Registration Dossier

Toxicological information

Repeated dose toxicity: dermal

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 19, 2011 to June 14, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study. Study well performed with good scientific standards.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TPI 1618
- Substance type: Formaldehyde releaser
- Physical state: Clear colourless liquid
- Analytical purity: 100%
- Composition of test material, percentage of components: Formaldehyde releaser (reaction product of ethylene glycol, urea and paraformaldehyde)
- Lot/batch No.: 1179742
- Expiration date of the lot/batch: June 2013
- Stability under test conditions: confirmed analytically in dosing solutions for at least over 24 hours
- Storage condition of test material: ambient
- Other: Dosing solutions were stored deep frozen (-80°C) until analyses

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: males 53 days; Females: 67 days
- Weight at study initiation: males: 233.9 - 268.2 g; females: 213.9 - 235.5
- Fasting period before study: no
- Housing: single in Macrolon Type III cages
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +-3
- Humidity (%): 55 +-15
- Photoperiod (12 hrs dark /12 hrs light)

IN-LIFE DATES: From: November 22, 2010 to December 20, 2010

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: dorsal body surface (8 x 5.7 cm to 8 x 12.9 cm, depending on body weight)
- % coverage: 20%
- Type of wrap if used: semi-acclusive sterile gauze, held in place with non-irritating tape
- Time intervals for shavings or clipplings: days -1, 6, 15, 22 and 28

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no washing

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0, 40, 80 and 160 mg/kg bw
- Concentration (if solution): 0, 0.2, 0.4 and 0.8%
- Constant volume or concentration used: yes, 20 mL/kg bw

VEHICLE
- Justification for use and choice of vehicle (if other than water): tap water was used to comply with mode of employ


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the dosing solutions were performed for concentration (at study start and termination) as well as for homogeneity and stability with a validated method. In principle, with this method, the gaseous formaldehyde was measured by HPLC/UV after derivatization with DNPH. Results showed that the formulations were prepared correctly.
Duration of treatment / exposure:
28 days. Animals were exposed by dermal application to a shaved dorsal skin area.
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 40, 80 and 160 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5 males and 5 females/group; 3 dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels had been selected based on to be investigated concentrations of dosing solutions of 0.2, 0.4 and 0.8% at an application volume of 20 mL/kg bw and on available toxicological data.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behavior patterns. The onset, intensity and duration of any signs observed were recorded

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed individually before and after dosing at each time of dosing for any signs of behavioral changes, reaction to treatment or illness

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Daily at the end of the 6 hours exposure period

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
The quantity of food left by individual animals was recorded on a daily basis throughout the experimental period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week.

FOOD EFFICIENCY:
- No data

WATER CONSUMPTION:
Drinking water consumption was monitored daily by visual appraisal throughout the study.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the end of the study
- Dose groups that were examined: all (all animals)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 28 days
- Anaesthetic used for blood collection: Yes (light ether anesthesia)
- Animals fasted: Yes, overnight prior to sampling
- How many animals: all
- Parameters checked: haemoglobin content, erythrocytes, leucocytes, differential blood count, reticulocytes, platelets, haematocrit value, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), thromboplastin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
-- Time schedule for collection of blood: after 28 days
- Anaesthetic used for blood collection: Yes (light ether anesthesia)
- Animals fasted: Yes, overnight prior to sampling
- How many animals: all
- Parameters checked: albumin, globulin, albumin/globulin ratio, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea (in blood), calcium, chloride, potassium, sodium, alanine aminotransferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH),

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals from all dose groups with examination of the external appearance, and the appearance of tissues and organs in the cranium, the thoracic and the abdominal cavity.

