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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to GLP and valid methods, therefore it is reliable, adequate and relevant.
Justification for type of information:
The DBEA dossier contains old in vivo studies , which are not allowed today.
The new legal requirements (21 June 2016) require an in vitro skin sensitisation study. However the previous data requirements were met with an in vivo study during dossier compilation for the <1000 T/y tonnage band. Therefore there is no need to repeat the study using the alternative test method.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Proviplast 0142
- Substance type: organic monoconstituent
- Physical state: liquid
- Purity test date: 2011-07-11
- Lot/batch No.: 11701913 SA
- Expiration date of the lot/batch: production date 2010-12-14; shelflife 2 years
- Stability under test conditions: the test substance formulations were made freshly on each dosing occasion
- Storage condition of test material: required quantity of the test substance was received from the test substance controller and was stored in a dry, cool, well-ventilated area in the utility room.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaCrl
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratory, UK
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: 19.5-21.4 g
- Housing: individual caging for pre-screen test and proup caging (5 animals/group) for main study
- Diet (e.g. ad libitum): standard gamma irradiated pellet feed supplied by Tetragon Chemie Pvt. Ltd, Bangalore, ad dibitum
- Water (e.g. ad libitum): reverse osmosis water, ad libitum
- Acclimation period: 5 days prior to the pre-screen and main experiment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2°C-21.3°C
- Humidity (%): 53.1%-59.8%
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: 2011-08-12 To: 2011-08-27

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25%, 50%, 100% v/v
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
To detemine the highest non-irritant and technically applicable test substance concentrations in main study, a pre-screen test was performed using three different concentrations (25%, 50% and 100% v/v) of the test substance by using three mice (one per concentration). Pre-screen test was performed under identical conditions as the main study, except there is no assessment of lymph node proliferation. All mice were observed daily for clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded on Day 1 (pre-dose) and prior to termination (day 6). Both ears of each mouse were obderved for erythema and scoring was given. Ear thickness was measured using digital micrometer on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6. Excessive local irritaton is indicated by an erythema score > or = 3 and / or ear swelling of > or = 25%. The top dos selected for the main study was the highest technically applicable concentration while avoiding systemic toxicity and excessive local irritation.

MAIN STUDY
TEST SUBSTANCE EXPOSURE
- No. of exposures: 3
- Exposure period: day 1, 2 and 3
- Test groups: 3
- Control group: 2 (negative and positive (25% HCA) control)
- Site: each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (right and left). The application volume, 25 µL, was spread over the entire dorsal surface of each ear lobe.
- Frequency of applications: once daily for three consecutive days
- Duration: 3 days exposure
- Concentrations: 25%, 50% and 100% v/v

TRITIATED METHYL THYMIDINE ADMINISTRATION
- No. of exposures: 1
- Day(s) of challenge: 1
- Exposure period: day 6
- Test groups: 3
- Control group: 2
- Site: five days after the topical application, all group of mice were administered 250 µL of phosphated-buffered saline (PBS) containing 20µCi of tritiated methyl thymidine by intervanous injection via lateral tail vein.
- Concentrations: 250 µL of phosphated-buffered saline (PBS) containing 20µCi of tritiated methyl thymidine
- Evaluation (hr after challenge): approximately 5 hours after treatment with tritiated methyl thymidine

DETERMINATION OF INCORPORATED TRITIATED METHYL THYMIDINE (TMT)
Approximately five hours after treatment with TMT all mice were euthanized using CO2. The draining auricular lymph node were excised bilaterally of each mouse in each experimental group (2 nodes per mouse). Single cell suspension was prepated by mashing the lymph between the frosted ends of micoscopic slides. After washing, each suspension was stirred and incubated. The precipitaes were re-suspended and transfeerd to glass scintillation vials. The level of TMT incorporation was measured and expressed as the number of radioactive disintegrations per minute (dpm).


Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The body weights and dpm data were analyzed by Modified Leven's test for homogeneity and subjected to one-way analysis of variance (ANOVA). A comparion of treated with control groups were done using Dunnett's test. The alpha level for body weight and dpm data were conducted at 0.05 and 0.01 respectively. Due to heterogeneity of dpm data, it was transferred to log to obtain normality and homogeneous variences. Transferred dpm data was subjected to Dunnett's test.

Results and discussion

Positive control results:
25% HCA (v/v) was used as positive control. The obtained value for desintegrations per minute- Back ground count (5% trichloro acetic acid) is 4049.6 +/- 560.16 or for stimulation index 9.43+/- 1.30.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: a dose level of 100%, 50% and 25% Proviplast 0142 produced SI of 2.29+/-0.21, 1.77+/-0.16 and 1.38+/-0.16
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: A dose level of 100%, 50% and 25% Proviplast 0142 produced DPM of 982.40+/-91.11, 758.60+/-68.78 and 593.60+/-70.21

Any other information on results incl. tables

Mortality: no mortality was observed in treated group of mice throughout the observation period.

Body weight: all the animals showed normal body weight gain during the experimental period except in the positive control group which showed decrease in body weight gain and no statistical significant difference was observed in treated groups from vehicle control group.

Clinical signs of toxicity: no clinical signs of toxicity were observed in treated group of mice for the entire observation period. No changes were observed at the treatment sites.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
A dose level of 100%, 50% and 25% produced stimulation indices of 2.29, 1.77 and 1.38. Stimulation indices of less than 3.0 were observed at all the three concentrations tested. Therefore, the EC3 value (the estimated concentration required to induce a threshold positive response) for Proviplast 0142 under the condition of this study was not calculated and hence test item Proviplast 0142 may be considered as a non sensitizer under present experimental condition.
Executive summary:

Five groups of female CBA/CaCrl mice (5mice/group) namely G1- negative control, G2-positive control (25% HCA). G3 - 25% of test substance, G4- 50% of test substance, G5 -100% of test substance received topical application (epidermal) on the dorsum of each ear with 25 µL of vehicle [acetone: olive oil (4:1) (v/v)]. HCA (25%), test substance at three different concentrations 25%, 50% and 100% respectively for three consecutive days followed by no treatment for two days. Known volume of the test substance was uniformly mixed with vehicle [acetone: olive oil (4:1) (v/v)J to obtain concentrations of 25% and 50%. 100% of test substance was applied as such. Similarly, known volume of HCA was mixed uniformly with vehicle to obtain concentration of 25%. On day 6, 250 µL of PBS containing 20 µCi of tritiated methyl thymidine was administered intravenously to all the groups of mice. All the animals were necropsied at the end of five hours of intravenous injection on day 6 and the draining auricular lymph nodes were collected. The following parameters like mortality, clinical signs of toxicity, erythema and edema at the site of application and body weight prior to topical application (day1) and prior to intravenous administration (day 6) were recorded.

No mortality, clinical signs of toxicity, erythema and edema at the site of application occurred in any group of mice. Body weight gain in all the test substance treated groups was comparable to Negative control animals. The DPM (Disintegrations per minute) value of positive control was 9.43 times more than the vehicle control.

In conclusion, dose levels of 100%, 50% and 25% of Proviplast 0142 produced stimulation indices of 2.29, 1.77 and 1.38 respectively. Stimulation indices of less than 3.0 were observed at all the three concentrations tested. Therefore, the EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI=3) for the test substance under the condition of this study was not calculable and hence test item Proviplast 0142 may be considered as a non sensitizer under present experimental condition.