Registration Dossier

Administrative data

Description of key information

In a subacute study with rats (Hilltop Research, 1997) the test substance did not cause any adverse effects up to the highest dose tested (1000 mg/kg), therefore, the NOAEL was set at 1000 mg/kg bw.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-07-25 - 1997-11-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study which is well documented and meets generally accepted scientific principles.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
Remarks: With regard to OECD TG 407, organ weights for thymus, spleen, and heart were not registered. Furthermore, the range of temperature (18 – 26 °C) slightly exceeded the recommended range (19 – 25 °C). These variations had no influence on the validity of the study.
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: 7-week old
- Weight at study initiation: 227 - 259 g in males; 154 - 196 g in females
- Housing: individually in stainless-steel, hanging, wire-mesh cages
- Diet: Pelleted, certified standard diet ad libitum
- Water: Tap water ad libitum
- Acclimation period: 10 days
- Duration of treatment: 1997-08-05 - 1997-09-02
- Observation period after the end of dosing: 1997-09-03 - 1997-09-22

ENVIRONMENTAL CONDITIONS
- Temperature: 18 – 26 °C
- Humidity: 50 +/- 20 %
- Air changes: ca. 11 changes/h
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other:
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared weekly. For each dosing formulation, the appropriate amount of test item was weighed and mixed into a paste with a small amount of methylcellulose vehicle. Additional methylcellulose vehicle was added until the desired volume was achieved. The formulations were mixed for approximately 15 minutes and transferred to jars that were suitable for the dosing procedure. For the Week 2 mix only, the mix was sonicated for approximately 15 minutes while mixing, and transferred into jars.

VEHICLE
- 0.5% methylcellulose in distilled water
- Amount of vehicle (if gavage): 10 mL/kg bw


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HOMOGENEITY
Duplicate samples were taken from the top, middle, and bottom of the low- and high-dose level formulations prior to initiation of dosing. Analyses were performed in duplicate from one sample, and the other sample was frozen (-20°C).

STABILITY
Prior to initiation of dosing, samples from the low- and high-dose level formulations were analyzed to assess 4-, 7-, and 10-day stability. Analyses were performed in duplicate from samples stored at room temperature.

CONCENTRATION
A sample of each weekly formulation for each dose level was analyzed in duplicate for concentration of the test substance.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
10, 100 and 1000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
4 groups:
10/sex/groups 1 and 4 [control, 1000 mg/kg/day] (incl. recovery group)
5/sex/groups 2 and 3 [10 mg/kg/day, 100 mg/kg/day]
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based upon the oral LD50 and test item structure
- Randomization: Computer-generated random method
- Recovery group: 14-day recovery period
Observations and examinations performed and frequency:
MORTALITY AND MORIBUNDITY: Yes
- Time schedule: evaluated twice daily

IN-LIFE OBSERVATIONS: Yes
- Time schedule:
On each day of dosing, each animal was observed approximately 1 hour postdose.
Once before treatment and weekly thereafter, each animal was removed from its cage and examined.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to treatment at randomization, on the first day of treatment, and at weekly intervals thereafter.

FOOD CONSUMPTION: Yes
- measured and recorded weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period; additionally at the end of the recovery period
- Anaesthetic used for blood collection: No data, blood samples were obtained via the jugular vein at termination
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined: Red blood (erythrocyte) count, hemoglobin, hematocrit, platelet count, white blood cell (leukocyte) count, differential blood cell count, blood cell morphology

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period; additionally at the end of the recovery period
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined: glucose, urea nitrogen, creatinine, total protein, albumin, globulin, albumin/globulin ratio, cholesterol, triglycerides, total bilirubin, alanine aminotransferase, gamma glutamyltransferase, aspartate aminotransferase, calcium, inorganic phosphorus, sodium, potassium, chloride

URINALYSIS: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- prior to treatment and terminal sacrifice

