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Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date: 20 November 2013; Experimental completion date: 26 November 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: CIM-25
Batch: MB-1
Expiry date: 31 March 2014
Storage Conditions: room temperature in the dark





In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd.
- Age at study initiation: Eight to twelve weeks old.
- Weight at study initiation: 15 to 23 g.
- Housing: The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): Free access to food (2014C Teklad Global Rodent diet) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains tap water was allowed throughout the study.
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was controlled to remain within target ranges of 19 to 25°C.
- Humidity (%): Relative humidity was controlled to remain within target ranges of 30 to 70%.
- Air changes (per hr): The rate of air exchange was approximately fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (LLNA)

Vehicle:
other: 1% pluronic L92 in distilled water.
Concentration:
Mice were treated with the test item at concentrations of 10%, 5% or 2.5% w/w in 1% pluronic L92 in distilled water.
No. of animals per dose:
Groups of four mice were treated with the test item at each concentration (including vehicle control).
Details on study design:
Test Item Formulation and Experimental Preparation:
For the purpose of the study, the test item was freshly prepared as a solution in 1% pluronic L92 in distilled water. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.

The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

Procedure:
Test Item Administration:
Groups of four mice were treated with the test item at concentrations of 10%, 5% or 2.5% w/w in 1% pluronic L92 in distilled water. Information available suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/ml, specific activity 2.0 Ci/mmol) giving a total of 20 µCi to each mouse.

Observations:
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.

Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures:
Termination:
Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension:
A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation:
After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by beta-scintillation counting. The "Poly Q" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system.














Positive control substance(s):
other: alpha-Hexylcinnamaldehyde, tech., 85%

Results and discussion

Positive control results:
Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of alpha-Hexylcinnamaldehyde, tech., 85% as an emulsion in 1% pluronic L92 in distilled water at concentrations of 5%, 10% or 25% v/v. A further group of five animals was treated with 1% pluronic L92 in distilled water alone.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation ofthe vehicle control group are as follows:

Concentration (%) v/v in 1% pluronic L92 in distilled water: 5%
Stimulation Index: 1.06
Result: Negative

Concentration (%) v/v in 1% pluronic L92 in distilled water: 10%
Stimulation Index: 1.88
Result: Negative

Concentration (%) v/v in 1% pluronic L92 in distilled water: 25%
Stimulation Index: 7.22
Result: Positive

alpha-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Concentration (% w/w) in 1% pluronic L92 in distilled water: 2.5%: SI 1.38 5%: SI 1.66 10%: SI 1.86
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Concentration (% w/w) in 1% pluronic L92 in distilled water: Vehicle: 11110.21 dpm 2.5%: 15339.52 dpm 5%: 18467.35 dpm 10%: 20638.14

Any other information on results incl. tables

Results:

Estimation of the Proliferative Response of Lymph Node Cells:

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 1.

Table 1: Disintegrations per Minute, Distinegrations per Minute/Node and Stimulation Index

Concentration (% w/w) in 1% pluronic L92 in distilled water

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

11110.21

1388.78

na

na

2.5

15339.52

1917.44

1.38

Negative

5

18467.35

2308.42

1.66

Negative

10

20638.14

2579.77

1.86

Negative

dpm = Disintegrations per Minute

a = Disintegrations per minute/node obtained by dividing the disintegrations per mnute value by 8 (total number of lymph nodes)

b = Stimulation Index of 3.0 or greater indicates a positive result

na = Not applicable.

Clinical Observations and Mortality Data:

Dark blue colored staining on the ears and fur was noted in all test animals post dose on Days 1 to 3.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body Weight:

Body weight changes of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be a non-sensitizer under the conditions of the test.
Executive summary:

Introduction:

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:

• OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitization: Local Lymph Node Assay" (adopted 22 July 2010)

• Method B42 Skin Sensitization (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

Methods

Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in 1% pluronic L92 in distilled water at concentrations of 10%, 5% or 2.5%w/w.A further group of four animals was treated with 1% pluronic L92 in distilled water alone.

Results:

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration % w/w) in 1% pluronic L92 in distilled water

Stimulation Index

Result

2.5

1.38

Negative

5

1.66

Negative

10

1.86

Negative

Conclusion

The test item was considered to be a non-sensitizer under the conditions of the test.

The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals.

The test item does not meet the criteria for classification according to the Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.