Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Experimental Toxicology and Ecology, BASF SE
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of test substance: Butandiolformal
Test item No.: 09/0372-1
Batch identification: 30133467J0
Purity: 97.2g/100g
Homogeneity: Homogeneous

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age (at supplied): 34-36 days
- Age (at study initiation): 41-43 days
- Supplier: Charles River Laboratories, Sulzfeld, Germany
- Weight at study initiation:
- Housing:
the rats were housed together (5 animals per cage) in H-Temp polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Bedding in the cages were Type Lignocel PS 14, dust-free bedding, supplied by SSNIFF, Soest, Germany.
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP” meal, Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
drinking water
Details on oral exposure:
The test-substance preparations were produced at least every 4th day a week.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in drinking water at room temperature for a period of 4 days was proven before the start of the administration period.
Homogeneity and concentration control analyses of the test-substance preparations were performed in samples of all concentrations at the start of the administration period.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0 , 100, 300 and 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
Butandiolformal was administered to groups of 5 male and 5 female Wistar rats at dose levels of 0 mg/kg bw/d (vehicle control; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3) over a period of 4 weeks by gavage.

Examinations

Observations and examinations performed and frequency:
Food consumption and body weights were determined weekly. Drinking water consumption was monitored daily.
The animals were examined for signs of toxicity or mortality at least once a day.
In addition, the animals were examined daily for any clinically abnormal signs before and after treatment.
Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter.
Moreover, a functional observational battery (FOB) as well as motor activity measurements were carried out at the end of the administration period.
Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period.
Blood was taken from the retroorbital venous plexus and urinalysis was performed in metabolism cages.

Following hematological parameters were determined:
Leukocyte and erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, hemoglobin and hemoglobin concentration, platelet count, differential blood count, reticulocytes, prothrombin time (Hepato Quick’s test).
Following clinical chemistry parameters were determined:
Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), gamma-Glutamyltransferase (GGT), Sodium, Potassium, Chloride, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol, Magnesium, Bile acids.
Urinalysis included:
pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, color, turbidity, volume.
Sacrifice and pathology:
After the administration period all animals were sacrificed and assessed by gross pathology, followed by histopathological examinations.
The animals were sacrificed by decapitation under isoflurane anesthesia.
Organ weights:
Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles with coagulation glands, Spleen, Testes, Thymus, Thyroid glands, Uterus with cervix.
Histopathology:
All gross lesions, Adrenal glands, Bone marrow (femur), Brain, Cecum, Cervix, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum, Jejunum(with Peyer’s patches), Kidneys, Liver, Lungs, Lymph nodes (mesenteric and axillary lymph nodes), Ovaries, Pituitary gland, Prostate, Rectum, Seminal vesicle with coagulation glands, Sciatic nerve, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cords), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.

THerefore all immunorelevant organs and tissues were evaluated and special attention was given for the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina.
Statistics:
Body weight, body weight change - DUNNETT's test (two-sided) for the hypothesis of equal means
Organ weights, Feces, rearing, grip strength length forelimbs, grip strength length hindlimbs, foot-splay test, motor activity, clinical pathology parameters, urine volume, urine specific gravity - Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided).
Urinalysis, except color, turbidity, volume and specific gravity - Pairwise comparison of each dose group with the control group using FISHER's exact test for the hypothesis of equal proportions.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see below
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Analytics:
The various analyses confirmed:
• the stability of the test-substance preparations over a period of up to 4 days at room temperature,
• the homogeneous distribution of the test article in the vehicle,
• the correctness of the prepared concentrations.

No test substance-related, adverse findings were observed.

Mortality:
One female rat of test group 100 mg/kg bw/d was found dead after the application on study day 3. This finding was clearly incidental.

Clinical examinations:
Salivation before and after treatment was seen in 3 males and all females of test group 1000 mg/kg bw/d. This finding was observed on day 6 for the first time and afterwards on several days of the study. This finding was assessed as non adverse, treatment independent conditioning effect.

Food consumption:
No test substance-related effects.

Body weight:
No test-substance related effects.

FOB and motor activity measurement:
No test-substance related effects.

Hematology:
No treatment-related changes among hematological parameters were measured.
(In males of test groups 100 and 1000 mg/kg bw/d the hematocrit values were higher compared to controls, but still within the historical control range (0.384-0.432 L/L). In females of test group 300 mg/kg bw/d the mean corpuscular hemoglobin concentration (MCHC) was lower compared to controls, but the mean was still in the historical control range (22.04-23.55 mmol/L). Both parameter values were not changed dose-dependently and, therefore, they were regarded as being not treatment-related. In males of test group 1000 mg/kg bw/d the relative monocyte counts were increased incedentaly.)

Clinical chemistry:
No treatment-related, adverse changes among clinical chemistry parameters were measured.
In males of test group 300 mg/kg bw/d the urea level was increased. In females of the same test group the sodium concentration was increased and in females of test group 1000 mg/kg bw/d the cholesterol concentration was higher compared to controls. The urea levels in male rats and the sodium concentration in females were not changed dose-dependently. All three parameters were altered without any change in other parameters and, therefore, these alterations were regarded as non-adverse (ECETOC Technical Report No. 85, 2002).

Urinalyses:
No treatment-related changes among urinalyses parameters were measured.
In male rats of test group 1000 mg/kg bw/d the incidences of transitional cells and casts in the urine sediment were higher compared to controls. These observations were confirmed by the histopathological findings of an alpha2µ globulinuria and were regarded as non-adverse.
In female rats of test group 100 mg/kg bw/d the urine volume was lower and the specific gravity was higher compared to controls, whereas in females of test group 1000 mg/kg bw/d the urine volume was higher and specific gravity lower compared to controls. These changes stood alone without any other indication of renal dysfunction and reflected the normal adaptation of the urinary flow correlating with the water intake. Therefore, a relation to treatment was excluded.

Absolute organ weights:
A significant, treatment-related increase of the absolute and relative mean liver weights in females of test group 1000 mg/kg bw/d was observed (see table 1).
A minimal, but significant decrease of the prostate weight was observed in males of test group 100 mg/kg bw/d (see table 1). This finding occurred without dose-relationship and was considered to be incidental.

Gross lesions
All gross lesions observed in test animals occurred singularly. They were considered to be spontaneous lesions in origin and not related to treatment.
Histopathology
In the kidneys of male rats, tubular eosinophilic droplets were minimally to slightly increased in test groups 300 and 1000 mg/kg bw/d when compared to controls. Hyaline droplets in the proximal convoluted tubules most likely reflect the presence of reabsorbed alpha2µ globuline, a poorly hydrolyzable, low molecular weight protein characteristic for male rats. However, an increase of hyaline droplets, as observed starting at 300 mg/kg bw/d, was considered to be a treatment-related effect. This finding is specific for male rats and not predictive for humans. Since no tubular injury was found in this study, the hyaline droplets increase was regarded as non-adverse.
No clear histopathological correlate was found in the liver of female rats of test group 3 (1000 mg/kg bw/d) that could explain the statistically significant increase of the absolute and relative weight. Nevertheless, the weight increase was regarded as a treatment-related adaptive effect due to an enzyme induction as detoxifying mechanism.


Effect levels

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no relevant signs of general systemic toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Relative liver and prostate weights.

 

Male animals

Female animals

Test group (mg/kg bw/d)

1

(100)

2

(300)

3

(1000)

1

(100)

2

(300)

3

(1000)

Liver

-

-

-

96%

111%

122%**

Prostate

85%*

98%

110%

-

-

-

* : p0.05; **: p0.01

Applicant's summary and conclusion