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY/NECROPSY: Yes
On Day 30, the first five animals/sex/group were food-fasted overnight, weighed the day of scheduled necropsy, given an intraperitoneal injection of sodium pentobarbital, and exsanguinated. After at least a 14 day posttreatment recovery period, the last five animals/sex in groups 1 and 4 were sacrificed in the same manner. A necropsy was performed on each animal and all findings were recorded.
The necropsy included examination of the following:
- all orifices
- cervical tissues and organs
- cranial cavity
- external surface of the brain; the external surface of the spinal cord and cut surfaces of the brain and spinal cord were examined whenever tissue trimming was performed
- external surface of the body
- nasal cavity and paranasal sinuses
- thoracic, abdominal, and pelvic cavities and viscera
Organ Weights
At each scheduled sacrifice, the following organs were weighed after careful dissection and trimming of fat and other contiguous tissue:
adrenal (2), brain, kidney (2), liver, ovary (2), testis with epididymis (2)
Paired organs were weighed together. Organ-to-body weight percentages and organ-to brain weight ratios were calculated.

Tissue Preservation
The following tissues from each animal were preserved in 10% neutralbuffered formalin:
adrenal (2), aorta, brain, cecum, colon, duodenum, esophagus, femur with bone manow (articular surface of the distal end), heart, ileum, jejunum, kidney (2), lesions, liver, lung, mesenteric lymph node, ovary (2), pancreas, pituitary, prostate, rectum, salivary gland (mandibular [2]), sciatic nerve, seminal vesicle (2), spleen, stomach, testis with epididymis (2), thymus, thyroid (2) with parathyroid, trachea, urinary bladder, uterus with cervix and vagina
The following tissues were preserved for possible future analysis: eye (2), lacrimal gland (exorbital), mammary gland (females only), skeletal muscle (thigh), spinal chord (cervical, thoracic, and lumbar), skin

HISTOPATHOLOGY: Yes
Tissue samples from animals in the control and high-dose group were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. Macroscopic lesions, lungs, liver, and kidneys were examined microscopically from each low- and mid-dose animal.
Statistics:
Absolute bw, bw change, food consumption, clinical pathology (except hemolysis and cellular morphology gradings), and organ weight data of the treated groups were compared statistically to the data from the same sex of the control group.
Tests for homogeneity of variances and ANOVA were evaluated at the 5% probability level. If the variances of untransformed data were heterogeneous, a rank transformation of the data was performed to achieve variance homogeneity. If the transformation did not achieve variance homogeneity, the analyses were still performed on the rank-transformed data.
Control vs. treated group mean comparisons were evaluated at the 5.0% two-tailed probability level. Statistical significance is designated throughout the text of this report by the term significant.
Details on results:
IN-LIFE OBSERVATIONS AND MORTALITY:
All animals survived to scheduled necropsy. In general, the findings observed occurred sporadically and/or were of the type commonly seen in this species. There were no distinct or pronounced test material-related differences between the control and test groups.

BODY WEIGHT:
There were no significant differences in the weekly mean bw, weekly bw change, and total bw change between the control and treated animals. Mean bw generally increased over the course of the study for all groups.

FOOD CONSUMPTION AND FOOD CONSUMPTION RATIOS:
There were no significant differences in the weekly and total mean food consumption between the control and treated groups. Weekly mean food consumption increased for all groups throughout the course of the study.

HAEMATOLOGY:
Some significant differences were observed in the hematology data in Group 4 animals (1000 mg/kg) only at recovery Day 49; they were considered incidental to treatment and were not itemized. The differential leukocyte counts, cellular morphology results, and remaining hematology data were generally unremarkable and comparable between all groups at Day 30 and Groups 1 and 4 at Day 49.There was no evidence in the clinical pathology data of an effect from the administration of the test substance.

CLINICAL CHEMISTRY:
Some significant changes were noted during clinical chemistry, however they could not be attributed to the administration of the test material due to the low magnitude of the changes, the lack of dose dependence, and/or the occurrence of the difference only at recovery Day 49; they were not characterized further in the report.

ORGAN WEIGHTS:
Organ weights were unaffected by exposure to the test substance.

GROSS PATHOLOGY:
There were few gross abnormalities noted; none suggested any effect from exposure to the test substance.

