, colon, rectum), heart, kidneys, liver, lungs, lymph node, mammary gland, ovaries with oviducts, pancreas, peripheral nerve, pituitary, prostate, salivary gland, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, testes with epididymides, thymus, thyroids, trachea, urinary bladder, uterus with vagina, all gross lesions.
The following organs were weighed from all animals and absolute weights, organ to final body weight and brain weight ratios were calculated: Adrenals, brain, kidneys, liver, lungs, ovaries and testes.

HISTOPATHOLOGY: Yes
Macroscopic examination of hematoxylin-eosin stained paraffin sections were conducted on all tissues fixed during gross pathology for all animals euthanized in extremis and for all animals in the control and high dose groups. The lungs, liver, kidneys, testes, epididymides and gross lesion were examined from all animals in groups 2 and 3.
Other examinations:
At study termination, 5 animals/sex/group were euthanized by carbon dioxide inhalation and then perfused in situ for neuropathological examination. The central and peripheral tissues were dissected and preserved. Brain weight excluding olfactory bulbs and brain dimensions were recorded. Any observable gross changes, abnormal coloration or lesions of the brain and spinal cord were recorded. The nerve tissues were embedded in plastic or paraffin, sections and the stained with hematoxylin and eosin. The following nerve tissues were sampled for a qualitative histopathological examination from animals in the control and high dose groups. 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, tail.
Statistics:
All statistical tests for data other than the FOB and Locomotor Activity were performed by a Digital MicroVAX 3400 computer with appropriate programming. All analyses were two-tailed (Except as noted) 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, food consumption, clinical pathology values, absolute and relative organ 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. Animals that were euthanized in extremis were not included in the calculation of the means for any test period. Contiguous FOB and locomotor activity 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:
effects observed, treatment-related
Description (incidence and severity):
Mean weekly body weights and cumulative mean body weight gains were decrease in the 5000ppm group and the 10000ppm group males and females during the study weeks 1 through 13.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption values were slightly reduced in the 5000ppm group females and the 10000ppm group males and females throughout the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Potential test article related decreases were observed in mean red blood cell count, haemoglobin and hematocrit values in the 10000ppm group males and females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Potential test article related decreases were observed in total protein, globulin and cholesterol levels in the 5000 and 10000ppm group males.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 10000 ppm
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
9/10 males in the 10000ppm group had small and/or soft testes
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
10000 ppm males had aspermatogenesis in the testes and 9 of these males had absence of spermatocytes in the epididymal tubules.
Histopathological findings: neoplastic:
no effects observed
Details on results:
BODY WEIGHT AND WEIGHT GAIN
Mean weekly body weights and cumulative mean body weight gains were decreased in the 5000ppm group and the 10000ppm group males and females during the study weeks 1 through 13.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption values were slightly reduced in the 5000ppm group females and the 10000ppm group males and females throughout the study.

HAEMATOLOGY
Potential test article related decreases were observed in mean red blood cell count, haemoglobin and hematocrit values in the 10000ppm group males and females.

CLINICAL CHEMISTRY
Potential test article related decreases were observed in total protein, globulin and cholesterol levels in the 5000 and 10000ppm group males.

ORGAN WEIGHTS
Mean absolute and relative (to brain and body weight) liver and testes weights were decrease in the 10000ppm group males. Potential test article related decreases in mean absolute and relative (to brain weight) kidney weights were also observed in the 10000ppm group males.

GROSS PATHOLOGY
At macroscopic examination 9/10 males in the 10000ppm group had small and/or soft testes. Microscopically, 10/10 males in this group had aspermatogenesis in the testes and 9 of these males had absence of spermatocytes in the epididymal tubules.
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reduced weight gain (females), clinical chemistry findings (males) at 5000 ppm
Dose descriptor:
NOAEL
Effect level:
10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Neurotoxicity: no effects observed
Critical effects observed:
not specified

No further data

Conclusions:
Based on the results of this study, the NOAEL for systemic toxicity was considered to be 1000 ppm and the NOAEL for neurotoxicity was considered to be 10000 ppm.
Executive summary:

Groups of 15 male and 15 female rats were fed for at least 91 days on weekly prepared diets that contained 1000, 5000, 10000 ppm Busan 11-M1. The potential subchronic toxicity (10 rats/sex/dose) and neurotoxicity (5 rats/sex/dose) related to the administration of Busan 11-M1 in rats were evaluated in this combined repeated dose study. No clinical effects or mortality attributable to treatment were encountered. Effects attributable to treatment were encountered in body weight, food intake, haematology, clinical chemistry, and organ weights. At the highest dose level 9/10 males had small and/or soft testes. Microscopically, all males in the highest dose level group had aspermatogenesis in the testes and 9 of these males had absence of spermatocytes in the epididymal tubules. It was concluded that 1000 ppm dose, providing an intake of 70 mg/kg/day for males and 80 mg/kg/day for females, was the NOAEL.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
70 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study is reliable for the determination of the subchronic endpoint

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Groups of 15 male and 15 female rats were fed for at least 91 days on weekly prepared diets that contained 1000, 5000, 10000 ppm Busan 11-M1. The potential subchronic toxicity (10 rats/sex/dose) and neurotoxicity (5 rats/sex/dose) related to the administration of Busan 11-M1 in rats were evaluated in this combined repeated dose study. No clinical effects or mortality attributable to treatment were encountered. Effects attributable to treatment were encountered in body weight, food intake, haematology, clinical chemistry, and organ weights. At the highest dose level 9/10 males had small and/or soft testes. Microscopically, all males in the highest dose level group had aspermatogenesis in the testes and 9 of these males had absence of spermatocytes in the epididymal tubules. It was concluded that 1000 ppm dose, providing an intake of 70 mg/kg/day for males and 80 mg/kg/day for females, was the NOAEL.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only one study available for this endpoint

Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: testes

Justification for classification or non-classification

Findings in the 90 -day toxicity study are not of sufficient severity and/or do not appear at sufficiently low dose levels to trigger classification for repeated dose effects (STOT-RE) under the CLP Regulation.