Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: dermal
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:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
adrenals were not weighed
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Irgasan DP300
- Physical state: Off-white powder
- Analytical purity: not specified in the report, however, according to " Triclosan supplement I to EU dossier submitted 18 August 2009", the purity of CIBA-produced Triclosan exceeded 99%, and Irgasan DP300 with the batch No. 5.2.0211.0 was described with a purity of 99%
- Lot/batch No.: 5.2.0211.0
- Stability under test conditions: the stability of triclosan in vehicle was analytically confirmed throughout the study period.
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Designation: Crl:CDBR (VAF/Plus) rats
- Source: Charles River Laboratories, Inc., Kingston Facility, New York, USA
- Age at study initiation: males 7-8 weeks, females 9-10 weeks
- Weight at study initiation: males 245.6-287.2 g,females 193.5-242.2 g
- Fasting period before study: no
- Housing: individually, in suspended stainless steel and wire mesh cages with absorbent paper below
- Diet (e.g. ad libitum): Purina Certified Rodent Chow (mash), ad libitum
- Water (e.g. ad libitum): from Elizabethtown Warer Company, at libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 40 - 70%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hrs / 12 hrs

Administration / exposure

Type of coverage:
occlusive
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF THE TEST MATERIAL
The test material was diluted in carrier at the scheduled concentrations; the mixture was prepared weekly and stored at room temperature

TEST SITE
Initially, approximately 24 hours prior to the first topical administration of the test material, the fur of each rat on the dorsal surface from the shoulder region to the lumbar region was closely clipped with an electric dipper. The skin was left intact. The animals were reclipped once per week and as needed, unless contradicted by the presence of severe dermal irritation.
The treated area was about 10% of the body surface and after application of the test material, it was covered by means of a gauze pad placed on the dose site, covered again with Blenderm tape, and wrapped with COBAN to prevent evaporation and ingestion of the test material.

REMOVAL OF TEST SUBSTANCE
After a daily contact exposure time of at least 6 hours, the dressing was removed. For removal of residual test material, the application site was gently cleaned by wiping with water and paper towel.

TEST MATERIAL
The test material was applied onto the skin at a volume of 2 mL/Kg bw, following test concentrations were used: 0, 0.5, 2.0, and 4.0% (w/v); control animals received carrier only at a volume of 2.0 mL/kg bw.

VEHICLE
Since Triclosan was soluble in Propylene glycol at the concentrations used, this vehicle was used.
Supplier: J. T. Baker Company, Phillipsburg, N.!.
Batch Number: C40638
Date Received: October 2, 1990
Description: Colorless liquid
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily, 6 h/day
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 40, 80 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10/sex/dose level
Control animals:
yes, concurrent no treatment
Details on study design:
The study comprised following 5 test groups:
Group 1 served as the carrier control group and received propylene glycol (PPG) only.
Groups 2, 3, and 4, received 10, 40, and 80 mg/kg of triclosan in PPG, respectively.
Group 5 served as a satellite recovery group. Satellite animals were treated the same as those of group 4 and were observed for reversibility, persistence or delayed occurrence of toxic effects for at least 28 days after treatment.

Examinations

Observations and examinations performed and frequency:
CLINICAL SYMPTOMS AND MORTALITY
The animals were checked twice daily (on weekends and holidays: once daily) for mortality, and once for clinical symptoms.

BODY WEIGHT
Body weights were recorded before treatment and once weekly thereafter.

FOOD CONSUMPTION
Food consumption was recorded once weekly.

WATER CONSUMPTION
Not measured

OPHTHALMOSCOPIC EXAMINATION
The eyes were examined prior test initiation and one week prior to test termination.

HAEMATOLOGY AND CLINICAL CHEMISTRY
At main study termination. blood was collected from the retroorbital sinus of all animals while under methoxyflurane anesthesia. All animals were fasted overnight prior to blood collection. Blood was collected again from the satellite animals in the same manner at satellite termination.
Following haematological parameters were examined: erythrocyte count, haemoglobin concentration, haematocrit, leukocyte count, differential leukocyte count MCH, MCHC, MCV, prothrombin time, activated partial thromboplastin time, thrombocyte count.
Following clinical chemical parameters were examined: albumin, glucose, cholesterol, urea, total bilirubin, calcium, creatinine, total protein, triglycerides, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), P, Cl-, Na+, K+, CO2.

