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Description of key information

Skin sensitisation:

In a Local Lymph Node Assay (LLNA) in mice (CBA/CaHsdRcc(SPF) strain) equivalent to OECD 429 Guideline, EU Method B.42 and EPA OPPTS 870.2600 Guideline, the test item was observed to be non-sensitizing to the skin (Wang-Fan, 2006).

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.

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:
2006-03-01 to 2006-03-15
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
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:
- Name of test material (as cited in study report): TIC2782 (T002907)
- Substance type: white solid
- Physical state: solid
- Analytical purity: 99.9%
- Purity test date: no data
- Lot/batch No.: 00467090 ( = charge 05C0836)
- Expiration date of the lot/batch: 2006-05-04
- Stability of test item: stable under storage conditions
- Storage condition of test material: at room temperature (range of 20 +/- 5 °C), light protected
Species:
mouse
Strain:
CBA
Remarks:
CaHsdRcc(SPF)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: 16 female (+ 3 females for pretest (non-GLP)) mice, CBA/CaHsdRcc (SPF) (nulliparous and non-pregnant); RCC Ltd, Laboratory Animal Servive, CH-4414 Füllinsdorf, Switzerland
- Age at beginning of acclimatization: 8-12 weeks
- Weight at treatment (day 1): 17.0 -21.3 grams
- Housing: Standard Laboratory Conditions; individual in Makrolon type-2 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet (e.g. ad libitum):ad libitum, pelleted standard Kliba 3433, batch no. 76/05 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst).
- Water (e.g. ad libitum): ad libitum, community tap water from Itingen
- Acclimation period: at least 6 days, 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): 10-15
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light/12 hour dark cycle with at least 8 hours music during the light period

IN-LIFE DATES: From: 2006-03-08 To: 2006-03-13
Vehicle:
dimethylformamide
Concentration:
0, 10, 25 and 50% (w/v)
No. of animals per dose:
4 females per group; 3 test groups and 1 negative control group
Details on study design:
RANGE FINDING TESTS:
- In non-GLP solubility pretest, the test item was tested in different vehicles: acetone/olive oil (4/1, v/v), ethanol/water (7/3, v/v) and N,N-dimethylformamide (DMF). N,N-dimethylformamide (DMF) was found to be a suitable vehicle and was selected and used in the main test. 50 % was the highest technically applicable concentration in the chosen vehicle.
- A non-GLP local toxicity pre-test was performed for determination of concentrations for the main test. Three single animals were each treated with one of three different concentrations: 10 %, 25 % and 50 %, in both ears on three consecutive days. No clinical signs were observed at these concentrations one day after each single application. One day after the third topical application, the residual test item was found at the dosing sites of 50 %.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with the test item at concentrations of 10 %, 25 % and 50 % in DMF. 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).
- Criteria used to consider a positive response: a test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled: first, exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the S.I.; second, 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.

ADMINISTRATION OF ³HTdR
- Five days after the first topical application, all mice were administered with 250 μl of PBS (phosphate buffered saline) containing 78.37 μCi/ml 3HTdR (equal to 19.6 μCi 3HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED ³HTdR
- Approximately five hours after treatment with 3HTdR all mice were euthanized by inhalation of CO2 (dry ice).
- The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing twice 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 glass scintillation vials with 10 ml of ‘Irga-Safe Plus’ scintillation liquid and thoroughly mixed.
- The level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated for the body weights.
Positive control results:
Stimulation indices of 1.8, 2.9 and 6.2 were determined with the test item at concentrations of 5%, 10% and 25%, respectively, in acetone:olive oil, 4:1 (v/v).
Alpha-hexylcinnamaldehyde was therefore found to be a skin sensitizer in the LLNA test and an EC3 value of 10.5% was derived.
Parameter:
SI
Value:
0.7
Test group / Remarks:
10% w/v group (based on results of 4 animals)
Parameter:
SI
Value:
1.3
Test group / Remarks:
25% w/v group (based on results of 4 animals)
Parameter:
SI
Value:
0.9
Test group / Remarks:
50% w/v group (based on results of 4 animals)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
control group: 351 dpm per node (8 lymph nodes in total)
10% w/v group: 257 dpm per node (8 lymph nodes in total)
25% w/v group: 441 dpm per node (8 lymph nodes in total)
50% w/v group: 322 dpm per node (8 lymph nodes in total)

DETAILS ON STIMULATION INDEX CALCULATION
see Results

EC3 CALCULATION : no EC3 value could be calculated as the SI values for all groups were below 3

