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Description of key information

Skin sensitization

In a Local Lymph Node Assay (LLNA) in mice (CBA/Ca strain), according to OECD Guideline 429 and SPL Standard Test method 595.12, T003063 has an EC3 value of 9.8% (w/v) (Honarvar, 2008). Therefore, the test item is classified as skin sensitizer category 1B according to the CLP Regulation (EC) No. 1272/2008.

Respiratory sensitization

No reliable respiratory sensitization study was available

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23/09/2008 to 08/10/2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 507652
- Expiration date of the lot/batch: April 03, 2009 (retest date)
- Purity: 100,4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands B.V., Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks (beginning of treatment)
- Weight at study initiation: 19.7 - 24.5 g
- Housing: Single housing in a Makrolon Type I cage with wire mesh top; granulated soft wood bedding
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 6.00 a.m. to 6.00 p.m.
Vehicle:
dimethylformamide
Concentration:
The test item in the main study was assayed at 1, 10 and 25%. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.
No. of animals per dose:
Number of animals for the pre-test: 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which can be technically used was a 25% dilution in dimethylformamide.
- Irritation: Two mice were treated with concentrations of 2.5, 5, 10 and 25% on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application. At the tested concentrations animals did not show any signs of irritation or systemic toxicity. The test item in the main study was assayed at 1, 10 and 25%. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.
- Lymph node proliferation response: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Determination of incorporated 3HTdR
- Criteria used to consider a positive response: The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fullfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3 HTdR at least 3-fold or greater than that recorded in control mice, as indicated by stimulation index (SI). The estimated concentration of the test item required to produce a SI of 3 is referred to as the EC3 value.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a volumetric flask on a tared balance and dimethylformamide was quantitatively added. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 1, 10, and 25% (w/v) in dimethylformamide. The application volume, 25 µL, was spread over the entire dorsal surface (diameter~ 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-Methyl Thymidine:
Five days after the first topical application, all mice were administered with 250 µL of 78.6 µCi/mL 3HTdR (corresponds to 19.7 µCi 3HTdR per mouse) by intravenous injection via a tail vein.

Determination of incorporated 3HTdR:
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium.
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing twice with phosphate buffered saline (approximately 10 mL), the lymph node cells were resuspended in 5% trichloroacetic acid (approximately 3 mL) and incubated (at ca. 4°C) for at least 18 hours for precipitation of macromolecules. Precipitates were resuspended oin 5% trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of 'Ultima Gold' scintillation liquid and thoroughly mixed.
The level of 3HTdR incorportation was measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5% trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated (body weights).
A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation:
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a,b) and (c,d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
Positive control results:
The EC3 value was estimated to be < 5%. A precise calculation of the EC3 value was not possible since the S.I. was above the threshold of 3 even at the lowest tested concentration. The obtained values are within the expected range and the positive control experiment is considered fully valid.
Parameter:
SI
Value:
2.04
Test group / Remarks:
4 animals of 1% w/v group
Parameter:
SI
Value:
3.02
Test group / Remarks:
4 animals of 10% w/v group
Parameter:
SI
Value:
4.03
Test group / Remarks:
4 animals of 25% w/v group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
DPM per lymh node (8 lymph nodes in total):
1% w/v group: 547.3
10% w/v group: 808.8
25% w/v group: 1077.3

DETAILS ON STIMULATION INDEX CALCULATION
In this study Stimulation Indices (S.I.) of 2.04, 3.02, and 4.03 were determined with the test item at concentrations of 1, 10, and 25 % in dimethylformamide, respectively.

EC3 CALCULATION
EC3 = (a-c) [(3-d)/(b-d)]+c = 9.8% (w/v)
test group 2: 1% = a, S.I. of 2.04 = b
test group 3: 10% = c, S.I. of 3.02 = d
a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

CLINICAL OBSERVATIONS:
No deaths occurred during the study period. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test item T003063 was found to be a skin sensitiser under the described conditions. The EC3 value calculated was 9.8% (w/v). As the EC3 value is > 2%, the substance is classified as skin sensitiser category 1B.
Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In a Local Lymph Node Assay (LLNA) in mice (CBA/Ca strain), the skin sensitization potential of T003063 was investigated following topical application to the dorsal surface of the ear of mice (Honarvar, 2008). Test item concentrations selected for the main study were based on the results of a preliminary sighting test. Three groups, each of four animals, were treated with 50 µL of the test material (25 µL per ear) as a solution in dimethyl formamide at concentrations of 1%, 10% and 25% w/w. A further group of four animals was treated with dimethyl formamide alone. Clinical observations and checks for mortality were recorded pre- and 1-hour post-dose on Days 1, 2, and 3 and on Days 4, 5, and 6. Body weights were recorded on Day 1 and 6. The proliferation 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 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

Positive control tests using hexyl cinnamic aldehyde (CAS 101-86-0) at 5, 10 and 25% v/v in acetone/olive oil 4:1 and 2,4 Dinitrobenzene sulphonic acid, sodium salt (DNBS), at 1, 10, and 20% v/v in 1% pluronic L92 in distilled water elicited a positive classification.

No mortality or signs of systemic toxicity were observed. The mean body weight gain shown by the animals over the study period was considered to be normal. Mean DPM/node (DPM value/8 total lymph nodes) for the experimental groups treated with test item concentrations 1, 10 and 25% were 547.3, 808.8 and 1077.3, respectively. The SI values calculated for the substance concentrations 1, 10 and 25% were 2.04, 3.02 and 4.03, respectively. The EC3 value was calculated to be 9.8. T003063 was found to be a Category 1B skin sensitiser based on GHS criteria.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitization:

The EC3 value calculated was 9.8% (w/v) for T003063. As the EC3 value is > 2%, the substance is classified as skin sensitiser category 1B.

Respiratory sensitization:

No data were available to decide on the classification for respiratory sensitisation.