URINALYSIS
Urine samples were collected from all animals prior to main study termination and all satellite animals prior to satellite termination. Animals were not fasted during the urine collection interval. Following parameters were examined: colour and appearance, sediment, ketones, pH, blood, bilirubin, urobilinogen, glucose, volume, density, protein.
Sacrifice and pathology:
SACRIFICE
For the purpose of necropsy, the main study was terminated after at least 13 weeks of dosing. Animals were sacrificed via exsanguination following methoxyflurane anesthesia. The satellite animals were terminated after at least a 28 day recovery period. The procedure was the same as in the main study termination. Animals which died or needed to be sacrificed in extremis also were subjected to necropsy.

ORGAN WEIGHT
From all animals sacrificed at termination, brain, gonads(testes/ovaries), liver, spleen and kidneys were weighed.

GROSS PATHOLOGY
Gross pathological examination was conducted on all sacrificed animals

HISTOPATHOLOGY
The following tissues were removed from an animals and preserved in 10% neutral buffered formalin: adrenal, aorta (thoracic), brain (cerebrum, cerebellum, brainstem), epididymides, oesophagus, exorbital lachrymal glands, eyes, femoris muscle with sciatic nerve, heart, kidneys, large intestine (sections from colon and caecum), liver, lungs (with mainstem bronchi), mammary gland (inguinal, female only), mesenteric lymph nodes, ovaries and oviducts, pancreas, pituitary, prostate, rectum, salivary glands, seminal vesicles, skin (treated and untreated) small intestine (sections from duodenum, jejunum, ileum), spinal cord (cervical, midthoracic, lumbar), spleen, sternum with marrow, stomach, testes, thymus, thyroid with parathyroids, trachea, urinary bladder, uterus (corpus, cervix), all gross lesions.
Tissues from the control group, the high dose group as weIl from all animals that died or were sacrificed in extremis during the study, were collected, processed. sectioned, stained (hematoxylin/eosin) and examined microscopically. Where changes or equivocal results were seen, then tissues affected were examined in the other groups. The lungs, liver, kidneys and gross lesions from the low and mid dose groups were also be processed and examined microscopically.
Other examinations:
Dermal responses were evaluated prior to dosing on days 0, 1, and 4, and twice weekly thereafter until study termination.
Statistics:
Statistical treatment of the results was conducted where appropriate.
Statistical evaluation of equality of means was done by an appropriate one way ANOVA and a test for ordered response in the dose groups. First, Bartlett’s Test was performed to determine if the dose groups have equal variance. If the variances were equal, the testing was done using parametric methods, otherwise nonparametric techniques were used.
For the parametric procedures, a standard one way ANOVA using the F distribution to assess significance was used. If significant differences among the means were indicated, Dunnett's Test was used to determine which treatment groups differed significantly from control. In addition to the ANOVA, a standard regression analysis for linear response in the dose groups was performed. The regression also tested for linear lack of fit in the model.
For the nonparametric procedures the test of equality of means was performed using the Kruskal-Wallis Test. If significant differences among the means were indicated, Dunn’s Summed Rank Test was used to determine which treatment groups differed significantly from the control. In addition to the Kruskal-Wallis Test, Jonckheere’s Test for monotonic trend in the dose response was performed.
Bartlett’s Test for equal variance was conducted at the 1% level of significance. All other tests were conducted at the 5% and 1% level of significance.
The statistical t-test was also used to compare the high dose and satellite animals data (where appropriate) to substantiate theft equivalence in order to accurately evaluate the recovery effect.