HISTOPATHOLOGY:
There were no histologic findings suggestive of any adverse effects resulting from exposure to the test substance.

OPHTHALMOSCOPIC EXAMINATION:
None of the observations noted at study termination were attributed to test substance administration.
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no test substance-related clinical observations and no effect on bw, food consumption, clinical pathology, organ weights, and macroscopic/microscopic appearance of tissues up to and including the high dose level of 1000 mg/kg bw/day
Critical effects observed:
not specified

DOSE ANALYSIS

Homogeneity analyses of the low- and high-dose levels indicated that the test item was homogeneously mixed; relative standard deviation values were within 3.84 and 2.38 % of the target concentration, respectively.
Results of stability analyses indicated that the formulations were stable for 10 days at room temperature. All values (average of duplicate sample) were within 7.5 % of initial concentration.

Results of routine concentration analyses (average of duplicate sample) indicated that all formulations were within 10 % of target concentration.

Executive summary:

In a 28-day repeated dose toxicity study according to OECD guideline 407 and in compliance with GLP, the test item was administered daily to five rats/sex/dose level by gavage (vehicle: 0.5 % methylcellulose) at dose levels of 0, 10, 100, and 1000 mg/kg body weight. A 14-day recovery period was performed with five additional male/female rats treated with 0 or 1000 mg/kg bw/d. Diet and water were provided ad libitum. The analytically determined test item concentrations indicated that all formulations were within 10% of target concentration. Formulations of the test item were determined to be homogeneous and stable at room temperature for up to 10 days. The animals were observed twice daily (a.m. and p.m.) for mortality and moribundity. At least once each week, each animal was removed from its cage and examined for abnormalities and signs of toxicity. Body weights and food consumption data were collected weekly. Ophthalmic examinations were done before initiation of treatment and on Day 29. Blood samples were collected for hematology and clinical chemistry at terminal and recovery sacrifices. After at least 28 days of treatment, the first five animals/sex/group were weighed, anesthetized, exsanguinated, and necropsied. After at least 14 days of recovery, the remaining animals in Groups 1 and 4 were sacrificed in the same manner. At necropsy, macroscopic observations were recorded, selected organs were weighed (limited to adrenals, liver, brain, kidneys and gonads), and selected tissues were collected and preserved. Microscopic examinations were done on tissues from each animal in the control and high-dose groups. Macroscopic lesions, lungs, liver, and kidneys were examined microscopically from each low- and mid-dose animal. All animals survived to their scheduled sacrifice. There were no test item-related clinical observations and no effect on body weight, food consumption, clinical pathology, organ weights, and macroscopic/microscopic appearance of tissues. No target organ was identified. The NO(A)EL was determined as 1000 mg/kg bw in males/females.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day

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

In a GLP-compliant subacute toxicity study (OECD 407), the test substance in 0.5% methylcellulose was administered daily by gavage to five Sprague-Dawley rats per sex and dose group at 0, 10, 100, and 1000 mg/kg body weight for a period of 28 days. Five additional male and female animals were assigned to the control and the high dose group for a 14-day recovery period. All animals survived to their scheduled sacrifice. There were no test substance-related clinical observations and no effects on body weight, food consumption, organ weights, and macroscopic/microscopic appearance of tissues. Some variations in hematology parameters in group 4 reached the level of statistical significance but were only present at the end of the recovery period and were therefore considered incidental and of no toxicological relevance. Some significant changes were noted during clinical chemistry observation, however they could not be attributed to the administration of the test material due to the low magnitude of the changes, the lack of dose dependence, and/or the occurrence of the difference only at recovery Day 49. No target organ was identified. Therefore, based on the results of this study and under the conditions chosen, the NO(A)EL was set at 1000 mg/kg bw in males/females.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP guideline study which is well documented and meets generally accepted scientific principles.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data is reliable and suitable for the purpose of classification under Directive 67/548/EEC. Based on the present data, classification for repeated dose toxicity is not warranted under Directive 67/548/EEC.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for repeated dose toxicity is not warranted under Regulation (EC) No.1272/2008.