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Repeated dose toxicity: dermal

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Administrative data

sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 1999 - May 2000
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
according to guideline
EPA OPPTS 870.3250 (Subchronic Dermal Toxicity 90 Days)
Version / remarks:
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
Specific details on test material used for the study:
- Glutaraldehyde 50% aq. sol.
- Supplier: sponsor
- Physical state: colorless clear liquid
- Analytical purity: 50.3%
- Lot/batch No.: 50-4402
- Stability under test conditions: the stability of the test substance was guaranteed until February 2000 by the sponsor
- Storage condition of test material: at + 4 °C, under nitrogen

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River, Saint-Aubin-lès-Elbeuf, France (Breeder)
- Age at study initiation: about 9 weeks old
- Weight at study initiation: mean body weight of the males, 340 g (320 g- 362 g); mean body weight of the females, 221 g (207 g – 243 g)
- Fasting period: at least 14 hours fasting prior sacrifice at test ending
- Housing: individually in suspended wire-mesh cages, with metallic tray, containing autoclaved sawdust placed under each cage.
- Diet (e.g. ad libitum): A04 C pelleted maintenance diet
- Water (e.g. ad libitum): filtered tap water (0.22 micron filter)
- Acclimation period: 6 days

- Temperature (°C): 21 +/- 2
- Humidity (%): 50 +/- 20
- Air changes (per hr): 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 hrs / 12 hrs

Administration / exposure

Type of coverage:
Details on exposure:
- The route of administration was dermal;
- Protectol GDA was diluted with water to give concentrations of 5, 10 and 15% test concentrations;
- The applied test volume was 2 ml/kg bw;
- The test solution was applied to the clipped dorsal skin of each animal;
- The site of application was about 10% of the total body surface;
- The application site was covered with a gauze held in place with a semi-occlusive dressing for 6 hours;
- Following removal of the dressing, residual test substance on the skin was removed using a gauze pad moistened with water;
- The control animals were treated similarly as above, but with the vehicle only (i.e. water).
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The concentration of a sample taken from each dosage form (including the control) prepared for use in weeks 1, 4, 8 and 13 was determined by means of UV spectrophotometry at 233 nm; the concentration of Protectol GDA was determnined from a calibration curve obtained by linear regression analysis of absorbance against concentration of Protectol GDA in standard solutions (external standard calibration).
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
The animals were treated 5 days a week over a period of 13 weeks, implying 67 days of test substance-administration.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day
(active ingredient); calculated on the basis of an application volume of 2 ml/kg bw;
5 % test material, 2.5% (active ingredient)
Dose / conc.:
100 mg/kg bw/day
(active ingredient); calculated on the basis of an application volume of 2 ml/kg bw;
10 % test material, 5% (active ingredient)
Dose / conc.:
150 mg/kg bw/day
(active ingredient); calculated on the basis of an application volume of 2 ml/kg bw;
15 % test material, 7.5% (active ingredient)
No. of animals per sex per dose:
Control animals:
yes, concurrent no treatment


Observations and examinations performed and frequency:
Starting from the day prior test initiation, the animals were checked at least once daily for clinical signs of toxicity (including mortality) and signs of skin irritation. In addition to these standard examinations, the animals also were subjected once a week to a more detailed examination of following parameters: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, autonomic activity, changes in gait and posture, reaction to handling, presence of clonic or tonic movements, and stereotypical or bizarre behaviour.

The body weight of each animal was recorded prior allocation of the animals to each test group, on the first day of treatment and thereafter once a week over the whole treatment period.

Food consumption for each animal was recorded once a week over a 7-day period, from test initiation until test ending.

Not considered

Not considered

All animals were subjected to ophthalmologic examinations prior test initiation. At test ending, the eyes of the control animals and of the animals of the high-dose group were examined.

Blood samples were collected via orbital sinus puncture from all surviving animals at the end of the experimental period (week 13). Prior to blood sampling, the animals were fasted overnight. The blood samples served for the evaluation of the haematological and clinical-chemical parameters.
Following haematological parameters were considered: erythrocytes, hemoglobin, mean cell volume, packed cell volume, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocytes, leucocytes, differential white cell count with cell morphology (neutrophils, eosinophils, basophils, lymphocytes, monocytes), and prothrombin time.
Following clinical-chemical parameters were considered: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total protein, albumin, albumin/globulin ratio, cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase.

Not considered

See clinicals symptoms

Sacrifice and pathology:
At the end of the treatment period the animals were fasted for at least 14 hours and were then sacrificed for necropsy. Animals that died during the experiment also were subjected to necropsy.

The animals were weighed a last time prior sacrifice, and after sacrifice, following organs were taken for weighing: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus. Both, the absolute and the relative organ weights were determined.

Following sacrifice, all animals were subjected to a complete macroscopic examination, including the external surfaces, all orifices, the cranial cavity, the brain surface, the surface of the spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues, and the neck with its associated organs and tissues.

