2-bromo-5-hydroxy-4-methoxybenzaldehyde

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-01-15 to 1998-05-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

July 27, 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

September 30, 1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

426-540-0

EC Name:

-

Cas Number:

2973-59-3

Molecular formula:

C8 H7 Br O3

IUPAC Name:

2-bromo-5-hydroxy-4-methoxybenzaldehyde

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00265786
- Color/physical form: gray to brown powder
- Expiration date of the lot/batch: 1998-07-01
- Purity test date: No data
- Purity: 91.3 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in the original container at room temperature away from direct sunlight
- Solubility and stability of the test substance in the solvent/vehicle: at least 3 hours in PEG 400 at room temperature (approx. 20°C)

FORM AS APPLIED IN THE TEST (if different from that of starting material)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HanIbm:WIST (SPF)

Details on species / strain selection:

The test system is recognized by the international guidelines as the recommended test system.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. (CH-4414 Füllinsdorf/ Switzerland)
- Age at delivery: 6 weeks
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: males: 133-175 g (mean 161 g); females: 105-132 g (mean 122 g)
- Fasting period before study: no data
- Housing: standard laboratory conditions; in groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding
- Diet: ad libitum, pelleted standard Kliba 3433 (Batch no. 94/97) rat maintenance diet
- Water: ad libitum, tap water in water bottles.
- Acclimation period: Seven days under test conditions following a health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 22 +/- 3 °C
- Humidity (%): 40-70%
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h/12h, with music played during light period

IN-LIFE DATES: From: 1998-01-08 To: 1998-02-12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Rationale for method of treatment: Accidental oral ingestion is a possible route of human exposure.

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test substance was weighed into a glass beaker on a tared Mettler balance and the vehicle was added. The mixtures were prepared using an ultra turrax. Homogeneity of the test article in the vehicle was maintained during treatment using a magnetic stirrer. Concentration, homogeneity and stability of the test substance dose preparations/vehicle mixtures were determined on samples taken during weeks 2 and 3 of the treatment. The analyses were performed in the Analytical Laboratories of RCC Umweltchemie AG.
- Dose levels are expressed in terms of the compound as supplied.

VEHICLE:
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): 10 mL/kg bw/day
- Lot/batch no. (if required): no data
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Test substance/vehicle mixtures were prepared and approx. 2 g of each mixture was weighed into a 100 mL volumetric flask by RCC Research & Consulting Ltd. These samples for analysis of concentration and homogeneity were delivered to the analytical laboratory, where they were immediately analyzed or stored deep-frozen (about -20°C) until analysis. Storage stability samples were collected after storage of the test substance/vehicle mixture at test conditions for about two or three hours. Then, the latter samples were delivered to the analytical laboratory where they were immediately analyzed or stored deep-frozen (about -20°C) until analysis.
- The delivered samples were dissolved with about 70 mL of dimethyl formamide (DMF) by means of an ultrasonic bath. Afterwards, the 100-ml volumetric flasks were filled to the mark with DMF. Depending on the dose group, the latter sample solutions were further diluted with DMF to yield concentrations within the calibration range. Finally, a defined aliquot was quantified by HPLC.
- Method of separation: High Performance Liquid Chromatography (HPLC)
- Apparatus: Merck-Hitachi
- Column: Hypersil C-18 BDS; 3 um; 100 mm x 4 mm (i.d.)
- Eluent: 7.1 g sodium sulfate in 1 L bi-distilled water adjusted to pH 2.0 with sulfuric acid (eluent A); acetonitrile (eluent B)
- Flow: 1.5 mL/min
- Detection: UV at 240 nm
- Injection volume: 20 uL
- Injected samples were quantified by peak areas of the test substance in counts with reference to the calibration curve, which was obtained by correlation of the peak areas of the standard solutions with their corresponding concentrations.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose selection was based upon data from an acute toxicity study (RCC level Project 675843) and the results of a 5-day dose-range-finding study (RCC Project 675900) in which the test substance was administered by gavage to 3 rats per group and sex. In males at 200 and 1000 mg/kg, slightly higher liver weights were noted (at 200 mg/kg: +9 %, at 1000 mg/kg: + 13 %).
- Rationale for animal assignment: A computer-generated random algorithm was used to assign animals to test or control groups.
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not applicable

Examinations

Observations and examinations performed and frequency:

MORTALITY/VIABILITY:
- Time schedule: twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first test article administration and once a week thereafter, approx. 1 hr after test article administration
- Outside the home cage in a standard arena

