Reaction mass of 1,2-Diacetoxy-3-butene and 1,2-Dihydroxy-3-butene, monoacetate

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:

2003-01-22 - 2003-06-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD), GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Experimental Toxicology and Ecology, BASF Aktiengesellschaft, 67056 Ludwigshafen/Rhein, FRG

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

445-630-0

EC Name:

-

Molecular formula:

Unspecified

IUPAC Name:

1-(acetyloxy)but-3-en-2-yl acetate; 2-hydroxybut-3-en-1-yl acetate

Details on test material:

- Name of test material (as cited in study report): Vinylglykoldiacetat ca. 75% ig
- Test substance No.: 02/0526-1
- Date of production: October 21, 2001
- Physical state: liquid / clear
- Analytical purity: about 77%
- Lot/batch No.: Kanister 1, 2, 4, 5
- Stability under test conditions: stable; proven by reanalysis (analytical report 031-00161)
- Storage condition of test material: Room temperature, under N2

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar rats CHGIxBdHan:Wl
- Source: Charles River, Sulzfeld, Germany
- Identification: ear tattoo
- Age at study initiation: 33 - 35 d
- Weight at study initiation: mean 156.2 g (male), 121.5 g (female)
- Fasting period before study: no
- Housing: singly in type DK III stainless steel wire mesh cages supplied by Becker & Co ., Castrop-Rauxel, FRG (floor area about 800 cm2). Underneath the cages, waste trays were fixed containing absorbent material (type 3/4 dustfree embedding, supplied by SSNIFF, Soest, FRG).
- Diet (e.g. ad libitum): Kliba maintenance diet rat-mouse meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): drinking water; ad libitum
- Acclimation period: 7 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: doubly distilled water containing 0.1 % Cremophor EL

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was weighed in, depending on the dose group, filled up to the desired volume with doubly distilled water containing 0. 1 % Cremophor EL and mixed using a magnetic stirrer. These emulsions were prepared in intervals of no longer than 7 days.

VEHICLE
- Concentration in vehicle: 0.5, 2, 8 g/100 ml
- Amount of vehicle (if gavage): 10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses were carried out at Analytical Department of BASF Aktiengesellschaft, Ludwigshafen, FRG .
The stability of the test substance in doubly distilled water containing 0.05% Cremophor EL over a period up to 7 days at room temperature was tested prior to the start of the study.
Homogeneity analyses of the test substance preparations were performed in samples of the highest and lowest concentration at the start of the administration period. These samples also served for concentration control analyses.
Additional concentration control analyses were performed with samples from the mid concentration.

Duration of treatment / exposure:

28 d

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 200, 800 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control:

In several positive control studies, behavioral and neuropathological sequelae of substances with nervous system effects were evaluated using Functional Observational Batteries (FOB), Motor Activity Measurements and Neuropathology. These substances were 3,3'-Iminodipropionitrile, Carbaryl, Nomifensin, Diazepam, Acrylamide, Trimethyltinchloride

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. Additionally, further clinical examinations were carried out daily just before treatment, less than 1 hour after treatment and between 3 and 4 hours after treatment.



DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals


BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period; During the administration period the body weight was determined
on day 0 (start of the administration period) and thereafter at weekly intervals


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 calculated as time-weighted averages from the consumption and body weight gain data: No


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: Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly over a period of 7 days (food) or 4 days (water)


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning at the end of the application period
- Anaesthetic used for blood collection: Yes, Isoflurane
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning at the end of the application period
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex


URINALYSIS: Yes
- Time schedule for collection of urine: over night at the end of study period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period starting at about 10:00 a.m
- Dose groups that were examined: all
- Battery of functions tested: home cage and open field observations in a standard arena, sensorimotor tests, reflex tests, motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weights: liver, kidneys, adrenal glands, testes, epididymides, ovaries, uterus, spleen, brain, heart, thymus
HISTOPATHOLOGY: Yes
- all gross lesions, brain, pituitary gland, thyroid glands with parathyroid glands, thymus, trachea, lungs, heart, liver, spleen, kidneys, adrenal glands, testes/ovaries, uterus/vagina, epididymides, prostate gland, seminal vesicles, stomach (glandular and non-glandular), duodenum, jejunum, ileum (with parts of the pancreas), cecum, colon, rectum, urinary bladder, lymph nodes (mandibular and mesenteric), sciatic nerve, bone marrow (femur), eyes, spinal cord (cervical, thoracic and lumbar cord).