CLINICAL OBSERVATIONS:
No deaths occurred during the study period.
Neither clinical/local signs nor other findings were observed in any animals of the control group or Group 2 (10%). On the second application day, the residual test item was found at all the dosing sites in all mice of Groups 3-4, persisting for a total of two days.
No findings were observed on the size of the draining lymph nodes.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.
Interpretation of results:
GHS criteria not met
Conclusions:
In this study S.I. of 0.7, 1.3 and 0.9 were determined with the test item at concentrations of 10 %, 25 % and 50 %, respectively, in DMF.
TIC2782 (T002907) was therefore found to be a non-sensitizer when tested up to the highest applicable concentration of 50 % in DMF.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2004-03-01 to 2004-03-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: SPL Standard Test Method 595.12
Deviations:
no
GLP compliance:
no
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
no data
Species:
mouse
Strain:
CBA
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: 16 mice, CBA/Ca strain, no further data on source
- Age at study initiation: no data
- Weight at study initiation: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data
Vehicle:
dimethylformamide
Concentration:
0, 5, 10 and 25% (w/w)
No. of animals per dose:
4 mice/ group; 3 test groups and 1 control group
Details on study design:
RANGE FINDING TESTS:
- A preliminary sighting test was performed, at which there were no signs of systemic toxicity at a concentration of 25% w/w.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- 3 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 5%, 10% and 25% w/w. A further group of four animals was treated with dimethyl formamide alone.
- Criteria used to consider a positive response: no data

TREATMENT PREPARATION AND ADMINISTRATION: no data
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
other: 2,4 Dinitrobenzenesulfonic acid, sodium salt, at 1%, 10%, 20% v/v in 1% pluronic F-68 in distilled water
Statistics:
no data
Parameter:
SI
Value:
0.71
Test group / Remarks:
5% w/w in dimethyl formamide (results of 4 animals)
Parameter:
SI
Value:
1.19
Test group / Remarks:
10% w/w in dimethyl formamide (results of 4 animals)
Parameter:
SI
Value:
1.58
Test group / Remarks:
25% w/w in dimethyl formamide (results of 4 animals)
Cellular proliferation data / Observations:
No further details available

Positive Control Local Lymph Node Assay in the Mouse (2004)

Start Date

Finish Date

Test Material

Concentration

Vehicle

Stimulation Indexa

Classificationb

29/04/2004

05/05/2004

α‑Hexylcinnamaldehyde, tech., 85%

5%, 10%, 25% v/v

acetone/olive oil 4:1

1.40, 2.23, 6.09

Positive

29/04/2004

05/05/2004

α‑Hexylcinnamaldehyde, tech., 85%

5%, 10%, 25% v/v

acetone/olive oil 4:1

1.74, 2.20, 8.89

Positive

14/10/2004

26/10/2004

α‑Hexylcinnamaldehyde, tech., 85%

5%, 10%, 25% v/v

tetrahydrofuran

1.97, 3.71, 7.82

Positive

29/09/2004

05/10/2004

2,4‑Dinitrobenzenesulfonic acid, sodium salt

1%, 10%, 20% v/v

1% pluronic F-68

in distilled water

1.03, 4.41, 13.55

Positive

27/10/2004

02/11/2004

α‑Hexylcinnamaldehyde, tech., 85%

10%, 25%, 50% v/v

cottonseed oil

1.52, 2.63, 5.07

Positive


a=        Ratio of test to control lymphocyte proliferation

b=        Stimulation index greater than 3.0 indicates a positive result

* =        Standard Test Method 595 (Pooled nodes)

·=        Standard Test Method 599 (Individual nodes)

Interpretation of results:
GHS criteria not met
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin sensitisation - in vitro

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.

Skin sensitisation - in vivo

One key study is available for skin sensitisation (Wang-Fan, 2006), performed according to the OECD Guideline for the Testing of Chemicals, Guideline 429: Skin Sensitization: Local Lymph Node Assay (adopted 24 April 2002). Three groups of four female mice were treated with the test item at concentrations of 10 %, 25 % and 50 % in DMF by topical application to the dorsal surface of each ear for three days. 50 % was the highest concentration in the chosen vehicle. A control group of four mice was treated with the vehicle DMF only. Five days after the first application, the mice were injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine, 3HTdR). Five hours after injection, the mice were sacrificed, and the lymph nodes excised, and pooled per group. Suspensions of lymph node cells were prepared from the pooled lymph nodes. The proliferative capacity of the pooled lymph node cells was determined by the incorporation of 3HTdR measured in a β-scintillation counter.

The test item was found to be a non-sensitizer when tested up to the highest applicable concentration of 50 % in DMF.

One additional supporting study was identified (Sanders, 2004). The test material was assessed for its skin sensitising potential using the LLNA in the CBA/Ca strain of mouse. The highest concentration tested was 25%, and the test item was considered to be a non-sensitiser under the conditions of the test.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation

The test item is considered to be non-sensitising, based on the available data and the criteria of the CLP Regulation.

Respiratory sensitisation

No reliable study is available.