diethylmethoxyborane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: EEC Directive 92/69/EEC, B.7 Repeated Dose (28 days) Toxicity (oral)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

425-380-9

EC Name:

-

Cas Number:

7397-46-8

Molecular formula:

C5H13BO

IUPAC Name:

methyl diethylborinate

Details on test material:

- Name of test material (as cited in study report): DEMB
- Physical state: Water-white liquid
- Analytical purity: 99.4 %
- Lot/batch No.: 183
- Expiration date of the lot/batch: August 01, 1997
- Stability under test conditions: in the vehicle dry propylene glycol stable for 16-20 h
- Storage condition of test material: At room temperature in the dark
- Other: Density 0.76 g/ml

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: female (mean): 170 g; male (mean): 202,7 g
- Housing: Group housing - 5 animals/cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 50%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Because DEMB is a highly flammable liquid in open air, all procedures were carried out in a glove-box under nitrogen. The test substance was taken from the original container into clean vials. The formulation was prepared an a volume/volume basis by adding the appropriate amount of the vehicle and subsequent mixing. Formulations may have been prepared in the afternoon, between 16.5 and 20.5 hours prior to use on the next morning. If applicable, the formulations were stored in closed containers at ambient temperature in the dark. Adjustment was made for specific gravity of vehicle and test substance. The formulations were prepared in dry propylene glycol. The propylene glycol (water contents <0.2%) was dried by addition of silica-gel at least several hours before use.

DIET PREPARATION
- Rate of preparation of diet (frequency): daily 16.5 and 20.5 hours prior to use
- Storage temperature of food: storage in closed containers at ambient temperature in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on the physical and chemical properties of DEMB.
- Amount of vehicle (if gavage): 5 ml/kg body weight

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

daily, 7 days/week for 28 days

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
5, 25, 125 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected on the basis of a 5-day dose range finding study

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily death was recorded; once daily clinical signs were recorded; the time of onset, degree and duration were recorded. All symptoms were recorded and graded according to fixed scales:
Maximum grade 4: grading slight (1) to very severe (4).
Maximum grade 3: grading slight (1) to severe (3).
Maximum grade 1: presence is scored (1).

BODY WEIGHT: Yes
- Time schedule for examinations: On days 1, 8, 15, 22 and 28.

FOOD EFFICIENCY:
- body weight gain data: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination, between 7.30 and 9.30 a.m.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all rats
- Parameters checked: erythrocyte count/RBC, haemoglobin/HB, haematocrit/HCT, mean corpuscular volume/MCV, mean corpuscular haemoglobin/MCH, mean corpuscular haemoglobin concentration/MCHC, platelet count, red cell distribution width/RDW, total leucocyte count/WBC, differential leucocyte count/SEG: neutrophils, eosinophils, basophils, lymphocytes, monocytes, prothrombin time/PT, partial thromboplastin time/PTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination, between 7.30 and 9.30 a.m.
- Animals fasted: Yes
- How many animals: all rats
- Parameters checked: alanine aminotransferase/(ALAT/GPT), aspartate aminotransferase/(ASAT/GOT), bilirubin, total/BILI T., creatinine, glucose, urea, protein, total/PROTEIN T., protein, albumin/ALBUMIN, alkaline phosphatase/ALP, sodlum, potassium, chloride, calcium, phosphorus/INORG.PHOSPH

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

The following statistical methods were used to analyse the data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
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-to-one rank test) was applied when the data could not be
assumed to follow a normal distribution. All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores). Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY: Two males and one female of the 125 mg/kg/day dose group were found dead on day 9 or 10 of treatment. The other animals of this dose group were sacrificed for humane reasons on day 10. No mortality occurred in the other dose groups during the study period.
Prior to death or sacrifice of the animals receiving 125 mg/kg/day, hunched posture or pale skin was observed among males and lethargy, ventra-lateral recumbency, hunched posture, uncoordinated movements, pale skin, quick breathing, ptosis and/or piloerection were observed in all females.
No treatment related clinical signs were observed in the animals of the 5 and 25 mg/kg/day dose groups. The findings such as alopecia, scabs and red staining around the eye noted among treated and/or control animals were considered to be of no toxicological significance as incidences remained within the range expected for rats used in this type of study. Salivation is commonly noted in rats of this age and strain following oral gavage and considered to be related to multiple intra-oesophageal intubation and/or the taste of the test substance. Therefore, this finding was considered
not to be a sign of systemic toxicity.