Other examinations:

The immunorelevant organs and tissues were evaluated according to the following parameters:
- Thymus: increased/decreased grade of cortico-medullar ratio (related only to area), increase of starry sky cells, changes of cellular density in the cortex, changes of cellular density in the medulla
- Spleen: altered cellular composition of follicles, changes of the cellularity of PALS, lymphoid follicles, marginal zone, red pulp, altered number of germinal centers
- Lymph nodes (mesenteric and mandibular lymph nodes): changes in the cellularity of follicles, interfollicular area, paracortical area, medulla, altered number of germinal centers, altered cellular composition of paracortex, changes of the cellularity of sinus
- Peyer´s patches (of the jejunum): changes of the cellularity of follicles (including mantle zone and germinal centers), changes of the cellularity of interfollicular area

Statistics:

Food/ water consumption, body weight/change, food efficiency: DUNNETT's test (two-sided)
Feces, rearing, grip strength forelimbs/hindlimbs, foot-splay test, motor activity: KRUSKAL-WALLIS test (two-sided) followed by Wilcoxon-test (two sided)
Clinical pathology parameters: KRUSKAL-WALLIS test (two-sided) followed by Wilcoxon-test (two sided)
Urinalysis: FISHER's exact test
Weight parameters: KRUSKAL-WALLIS test (two-sided) followed by Wilcoxon-test

Results and discussion

Results of examinations

Details on results:

Group 0: vehicle control
Group 1: 50 mg/kg bw/day
Group 2: 200 mg/kg bw/day
Group 3: 800 mg/kg bw/day


CLINICAL SIGNS AND MORTALITY
No animal died prematurely.
Anogenital region smeared with urine, slight to moderate, was observed in 3 female rats of group 3 towards the end of the study.
Piloerection was seen in all animals of group 3 and in 4 males of group 2 on several days of the study, starting on day 9.
The above-mentioned observations were signs of general systemic toxicity and therefore related to the test compound .
Slight to severe salivation was observed in almost every dosed animal throughout the entire study, predominately after treatment. This finding was most probably related to the physical property (acidity) of the test article.
Due to the isolated occurences and or the lack of a dose-response relationship the findings alopecia, eyelid halfclosure, slight extended abdomen and soft feces were assessed as not substance-related.

BODY WEIGHT AND WEIGHT GAIN
Body weight in males of group 3 was reduced, statistically signifcantly on days 21 and 28 (-16.1%).
Body weight change in male rats of dose group 3 was also statistically significantly decreased during the whole study period, up to -36 .6% at the end of the study.
These impaired body weight data were assessed as being treatment-related .

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption was reduced in males of group 3 up to -14.7% on day 28 of the study.
This finding was clearly substance-related.
The slight impairment of food consumption in females of all dose groups was not statistically significant, without a dose-response relationship as well as without a correlation to body weight data. It was therefore assessed as being incidental and not related to the test article.

FOOD EFFICIENCY
Food efficiency in males of group 3 was statistically significantly decreased over the entire study, with the exeption of study day 14.
This finding is explainable by the fact that food efficiency was calculated based upon values of body weight and food consumption and were therefore assessed as a result of the compound-related impairment of both parameters.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Water consumption was increased throughout the whole study in animals of dose group 3 each sex, statistically significantly on days 7 (+31 .7%) and 14 (+33 .7%) in males as well as day 7 (+ 38.6%) and 21 (+25.9%) in females.
This finding was assessed as related to the test compound .

HAEMATOLOGY
At the end of the study slight, but statistically significant decreases in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were found in the peripheral blood of the high-dose males .
No corresponding findings were seen in the erythrocyte indices of the females and no treatment-related changes were observed in the other hematology parameters of either sex.

