Registration Dossier

Administrative data

Description of key information

Skin Sensitisation:

In a Local Lymph Node Assay (LLNA) in mice (CBA/CaOlaHsd strain) according to OECD Guideline 429 and EU Method B.42 the substance was observed to be non-sensitizing to the skin (Meurer, 2007).

An in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study (initiated before October 11th, 2016) is available.

Respiratory Sensitisation:

No data was available to decide a classification for respiratory sensitisation.

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
Study period:
2007-03-21 to 2007-06-18
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
Qualifier:
according to
Guideline:
EU Method B.42 (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: Tibotec Pharmaceuticals; 603T-1
- Expiration date of the lot/batch: December 19, 2007

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: N/A
- Solubility and stability of the test substance in the solvent/vehicle: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was placed into a volumetric flask glass beaker on a tared balance and the vehicle (DMF) was quantitatively added. The low dose of the test item concentration was prepared serially from the mid dose. For the high concentration the test item was used undiluted. Homogeneity of the test item in the vehicle was maintained during treatment
with the magnetic stirrer.



Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands
- Age at study initiation: 7-12 weeks at start of acclimatisation
- Weight at study initiation: 18.0-20.9 g (mean=19.0 g)
- Housing: Makrolon Type 1 cages with wire mesh top (EHRET GmbH, D-79302 Emmendingen) with granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen)
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water (e.g. ad libitum): Rossdorf community tap water, ad libitum
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness were used. No time of acclimatization provided.


ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 22 + /- 3
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 2007-03-29 To: 2007-04-04
Vehicle:
dimethylformamide
Concentration:
25, 50, and 100%
No. of animals per dose:
4 animals per dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: no data
- Irritation: A pre-test in 2 mice, test substance concentrations of 12.5, 25, 50, and 100% (w/v) were tested on one ear each. No irritation effects were observed at these concentrations after a single application.
- Lymph node proliferation response: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
-Criteria used to consider a positive response: Exposure to at least one test concentration must result in incorporation of 3HTdR at least three times or greater than that recorded in the control mice, as indicated by the SI, and the data must be compatible with a conventional dose reponse, although allowances must be made (especially at higher concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
- Each test group of mice was treated by topical application to the dorsal surface of each ear lobe wth the test substance at concentrations of 25%, 50%, and 100% in DMF. 25 uL was spread over the entire surface of each ear lobe once daily for three days. Another group of mice was treated with an equal volume of DMF alone.
- Five days after the first application, all mice were administered 250 uL of PBS containing 79.8 uCi/mL 3HTdR (equal to 19.95 uCi 3HTdR per mouse) by IV injection via a tail vein.
- Approximately five hours after treatment with 3HTdR, all mice were euthanized by intraperitoneal injection of Na-thiopental.
- The draining lymph nodes were rapidly excised and pooled for each group (8 nodes per group).
-Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 Rm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the
lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 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 HTdR incorporation was measured on a B-scintillation counter. Background levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid.
- HTdR incorporation is expressed as the number of disintegrations per minute (DPM)
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables. A statistical analysis was conducted for assessment of the dose-response relationship. An EC3 value could not be calculated.
Positive control results:
Stimulation index
5% (w/v) -2.04
10% (w/v) - 6.31
25% (w/v) - 12.45

Measurement DPM
5% (w/v) - 8152.94
10% (w/v) - 24974.40
25% (w/v) - 49155.3

EC3=6.1%. Considered to be a skin sensitizer.
Parameter:
SI
Value:
2.26
Test group / Remarks:
4 animals in 25% w/v in DMF
Parameter:
SI
Value:
0.8
Test group / Remarks:
4 animals in 50% w/v in DMF
Parameter:
SI
Value:
1.49
Test group / Remarks:
4 animals in 100% w/v
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
- DPM: 25% w/v = 9967.54
50% w/v = 3563.36
100% w/v = 6594.59
Control = 4437.49

DETAILS ON STIMULATION INDEX CALCULATION
-Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

EC3 CALCULATION
- The EC3 Value could not be calculated, since all SI´s are below 3.

CLINICAL OBSERVATIONS:
-No mortality occurred during the study period.
- On day five, all animals showed reduction of spontaneous activity, eyelid closure, and ruffled fur.

BODY WEIGHTS
-The body weight of the animals, recorded prior to the first application was within the range commonly recorded for animals of this strain and age. The body weight of the animals, recorded on the day of preparation was reduced.
Interpretation of results:
GHS criteria not met
Conclusions:
In this study Stimulation Indices of 2.26, 0.80, and 1.49 were determined with the test item at concentrations of 25, 50, and 100% in DMF. The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.The test substance was not considered to be a skin sensitizer under the describer conditions.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A local lymph node assay (LLNA) was performed to assess the skin sensitisation potential of T002675 in the CBA/CaOlaHsd strain of mouse, when administered to the dorsum of both ear lobes of mice after dissolution in DMF. The following test concentrations were tested: 25%, 50% and 100%. On the day of preparation, all animals showed reduction of spontaneous activity, eyelid closure, and ruffled fur. The weight of animals was also reduced. Cases of mortality were not observed. In this Stimulation Indices (S.I.) of 2.26, 0.80 and 1.49 were determined with the test item at concentrations of 25%, 50% and 100% in DMF respectively.

The test item T002675 was not a skin sensitiser in this assay.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the study results and the criteria of the CLP Regulation, T002675 should not be classified as a skin sensitising substance.

No data was available to decide a classification for respiratory sensitisation.