Following organs/tissues were preserved for the purpose of histopathology: all gross lesions, adrenals, aorta, brain (cerebellar and cerebral cortex, medulla/pons), caecum, colon, duodenum, epididymes, eyes with Harderian glands, femoral bone with articulation, heart, ileum, jejunum, kidneys, larynx, liver, lung with bronchi, lymph nodes, mammary glands, nose, oesophagus, optic nerve, ovaries, pancreas, pharynx, pituitary gland, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroids and parathyroids, tongue, trachea, urinary bladder uterus and vagina.
Following organs/tissues were embedded in paraffin wax, sectioned and stained with hematoxylin-eosin for microscopical examination:all organs/tissues listed above for all sacrificed animals of the control group and of the high-dose group (excepted for the femoral bone, the skeletal muscle, the tongue and the vagina), all organs/tissues listed above for all animals that died or had to be killed prematurely during the experiment, and all gross lesions.
A series of tests were used for statistical assessment of the findings, including among other Kolmogorov-Lilliefors´ test, Fisher´s test, Bartlett´s test and Student´s test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Poor condition including pallor of eyes and/or extremities, piloerection, round back, tremors, staggering gait, half-closed eyes and/or dyspnea, was seen in week 11 or 13 in 4 animals (1 male, 3 females) of the 100 mg/kg bw group. Soft feces were seen in one male of the 50 mg/kg bw group, cutaneous lesions on the abdomen were reported for a further male of this group (from week 12). Exophthalmus, and hair loss on one forelimb were respectively reported for one male (from week 11) and one female (from week 10) of the 150 mg/kg bw group. The low incidence of clinical signs and the absence of a dose-response relationship indicated that the clinical effects were not treatment-related.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Signs of local irritation, including scabs, desquamation and very slight or well-defined erythema, were noted at the application site of almost all treated animals.
mortality observed, non-treatment-related
Description (incidence):
In the control group, one male rat died on day 44 from acute rhinitis. A female was killed in extremis on day 46 for ethical reasons.
In the 50 mg/kg bw group, one male was found dead on day 86; no clinical signs had been seen prior to death. The low incidence of mortality, the absence of a dose-relationship and the occurrence of mortality in the control group indicated that mortality was not treatment-related.
In the 100 mg/kg bw group, one male was found dead on day 80; prior to death the animal showed poor clinical condition.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
For the males, no significant differences between treated and control groups were seen. In fact, individual differences in body weight were observed in all control and treated groups, which were not considered to be treatment-related.
For the females, the mean final body weight was slightly decreased compared to control in both the 100 and the 150 mg/kg bw/day groups. In fact, the mean body weight of females given 150 mg/kg bw/day was slightly lower than that of control animals. The observed difference was statistically significant on weeks 2 and 5 and decreased thereafter. Except for week 13, when a statistically significant lower mean body weight was recorded, the mean body weight of females given 100 mg/kg bw/day was similar to that of controls. The mean body weight of females given 50 mglkglday was similar to that of controls. Thus, the slight effect seen here were not considered to be treatment-related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption for both sexes was similar for all groups including control.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effects were seen. In fact, ophthalmologic examinations prior test initiation revealed findings such as variation in corneal thickness and vacuolization of the cornea, which mainly were seen in the control group. On week 13, ophthalmological examination revealed hyphemia, exophthalmia and luxation of the lens in one male of the 150 mg/kg bw/day group.
Haematological findings:
no effects observed
Description (incidence and severity):
In males of the 50 mg/kg bw/day group, slightly increased mean cell volume and mean cell haemoglobin as well as slightly decreased erythrocyte count and mean cell haemoglobin concentration were reported.
In males of the 100 and 150 mg/kg bw/day groups and in females of the 50 mg/kg bw/day group, increased eosinophil count was reported.
In females of the 150 mg/kg bw/day group, increased thrombocyte count was reported.
All these effects however were slight, without any dose-response relationship and had values in the range of historical control data. They were therefore considered to be of no biological/ toxicological relevance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
In males and females of the 100 and 150 mg/kg bw/day groups, lower glucose levels were reported.
In females of the 100 and 150 mg/kg bw/day groups, lower calcium levels were reported.
These effects were slight, without any dose-relationship and without any pathological significance. They therefore were considered to be of no biological/ toxicological relevance.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Differences in organ weights were minimal and without any dose-relationship or correspondence with microscopical findings.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Yellowish coloration of the skin was seen in control and test animals. Due to lack of corresponding microscopic findings this was regarded as being of no toxicological importance. Scabs were seen in all treated animals; they were associated with microscopic findings and were considered to be treatment-related. Changes in liver (yellowish or greyish areas, colored foci, changes in size or consistency) were seen both in control and treated animals (similar incidence and degree of severity). These changes were therefore not considered to related to the treatment.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Slight to moderate acanthosis together with the presence of inflammatory cells in the dermis was reported for the skin of treated animals. Furthermore, ulceration and scabs also were reported for the treated animals. The incidence and severity of these skin findings did not differ notably between the individual treated groups. These skin findings were considered to be treatment-related.
Lesions in the liver included coagulative hepatocellular necrosis, interlobular fibroplasia, tension lipidosis and macrophages with yellow pigment contents. These findings were in accordance with the macroscopical findings in the liver and were found with similar incidence and severity in both, treated and control animals. The effects affecting the liver possibly were related to the wearing of bandage and slight compression at the abdomen.

Effect levels

Dose descriptor:
Effect level:
150 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: no adverse effects observed up to the highest tested dose.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Analytical monitoring revealed a good agreement between the nominal and measured concentrations of the test substance in all dosage forms. In fact the deviation of the measured from the nominal values did not exceed 6%. 

Summary of the mean body weight data:


Body weight

Dose level (mg/kg bw/day)






Initial body weight (g/animal)





Final body weight (g/animal)





Body weight gain (g/animal; week 1 to13)





Body weight gain (%)






Initial body weight (g/animal)





Final body weight (g/animal)





Body weight gain (g/animal; week 1 to 13)





Body weight gain (%)





*, p<0.05


Applicant's summary and conclusion