HISTOPATHOLOGY: Yes
Organs removed and preserved: adrenal gland (2), aorta abdominalis, bone (os femoris with joint), bone marrow (os femoris), brain (cerebrum, cerebellum, brain stem), caecum, epididymis (2), eye with optic nerve and Harderian gland (2), gross lesions observed, heart (left and right ventricle, septum) intestine, large (colon, rectum), intestine, small (duodenum, jejunum, ileum, incl. Peyer´s patches (Swiss roll method), kidney and ureter (2), liver, lungs (with mainstem bronchi and, bronchioles), lymph node (cervical) (1), lymph node (mesenteric) (1), mammary gland, muscle (skeletal, leg), nerve (sciatic), oesophagus, ovary (2), pancreas, pituitary, prostate, salivary glands (mandibular, parotid, sublingual), seminal vesicle, skin (left flank, treated and untreated, 3 sections each), spinal cord (3 sections), spleen, stomach, testicle (2), thymus, thyroid (2) (incl. parathyroids), tissue masses or tumors, (including regional lymph nodes), tongue (incl. base), trachea (incl. larynx), urinary bladder, uterus (incl. cervix and oviducts) and vagina.
The afore-listed organs of all animals of groups 1 and 4 were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining.
In addition, frozen sections of the heart, liver and one kidney were made and stained with Oil Red O and examined microscopically.
Furthermore, three subsections per administration site (treated and untreated) were prepared from all animals, stained with haematoxylin-eosin after preparation of paraffin sections and examined histologically.
Statistics:
The test item-treated groups 2 - 4 were compared to the control group 1. The following statistical methods were used:
Multiple t-test based on DUNNETT, C. W. New tables for multiple (p  0.01) comparisons with a control, Biometrics, 482-491 (September 1964) was used for body weight / food consumption, relative organ weights. The following limit was used: p  0.01 ^ t = 3.39 (for 16 degrees of freedom)
Exact test of R. A. FISHER for histology (p  0.05)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No effects

SKIN IRRITATION
No local intolerance reactions were noted for the female animals treated with 40 mg TPI 1618/kg b.w./day and for the male animals treated with 160 mg TPI 1618/kg b.w./day.
A very slight erythema was noted for one of 5 male animals treated with 40 mg TPI 1618/kg b.w./day on test day 25. Furthermore, very slight erythemas were noted for 2 of 5 males and 4 of 5 females treated with 80 mg TPI 1618/kg b.w./day on 1 to 7 test days starting on test day 4 and for all female animals treated with 160 mg TPI 1618/kg b.w./day on 5 to 12 test days starting on test day 4. At termination, only 2 of 5 females treated with 80 or 160 mg TPI 1618/kg b.w./day showed very slight erythemas. No such findings were seen in male animals at termination. Therefore, TPI 1618 had no consistent effect on the skin as the findings were neither time- nor dose-related and occurred only sporadically. No histopathological changes of the treated skin sites were detected.

BODY WEIGHT AND WEIGHT GAIN
No effects

FOOD CONSUMPTION
No effects

FOOD EFFICIENCY
Not determined

WATER CONSUMPTION
No effects

OPHTHALMOSCOPIC EXAMINATION
No effects

HAEMATOLOGY
No effects

CLINICAL CHEMISTRY
No effects

URINALYSIS
Not determiend

NEUROBEHAVIOUR
Not determined

ORGAN WEIGHTS
No effects

GROSS PATHOLOGY
No effects

HISTOPATHOLOGY: NON-NEOPLASTIC
No effects

Effect levels

Dose descriptor:
NOAEL
Effect level:
> 160 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical signs; mortality; body weight; food consumption; ophthalmoscopic examination; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology;

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The Low-Observed-Adverse-Effect-Level (LO(A)EL) for systemic effects was above 160 mg/kg b.w./day after a daily dermal administration to rats over 28 days
The No-Observed-Adverse-Effect-Level (NO(A)EL) for systemic effects was above 160 mg /kg b.w./day after a daily dermal administration to rats over 28 days
Executive summary:

The subacute toxicity of EUF (TPI 1618) was investigated in rats in a 28-day repeated dose dermal toxicity study (semi-occlusive) according to OECD guideline 411. Groups of 5 male and 5 female rats received dosages of 0, 40, 80 or 160 mg/kg bw via concentrations of 0, 0.2, 0.4 and 0.8% of EUF (TPI 1618). The volume of application was 20 mL/kg bw.The results of analyses of the dosing solutions showed that the test item-formulations were correctly prepared.

No changes in behavior, external appearance as well as in body weight, body weight gain and food and drinking water consumption were noted. None of the animals died prematurely. No test item-related systemic changes were seen in laboratory examinations, and at macroscopic examinations or for organ weights.

Treatment with EUF (TPI 1618) caused very slight and transient erythemas to the treated skin sites in individual animals, but no such findings were seen at treatment termination.Therefore, EUF (TPI 1618) had no consistent effect on the skin as the findings were neither time- nor dose-related and occurred only sporadically.

No test item-related histopathological changes were noted for any of the examined organs including the treated skin sites. The systemic NOAEL was > 160 mg/kg bw.