CLINICAL CHEMISTRY
After 4 weeks of test substance administration alanine aminotransferase activities were increased in the sera of the high-dose animals of either sex.
In addition, increased gamma- glutamyltransferase activities were observed in the high-dose females.
No treatment related changes were seen in the other serum enzyme examinations.
Blood chemistry investigations revealed increased total bilirubin levels and decreased globulin concentrations in the high-dose males and females. Furthermore, in the serum of the high-dose females inorganic phosphate, calcium and triglyceride concentrations were increased and chloride level was decreased. Significantly increased triglycerides were also found in the serum of the mid-dose females.
No test substance-related changes were noted in the other blood chemistry examinations .

URINALYSIS
High-dose males produced decreased amount of urine with increased specific gravity, whereas high-dose females excreted increased amount of urine with decreased specific gravity. No treatment-related changes were seen in the other urine parameters of either sex.

NEUROBEHAVIOUR
Regarding the overall motor activity (summation of all intervals) no substance-related findings were observed in both sexes.
Comparing the single intervals with the control groups no substance-related findings were obtained in treated males as well as females.

ORGAN WEIGHTS
- Absolute weights:
In males of the high dose group, the mean terminal body weight was slightly although significantly decreased (- 19 .9%). In females of the high dose group, the mean terminal body weight was also slightly decreased (-7.1 %), however, this was not significant.
As a consequence of the significantly decreased mean terminal body weight in males of the high dose group, all mean organ weights were lower as compared to the control group.
However, only the mean weights of spleen (-20.7%) and thymus (- 31 .2%) were slightly although significantly decreased.
In females of the high dose group, the mean liver weight was slightly although significantly increased (+ 27.2%). A treatment-related effects was assumed for this.
The other mean absolute weight parameters did not show significant differences when compared with the control group .

- Relative weights (related to terminal body weight):
As a consequence of the significantly decreased mean terminal body weight in males of the high dose group, the following weights were significantly increased: liver (+ 21 .3%), testes (+ 20.4%), heart (+ 12.7%) and brain (+ 17.3%).
In female rats, in the mid and high dose groups, the mean liver weights were significantly increased (+ 10 .0% or + 36.9%, respectively).
A treatment-related effects was assumed for this.
In females of the low dose group, the mean weight of the kidneys was slightly although significantly increased (+ 11 .8%).
This was regarded incidental.
The other mean relative weight parameters (when related to terminal body weight) did not show significant differences when compared with the control group.

GROSS PATHOLOGY
No treatment related effects were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related findings were recorded in the pancreas. They consisted of slight, moderate or severe degeneration of the exocrine cells - represented as apoptotic exocrine epithelial cells without any further reaction or of degeneration of the exocrine cells (apoptosis) in combination with reactive soft tissue cell proliferation representing the picture of a progressive cirrhosis. The latter finding was noted in two female rats of the high dose group, only. Minimal degeneration of the exocrine cells of the pancreas - represented by only one focus of apoptotic cells in the whole tissue or only very few individual apoptotic exocrine cells distributed over the whole pancreas tissue was also noted in two control females.
This is indicative for the possibility of the spontaneous occurrence of such a finding in control and treated animals, and, hence, the presence of very few degenerative (apoptotic) exocrine cells as seen for example in a low dose male und in three females of the low dose group (as compared to no control male or two control females, respectively) was interpreted to be incidentally and not treatment-related.
The pathogenesis of the degenerative findings in the exocrine pancreas of male and female rats of the mid and high dose groups is not known.
Its treatment-related nature, however, is not doubted .
All other microscopic findings recorded were either single observations, or they occurred in control animals only, or they were recorded at comparable incidence and graded severity in control and high dose males and/or females. They were, hence, regarded fortuitous in nature and unrelated to the administration of the test article.
No morphologic correlate was obtained in the liver of high dose male and female rats that may explain its significantly increased mean absolute (females) or relative weights (males and females) .
Also, there was no morphologic correlate for the slight although significant decrease of the mean absolute weights of spleen and thymus as well as for the significantly increased mean relative weights of testes, heart and brain in male rats of the high dose group.
After a 4-week administration period, there was no indication of a morphologic affection of the organs of the central or peripheral nervous system, the reproductive system, and the immune system by the test article.

Effect levels

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: degeneration of the exocrine pancreas

Target system / organ toxicity

Critical effects observed:

not specified