BODY WEIGHT AND WEIGHT GAIN: On day 8, the body weights and body weight gain of males receiving 125 mg/kg/day were significantly lower than that of control animals. No such effects were seen in females of the same dose group. The body weights and body weight gain of animals receiving 5 or 25 mg/kg/day remained in the same range as controls over the 4-week study period, although a tendency to low values for body weights and body weight gain of females receiving 25 mg/kg/day was noted an day 28.

FOOD EFFICIENCY: Slightly reduced food consumption was noted in males receiving 125 mg/kg/day over the first week of treatment. No such effect was seen in females of the same dose group. The food consumption data over the second week were affected by the mortality of the animals during that week and could not be interpreted. There were no differences in food consumption, before or after allowance for body weight, between the 5 and 25 mg/kg/day treated animals and control animals.

HAEMATOLOGY: A statistically significant decrease in erythrocyte count, haemoglobin and haematocrit values among females receiving 25 mg/kg/day was found after the 4-week study period. The haematological parameters of males receiving 25 mg/kg/day and those in males and females receiving 5 mg/kg/day were not affected by treatment.

CLINICAL CHEMISTRY: There were no differences in the clinical biochemistry parameters noted between control and treated rats that were considered to be related to treatment with DEMB.
Clinical biochemistry values in treated males and females achieving a level of statistical significance when compared to controls, were considered to have arisen as a result of slightly high control values and, in the absence of treatment-related distribution or corroborative findings in the opposite sex, considered to be of no toxicological significance. The decrease in alanine aminotransferase (ALAT) value of females receiving 25 mg/kg/day was considered to be of no toxicological relevance, since target organ toxicity would result in elevated enzyme levels.

ORGAN WEIGHTS: Organ weights and relative organ weights of treated animals were considered not to be affected by treatment with DEMB. The statistically significant changes between adrenal weights of treated and control females were considered to have arisen by chance, and in the absence of a dose response relationship and of microscopic findings in the adrenals, considered not to be of toxicological significance. The statistically significant change in heart:body weight ratio was considered not to represent a clear sign of toxicity, as the value remained within the range of variation that can be expected for rats in this type of study, and the relevance of this finding was doubted.

GROSS PATHOLOGY: Macroscopic post mortem examination of the 125 mg/kg/day treated animals that died or were killed in extremis during the study revealed changes in the stomach, liver, kidneys, adrenal glands, spleen, mesenteric lymph node and lungs among the animals. Partial cannibalism prohibited complete post mortem examination of one male that died during the study. Macroscopic observations at necropsy of the control animals and animals receiving 5 and 25 mg/kg/day did not reveal any alterations that were considered to have arisen as a result of treatment. The scab formation and alopecia found in two females receiving 25 mg/kg/day confirmed the clinical data obtained during the observation period.

HISTOPATHOLOGY: NON-NEOPLASTIC: Microscopic correlates to the findings noted above at necropsy in rats receiving 125 mg/kg/day were as follows. In the liver slight to severe grades of hepatocellular vacuolation and slight to moderate centrilobular hypertrophy were seen in all rats of both sexes. Minimal to moderate degrees of dark brown pigment were seen in all rats of both sexes. Minimal to moderate degrees of a dark brown pigment was noted in the bile ducts of two males and two females. An additional finding was minimal to moderate coagulative necrosis in two females. In the stomach, hyperplasia with hyperkeratosis accompanied by submucosal inflammation was seen in four rats of each sex at minimal to moderate degrees.
Vacuolation of the adrenal cortex of a moderate to severe degree was noted in all rats of both sexes. Moderate to severe grades of haemopoiesis were observed in the spleen of all rats of both sexes. Minimal to slight degrees of reactive hyperplasia were recorded in the mesenteric lymph nodes of a total of four rats. An additional treatment related finding in the form of renal tubular degeneration, minimal to moderate, was seen in four females.
The microscopic findings recorded in control rats and rats receiving 25 mg/kg/day were considered to be spontaneous in nature and within the range of background morphological alterations encountered in Wistar rats of this age and strain. They occurred at similar incidence and severity in both dose groups.

HISTORICAL CONTROL DATA (if applicable)

OTHER INFORMATION: No clinical laboratory investigations were performed on the animals receiving 125 mg/kg/day, due to mortality or interim sacrifice of the animals of this dose group during the study.

Effect levels

Dose descriptor:

NOAEL

Effect level:

25 other: mg/kg/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on minimal changes that were found in body weight and erythrocyte parameters

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Executive summary:

According to Annex VI of the EU Regulation (EC) N0. 1272/2008 (CLP) and Annex I of the EU directive 67/548/EEC, boronic acid is classified as STOT rep Cat 2 and R43, respectively.