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Description of key information

The test substance was tested for skin sensitisation according to OECD TG 429 and under GLP. This study identified a potential for dermal sensitization and resulted in obligatory labelling requirement for skin sensitisation and classification into Category 1.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study was carried out or initiated before 11 October 2016 and addresses the standard information requirement. In vitro testing was therefore not considered.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 22, 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo (formed partially from Harlan in September 2015), 5800 AN Venray, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF, animals were derived from a controlled full-barrier maintained breeding system
- Age at study initiation: 8–9 weeks
- Weight at study initiation: 19-22 g
- Housing: full barrier in an air-conditioned room, animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Diet (e.g. ad libitum): ad libitum, Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): ad libitum, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least five days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
6.25%, 12.5% and 25% (w/v)
No. of animals per dose:
5 mice / dose group and 10 mice / prescreen test
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: the maximum technically applicable concentration of the test item in the vehicle was found to amount 25% in AOO
- Irritation: signs of excessive irritation were not detected in any animal treated with either 25 or 12.5% at any application site
- Systemic toxicity: signs of systemic toxicity were not detected in any animal treated with either 25 or 12.5%
- Ear thickness measurements: ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6
- Erythema scores: both ears were observed for erythema and scored according to Table 1 of the attached file

Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of ≥ 25% and absent in this test
. Please see attached file for detailed data of the prescreen test

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.

EC3 values, calculated concentrations which induce stimulation indices of three, were determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a-c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points were above or below the stimulation index of three, no EC3 value could be stated.

A substance was regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item resulted in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).


TREATMENT PREPARATION AND ADMINISTRATION:
3 test groups (3 different concentrations), 1 positive control group (1% phenylene-diamine in AOO) and 1 negative-control group (vehicle) were tested.

Topical Application:
Each mouse was treated by topical application of 25 μL of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days. The first treatment day was defined as study day 1.

Administration of 3H-Methyl Thymidine:
Five days after the first topical application, all mice were dosed with 20 μCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 μl of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 μCi/ml.

Preparation of Cell Suspension:
Approximately 5 hours after the injection of 3H-methyl thymidine, all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash, each pellet was resuspended in approx. 1 ml 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 ml 5% TCA and 7 ml scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H -Methyl Thymidine:
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.
Positive control substance(s):
other: phenylenediamine
Positive control results:
The stimulation index of the positive control (1% phenylenediamine in AOO) was 5.6 and therefore the test was considered to be valid.
Key result
Parameter:
SI
Value:
3.2
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
4.2
Test group / Remarks:
12.5%
Key result
Parameter:
SI
Value:
4.7
Test group / Remarks:
6.25%
Key result
Parameter:
EC3
Remarks on result:
not determinable
Remarks:
inverse dose-response relationship
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA and DETAILS ON STIMULATION INDEX CALCULATION
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.

EC3 CALCULATION
EC3 values, calculated concentrations which induce stimulation indices of three, were determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a-c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points were above or below the stimulation index of three, no EC3 value could be stated.

CLINICAL OBSERVATIONS:
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.

BODY WEIGHTS
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

Please see attached file for results.

Please see attached file for results.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Each of the tested concentrations exceeded the stimulation index of 3. The EC3 value could not be calculated due to an inverse a dose-response relationship of the data obtained.
Consequently, the test item is considered to be a dermal sensitiser. According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System), the test item has obligatory labelling requirement for skin sensitisation and is classified into Category 1.
Executive summary:

A valid test for skin sensitisation according to OECD Guideline and under GLP was carried out with the test substance and identified the substance as a skin sensitiser of GHS Category 1.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Each of the tested concentrations exceeded the stimulation index of 3. Consequently, according to OECD TG 429, the test item is considered to be a dermal sensitiser.

The EC3 value could not be calculated due to an inverse dose-response relationship of the data obtained. According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System), the test item has obligatory labelling requirement for skin sensitisation and is classified into Category 1.