Results and discussion

Results of examinations

Details on results:
Five animals died prior to scheduled termination. One control male was found dead on day 22, one 80 mg/kg bw/day male was found dead on day 84, one satellite (80 mg/kg bw/day) male was euthanized on day 84, one 40 and one 80 mg/kg bw/day female were found dead on day 7. All mortalities were considered unrelated to the test material.
Clinical signs observed during the test period were minimal with the majority of animals being free of abnormalities during the study. There was a low incidence of scabs, sores, alopecia, dental abnormalities, ocular discharge, and/or abnormalities of the tail observed intermittently in one or more groups. Additionally, one 10 mg/kg female was observed as emaciated, one satellite female had a swollen limb, one satellite female had a swollen ear, one control male was observed with soft stool, and one satellite male had red material adhering to the penis, hypothermia, and pale extremities. All of these findings were incidental and were not considered to be treatment-related.
Repeated topical applications of triclosan elicited erythema and/or oedema in all treated groups (10, 40, and 80 mg/kg bw/day; and satellite recovery). Erythema was not observed in any control animal.
Erythema was first observed on day 4 in the 40 mg/kg bw/day dose group (mid dose) and in both 80 mg/kg bw/day groups (high dose and satellite group). Erythema ranged from absent to severe. The severity of the erythema increased as the study progressed, and the majority of animals in the mid, high, and satellite dose groups were observed with severe erythema from day 7 until dosing termination.
Erythema also was observed in the low dose group (10 mg/kg bw/day) but the onset of erythema did not occur until day 21. Erythema ranged from well-defined to severe in several animals, but the frequency of severe erythema scores of the low dose group was much less than observed in the higher dose groups. Erythema was also observed in one control animal. However, this erythema was judged to be mechanically induced.
Oedema was observed sporadically in the mid, high and satellite group animals. Oedema ranged from very slight to slight and was observed primarily during the latter part of the study. Very slight oedema was observed in one low dose animal. Oedema was not observed in any control animal.
Supplemental dermal observations consisting of eschar and desquamation were observed in all treated groups. More severe signs of dermal irritation, characterized by exfoliation and atonia, were generally limited to the mid, high and satellite recovery groups. Desquamation was also observed in two control animals at a very low frequency. Eschar was observed in one control animal but this irritation was considered mechanically induced.
Dermal irritation decreased dramatically in the satellite recovery group following dosing cessation. By study termination on day 121, only one recovery animal was observed with erythema (severe) and eschar.
Body weight and food consumption showed no treatment-related effects, and ophthalmoscopy was inconspicuous.
No treatment-related effects of toxicological significance were reported for the haematological and clinical-chemical parameters, and for urinalysis. In fact, urinalysis had revealed occult blood in the urine of a small number of high dose and satellite male animals, and to a lesser degree in the mid dose males, at main study termination. This was observed in the female animals, but with decreased severity and incidence. In the absence of any other significant clinical chemistry or haematological changes or correlation to microscopic findings, the significance of this finding is unknown.
Organ weighing revealed no differences between control and treated groups, indicating no dose-related effects.
Referring to gross pathology, dermal irritation (desquamation, exfoliation, and/or eschar) was the only treatment-related macroscopic finding.
Histopathological treatment-related changes were confined to the treated areas of the skin. Microscopic examination of sections of the treated skin showed several changes, the most common of which were hyperplasia/hyperkeratosis of the epidermis, sebaceous gland hyperplasia, dermal inflammation, focal epidermal necrosis, and exudates.
In the mid dose male rats (40 mg/kg bw/day group), there was a marginal increase in the severity of sebaceous gland hyperplasia and hyperplasia/hyperkeratosis of the epidermis, as compared with controls. The same changes were more pronounced (increased severity) in the high dose male rats (80 mg/kg bw/day), as compared with controls. Male rats from both Groups 3 and 4 also showed increased incidences of dermal inflammation focal epidermal necrosis, and exudate formation. Examination of treated skin areas from satellite recovery males showed a return to near control levels for the incidence and/or severity of these treatment-related changes; the satellite animals, however, did show increased dermal fibrosis, as compared with controls and other treatment animals.
Examination of the treated areas of the skin from female rats from treatment Groups 2, 3, and 4 in general showed a spectrum of changes similar to those described for the male rats. In particular, there was a dose-related increase in the severity of sebaceous gland hyperplasia, hyperplasia/hyperkeratosis of the epidermis, dermal inflammation, and focal epidermal necrosis as well as an increased incidence of exudate formation, as compared with controls. Microscopic examination of treated skin areas from the female satellite recovery animals showed a decrease in the incidence and/or severity of these changes as compared with non-recovery animals. As with the mate animals however there was an increase in the incidence of dermal fibrosis with recovery.
Although changes were found in the "treated" skin from controls (sebaceous gland hyperplasia and hyperplasia/hyperkeratosis of the epidermis), the degree of severity was less than that observed for the treated animals. The changes in the skin from the control animals were considered to have been the result of the clipping, application procedures, and the wrapping of the skin.
Sections of the untreated skin examined microscopically showed an increased incidence of epidermal hyperkeratosis in the satellite male rats. In general this change was minimal and considered to be the result of the clipping procedure and possible contamination of the untreated site and not related to test material administration. Also, in the female rats, the incidence and severity of the epidermal hyperkeratosis was similar between the control, high dose, and satellite animals.
Findings associated with non-dermal tissues were considered to be isolated incidental findings unrelated to the test material.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Effect level:
> 80 other: mg/kg
Sex:
male/female
Basis for effect level:
other: see `Remark´

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion