reaction product of: saturated, monounsaturated and multiple unsaturated long-chained partly estrified alcohols of vegetable origin (Brassica napus L., Brassica rapa L., Helianthus annuus L., Glycine hispida, Gossypium hirsutum L., Cocos nucifera L., Elaeis guineensis) with O,O-diisobutyldithiophosphate and 2-ethylhexylamine and hydrogen peroxide

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:

Between Dec 15th - 1997 to Jan 29th -1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guideline

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:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

428-630-5

EC Name:

-

Molecular formula:

Not applicable

IUPAC Name:

reaction product of Z-9-octadecen-1-ol and O,O-diisobutyl hydrogen dithiophosphate

Details on test material:

- Name of test material (as cited in study report): Becrosan 6920
- Physical state:orange-yellow clear liquid
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Purity test date: not exactly defined mixture
- Lot/batch No.:12477
- Expiration date of the lot/batch: till December 1998
- Stability in solvent: stable in oil
- Storage condition of test material: The test article was stored at room temperature in the dark.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GrnbH, 97320 Sulzfeld, Germany

- Age at study initiation: 39 - 43 days

- Weight at study initiation:
males: 179 - 229 g; = -13.3% and +10.9% of the mean value of 206 g
females: 138 - 164 g = -9.2% and +7.9% of the mean value of 152 g

- Fasting period before study: Overnight


- Housing: The animals were housed up to 3 to a cage (Makrolon@T ype 3) separated by sex during acclimatisation. ALTROMIN Type S8/15, granulated soft wood bedding
- Diet (e.g. ad libitum): ALTROMIN 1326, pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water,@adli bitum (municipal supply)
- Acclimation period: 6-8 days before start of dosing


ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 22 °C
- Humidity (%): 30 - 60%
- Air changes (per hr): no data.
- Photoperiod (hrs dark / hrs light): artificial light is set to give a cycle of 12 hours light and 12 hours dark; light from 6.30 a.m. - 6.30 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

maize oil

Details on oral exposure:

Formulation of Test Article
The test article was dissolved in maize oil (refined by Caesar & Lorentz GmbH, D-40721 Hilden; Priifiorschrifi: DAC 1986, 6. Erg. 94; batch 71672187, shelflife April 1999) prior to use. According to the guidelines the solvent, maize oil" is the solvent of choice, if the test article is insoluble in water. Separate solutions were prepared for each dose level by combining a weighed quantity of test article in the appropriate volume of solvent. The solutions were prepared weekly.
All doses were expressed in terms of test article as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): According to the guidelines the solvent, maize oil" is the solvent of choice, if the test article is insoluble in water.

- Concentration in vehicle: Dose-dependent


- Amount of vehicle (if gavage): AA constant dose volume of 3 mvkg body weight was used. Individual doses was adjusted according to the weekly recorded body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of Test Article Formulation
To determine actual concentrations, 1 ml samples of each solution (including that for the solvent control group), prepared for use in week 1 and 4, were taken and stored in refrigerator until analyses.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

The substances was administered daily, for 28 consecutive days, by gavage.

Control animals were treated in an identical manner with 3 ml/kg/day of maize oil.

Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment.

The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Vehicle Control (0mg/kg/day test substance)
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Low Dose ( 100 mg/kg/day test substance)
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
Intermediate Dose (300 mg/kg/day test substance)
Basis:
actual ingested

Doses / concentrations 4

Remarks:

Doses / Concentrations:
High Dose (1000 mg/kg/day test substance)
Basis:
actual ingested

Doses / concentrations 5

Remarks:

Doses / Concentrations:
Recovery vehicle control group and High Dose (1000 mg/kg/day test substance)
Basis:
actual ingested

No. of animals per sex per dose:

5 animals of each sex (m/f) were allocated to each dose group.

In addition, 5 animals of each sex (m/f) were allocated to each control goup

Control animals:

yes, concurrent vehicle

other: recovery control (concurrent vehicle)

Details on study design:

- Dose selection rationale: Results of Preliminary Range Finding study.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Regulatory requirement
[- Post-exposure recovery period in satellite groups: 14 days, treatment free]

Positive control:

Not Used

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: carried out daily during the administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: carried out daily during the administration.


BODY WEIGHT: Yes
- Time schedule for examinations: body weights were recorded at the start of the study and then weekly thereafter and at the termination of the study before food withdrawal.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Not applicable

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

Food consumption was recorded weekly throughout the treatment and recovery period and group mean daily intakes were calculated.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
Not applicable


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples for the examinations to 6.2 - 6.4 were taken from the retroorbitd plexus of the animals of the dose groups 1 to 4 with ether anaesthesia after an overnight fast just prior the killing at the termination of the study one day after the last administration of test article.
- Anaesthetic used for blood collection: yes
- Animals fasted: overnight fast
- How many animals: dose groups 1 to 4
- Parameters examined:
erythrocyte count (RBC)
haemoglobin concentration (HB)
packed cell volume (HCT)
platelet count (PLT)
total leukocyte count (WBC)
leukocyte differential count

Coagulation Test
Parameters
prothrombin time (PT)
fibrinogen concentration (FIB)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: ADD
- Animals fasted: overnight fast
- How many animals: dose groups 1 to 4
- Parameters examined:
alanine aminotransferase (ALT)
alkaline phosphatase (AP)
aspartate aminotransferase (AST)
creatinine (Crea)
glucose (Gluc)
total cholesterol (Chol)
total protein (Prot)
urea nitrogen (UreaN)
albumin (Alb)
potassium (Pot)
sodium (Sod)
* The parameters aspartate aminotransferase and glucose were also determined from the animals of the dose groups 5 and 6 at the end of the recovery period.

URINALYSIS: no


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: A detailed off-cage clinical examination was carried out daily during the administration.
- Dose groups that were examined: all
- Battery of functions tested: ADD
- Behavioural Assessments: A detailed off-cage clinical examination was carried out daily during the administration.

Functional Performance Tests: yes

Sensory Reactivity Tests: yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the treatment or recovery period, respectively, all animals were killed by CO2 asphyxiation.
Macroscopic pathology
All animals were examined externally. The cranial, thoracic and abdominal cavities were opened and examined macroscopically.

Organ weights
The following organs of all animals were weighed after trimming of fat and other contiguous tissue: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes and thymus.


HISTOPATHOLOGY: Yes
Whole organs or samples of the tissues of all animals listed below were fixated in Formol-Alcohol and preserved in 4 percent formaldehyde:
adrenals
bone marrow (fiom sternum)
brain (3 sections)
epididymides
heart
kidneys
liver
lungs
lymph nodes (mesenteric and submandibular)
ovaries
Prostate
sciatic nerve
small and large intestine (including Peyer’s patches)
spinal cord (fiom second trachelo-vertebra [Axis])
spleen
stomach
testes
thymus
thvroid
trachea
urinary bladder
uterus

Other examinations:

None

Statistics:

Group means and standard deviations were calculated for each numerical data. Analysis of variance (ANOVA) was performed on all parameters, using treatment group as the factor in the analysis.
Analysis of variance followed different statistical methods. The 95% significance level was consider:

Body weights, food consumption: Welch-t-test
Rasch et al., Verfahrensbibliothek Versuchsplanung und-auswertung, Berlin 198 1.

Parameters of haematology (excluding differential leukocyte count), coagulation and clinical biochemistry and absolute and relative organ weights: Dunnett-test
Rasch et al., Verfahrensbibliothek Versuchsplanung und -auswertung, Berlin 198 1.

For differential leukocyte count standard deviation and ranges were calculated for each parameter .

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY

No animal died in the course of investigation.
Only in four animals not substance dependent clinical observations (slight nosebleeding, scratch at the right eye and excessive salivation) were made for a short time.
All other animals didn’t show any alterations of general state of being and behaviour at any observation time. Also no changes of sensory reactivity, grip strength and motor activity were observed.


BODY WEIGHT AND WEIGHT GAIN

The body weights and the body weight gain of the animals were not influenced by administration of the test substance.
The observed significant differences are accidentally.

HAEMATOLOGY

All haematological and coagulation parameters were not influenced by administration of the test substance.