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during pretest, treatment and before necropsy
- an online electronic recording system consisting of a Mettler balance connected to the RCC computer was used
- The determination of the terminal body weight was performed immediately prior to necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: once during the pretest period and weekly thereafter
- an online electronic recording system consisting of a Mettler balance connected to the RCC computer
- The food consumption was calculated per rat and per food consumption interval. It expresses the average food consumed per animal and per day over the food consumption interval.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pretest and after 4 weeks of treatment
- Dose groups that were examined: all
- The ophthalmoscopic examinations of both eyes of all animals were performed after the application of a mydriatic solution using a Miroflex 2 Ophthalmoscope. A description of any abnormality was recorded. For unilateral findings, unless otherwise indicated in the tables, the contralateral eye was without abnormalities.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy, early in the working day between the hours of 6.30 and 7.20 to reduce biological variation caused by circadian rhythms
- Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube
- Anaesthetic used for blood collection: yes (light ether anesthesia)
- Animals fasted: yes, in metabolism cages for approx. 18 hrs before blood sampling but allowed access to water ad libitum
- How many animals: all animals
- Parameters examined included: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, reticulocyte fluorescence ratios, nucleated erythrocytes (normoblasts), total leukocyte count, differential leukocyte count, red blood cell morphology, thromboplastin time (prothrombin time) and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy, early in the working day between the hours of 6.30 and 7.20 to reduce biological variation caused by circadian rhythms
- Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube
- Anaesthetic used for blood collection: yes (light ether anesthesia)
- Animals fasted: yes, in metabolism cages for approx. 18 hrs before blood sampling but allowed access to water ad libitum
- How many animals: all animals
- Parameters examined included: glucose, urea, creatinine, total bilrubin, total cholesterol, triglycerides, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatinine kinase, alkaline phosphatase, gamma-glutamyl transferase, calcium, phosphorous, sodium, potassium, chloride, albumin, total protein, globulin and the albumin/globulin ratio.

URINALYSIS: Yes
- Time schedule for collection of urine: during the 18-day fasting period
- Metabolism cages used for collection of urine: yes
- Animals fasted: yes, in metabolism cages for approx. 18 hrs before blood sampling but allowed access to water ad libitum
- Parameters examined included: volume (18-hour), specific gravity, osmolality, color, appearance, pH, protein, glucose, ketone, bilirubin, blood, nitrite, urobilinogen, urine sediment and crystals.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4
- Dose groups that were examined: all groups
- Battery of functions tested: a modified Irwin screen test was performed on 5 rats per group and sex with the highest allocation number. The following tests were performed:
- Home cage: increased locomotor activity, abnormal behavior (restlessness/writhing), alertness (increased/decreased) and the startle response (increased/decreased).
- In the arena: pilo-erection, tremor, twitches, respiration (abdominal/gasping/slower/faster), spasms (clonic/tonic), abnormal behavior (apathy/darting), abnormal body carriage (straub tail/hunched/hind quarters raised/shuffling or dragging of hind limbs), abnormal gait (limbs spread further apart/standing or walking on its toes/waddling or rolling from side to side/walking low on limbs/animal incapable of motor activity), exploratory activity (increased/decreased) and sedation.
- In the hand: fearfulness, passivity, exophthalmos, pupil diameter, responses of pupil to light, pinna reflex, corneal reflex, hypothermia (touch detection only), cutaneous blood flow (increased/decreased), cyanosis, ptosis, lacrimation, salivation, body tone (increased/decreased), pain response (increased/decreased), aggressiveness, vocalization and diarrhea.
- On return to the home cage: grooming (increased/decreased).
- Forelimb/hindlimb grip strength: Forelimb and hindlimb grip strength measurements were performed using a push-pull strain gauge (chatillon, DPP - 1.0 kg). The animals were placed with the forepaws inside the triangular grasping ring and with the hind paws outside the triangular grasping ring. Using one hand, the animals were held about 3/4 of the way up towards the base of the tail and steadily pulled away/towards the ring until the grip was broken. Then the measured value was recorded.
- Locomotor activity: During week 4, the animals were randomized and monitored for a 60-minute period. Locomotor activity was measured quantitatively. Decreased or increased activity was recorded. Activity was measured with Activity Monitor AM 1052 by interruptions of infrared light beams caused by movements of the animal. Low beams count was reported in 10-minute intervals, as well as the total activity of the measuring period.

OTHER: The assays of blood and urine parameters were performed at RCC, c/o BRL Biological Research Laboratories Ltd. under internal laboratory quality control conditions to assure reliable test results.

Sacrifice and pathology:

NECROPSY
- All animals were weighed and necropsied and descriptions of all macroscopic abnormalities were recorded
- At the end of the treatment period, the animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.

GROSS PATHOLOGY: Yes
- Descriptions of all macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
- Organ weights were recorded on the date of necropsy
- Organs: brain, heart, thymus, kidney, adrenals, spleen, testes, epididymides
- The organ to terminal body weight ratios as well as organ to brain ratios were determined


HISTOPATHOLOGY: Yes
- tissues and orans were collected and fixed in neutral phosphate buffered 4% formaldehyde solution
- Slides of selected organs and tissues were collected at terminal sacrifice from the animals of group 1 (control) and group 4 (high-dose), as well as all gross lesions from all animals were processed, embedded and cut at a thickness of 2 to 4 micrometers and stained with hematoxylin and eosin
- examination by light microscopy
- adrenal glands, bone marrow (sternum, femur), brain (cerebrum, cerebellum, pons), cecum, colon, duodenum, epididymides, heart, ileum with Peyers patches, jejunum with Peyer’s patches, kidneys, liver, lungs infused with formalin, lymph nodes (mesenteric, mandibular), ovaries, prostate gland, rectum, sciatic nerve, seminal vesicles, spinal cord (cervical, midthoracic, lumbar), spleen, stomach, testes, thymus, thyroid (incl. parathyroid gland), tibial nerve, trachea, urinary bladder infused with formalin, uterus, vagina and gross lesions.