CLINICAL CHEMISTRY

The activity of aspartate aminotransferase was dose dependent decreased, in the male animals of the high dose was this effect significantly. After the recovery period this effect was also observed, but it was not significantly.
The glucose content was dose dependent slightly increased without reaching pathological values*. In the female animals of the high dose was this effect significantly. After the recovery period this effect was not more observed.
* Normal range of glucose content: males: 5.5 - 7.2 mmovl; females 4.7 - 7.3 mmol/l
Baseline Haematology for Charles River Wistar Rats (CRL:(W)BR) as a Function of Sex and Age
The Charles River Breeding Laboratories, Inc., 1982
All other examined parameters of clinical biochemistry were not influenced by administration of the test substance.


NEUROBEHAVIOUR
Behavioural Assessment. normal
Functional Performance Tests. normal

ORGAN WEIGHTS
Significant differences to the control were only observed at relative weights of kidneys in males. A not dose dependent decrease was registered. After the recovery period this effect was not more observed. A slight, but not significant decrease of absolute weights of kidneys and also adrenals corresponds with the decreased relative weights.
A slight but not significant decrease of the absolute and relative weights of the liver was indicated dose dependent in male animals.
All other organ weights were not influenced by administration of the test substance.

GROSS PATHOLOGY

No changes of the organs and tissues were observed at the macroscopic examination of all 60 animals.

HISTOPATHOLOGY:
In three of the twenty examined animals (animals of the control and high dose group) the following not substance dependent histological findings were observed in control animals: - Central located hemorrhagic-purulent softenings were observed in Peyer’s patches of large intestine in one male. These softenings are observed from time to time also in the physiological digestive process.
- An edematous Adventitia was observed in some vessels of lungs in one male. This effect was caused presumably by a shock in the course of agony. - Hepatic tissue including choledochus was coalesced at hilus with the capsule of the right kidney in one female. Hepatic tissue is found often in this area, because the right kidney is connected with the liver by the Ligamentum hepatorenale.

Effect levels

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The observations show that Becrosan 6920 is well tolerated of the rats also in the high dose of 1000 mgkg body weight (maximum limit dose for repeated studies).

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

See above section, further details not considered necessary .

Applicant's summary and conclusion

Conclusions:

The observations show that Becrosan 6920 is well tolerated of the rats also in the high dose of 1000 mgkg body weight (maximum limit dose for repeated studies). The liver and kidneys were determined as target organs.

Executive summary:

Within a 28-Day Oral (Gavage) Toxicity Study with a 14-day treatment-fiee recovery period Becrosan6920was daily administered in the doses of 100, 300 and 1000mgkgbody weight to each5male and5femaleWISTARrats.

The following parameters were used for the evaluation of the substance effect:

Mortality, clinical signs, body weight development, food consumption, haematological parameters (erythrocyte count, haemoglobin concentration, packed cell volume, platelet count, leukocyte count and differential leukocyte count), coagulation parameters (prothrombin time and fibrinogen content), clinical chemistry parameters of the serum (alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase and protein, albumin, glucose, cholesterol, urea nitrogen, creatinine, sodium and potassium content), macroscopic pathological assessment of all animals, absolute and relative organ weights of adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes and thymus and histopathological assessment of these and fbrther organs and tissues.

The daily oral administration was tolerated without strong effects,onlythe following substance dependent effects were observed:

• The activity of aspartate aminotransferase of serum was dose dependent decreased, significantly in the males of the high dose group and not complete reversible after the 14 day recovery period. This corresponds to a slight but not significant decrease of the liver weights.
• The relative weights of kidneys in the males were not dose dependent decreased, significantly for the right kidney in the male animals of all dose groups.
• The glucose content of serum was dose dependent slightly increased without reaching pathological values, significant in the females of the high dose group. After the recovery period this effect was not more observed.

The general state of being and behaviour, body weights, food consumption and the

haematological and coagulation parameters were not influenced by administration of the test substance. No substance dependent macroscopic pathological and histological findings were observed.

The observations show that Becrosan6920is well tolerated of the rats also in the high dose of 1000 mgkg body weight (maximum limit dose for repeated studies). The liver and kidneys were determined as target organs.