Statistics:

Statistical methods used to analyze the body weight, organ weight and all ratios, data of grip strength measurement, data of locomotor activity, as well as clinical laboratory data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher’s exact-test was applied to the ophthalmoscopy data and macroscopic findings.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

- Soft feces were observed in all groups from study day 3 until the end of the treatment period. This was considered to be caused by treatment with the vehicle PEG 400.
- Outside cage observation revealed temporarily salivation and dyspnea in one group 2 male (on 1-2 days resp) and in one group 4 female an enlarged left eye. These findings were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

- All animals survived the scheduled study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- no differences from controls of statitical or biological significance were noted

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- there was no treatment-related effect observed on absolute and/or relative food consumption between the animals of the control and test article groups.
- overall mean food consumption (relative and absolute) was similar for treated groups and controls. The marginal differences reported are within the ranges knwon for this species of this age and body weight.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- no treatment-related changes were seen
- Ophthalmologic findings were noted in a small proportion of all animals from groups 1, 2 and 3. They included corneal opacity and persistent pupillary membrane. These findings occurred at similar incidences in the control and treated groups. Therefore, they are considered to be unrelated to treatment with test substance.

Haematological findings:

no effects observed

Description (incidence and severity):

- There were no adverse effects on hematology at termination of the treatment which could be considered of toxicological significance.
- no remarkable findings were noted

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- There were no adverse effects in clinical biochemistry at termination of the treatment which could be considered of toxicological significance.
- However, a few minor changes recorded between treated rats of group 4 (1000 mg/kg) and controls in the clinical biochemistry data may be related to the test item administration:
- glucose level slightly increased in females (p<0.05);
- triglyceride level slightly increased in females (p<0.05);
- alanine aminotransferase (ALAT) activity slightly increased in both sexes (p<0.01);
- gamma-glutamyltransferase (G-GT) activity slightly increased in females (p<0.05); and
- phosphorus level slightly increased in males (p<0.05).
- Although these findings attained statistical significance, the changes were of minor degree and may be regarded as minimal adaptive changes (of the liver) to treatment. In addition, the findings were generally within or only slightly outside the range of the normal background data and therefore, of no toxicological relevance.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Urinalysis data was generally unremarkable
- The slightly higher specific gravity and osmolality measurements recorded in males of groups 3 (200 mg/kg) and 4 were considered to be unspecific findings.
- Furthermore, there was no significant reduction in the overmight urine output and no histopathological evidence for adverse changes which may have affected the renal concentrating ability of the kidneys. The brown discoloration and/or turbid appearance of the urine in several animals of the control and treated groups was considered to be related to contamination with feces as a result of the increased incidence in diarrhea. This as a result of treatment with the vehicle PEG 400

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- No quantitative or qualitative observations attributable to the administration of the test article occurred in either male or female animals treated at dose levels up to and including the highest daily dose level, 1000 mg/kg.
- all test article treated groups, including animals treated at 1000 mg/kg, had a mean grip strength and locomotor activity similar to and not significantly different from the control groups (p >0.05)

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- there were no treatment related changes in organ weights as well as in organ weight ratios in all animals of all groups.
- Brain/body weights and spleen/brain weight ratios in the female group 2 showed minor, but statistically significant differences from the controls (p <0.05). This is considered incidental to treatment with test substance since, in all instances, their occurrence did not show a relationship to the dose level administered.
- All organ weights and ratios of the male groups were not significantly different from the control values (p>0.05).

Gross pathological findings:

no effects observed

Description (incidence and severity):

- A few macroscopic findings were recorded. These findings did not ditinguish treated rats from controls.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

- A few microscopic findings were noted. The type and severity of these findings did not distinguish treated rats from controls.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

No data

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

2

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

The overall mean concentrations of the homogeneity samples were found to be 100.2%, 96.6%, and 89.6% of the nominal concentrations of the 5, 20 and 100 mg/mL dose groups, respectively. The individual concentrations varied in the range from -4% to +3% of the mean concentrations. Therefore, the test substance was found to be homogeneous. The test substance was found to be stable in PEG 400 at room temperature for at least three hours.

Applicant's summary and conclusion

Conclusions:

The oral administration of the test substance to Wistar rats at doses of 0, 50, 200 and 1000 mg/kg bw/day for 28 days resulted in minimal adaptive changes of some clinical biochemistry parameters in high-dose (1000 mg/kg) males and females. Based on the results of this study, 1000 mg/kg bw/day was established as the no-observed-adverse-effect-level (NOAEL) and 200 mg/kg bw/day as the no-observed- effect-level (NOEL).