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Description of key information

Mouse Local Lymphnode Assay (LLNA) (in vivo), Mouse (CBA/Ca strain) f - (GLP, OECD Guideline 429, EU Method B42): not sensitising (average SI-value for 2.5 - 10 % doses: 1.42-0.93) (female)

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:
11.11.2014 - 25.11.2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed according to the recommended Guidelines (OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitization: Local Lymph Node Assay" (adopted 22 July 2010) and Method B42 Skin Sensitization (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008) and GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Adopted 22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(EC) No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate is as an attachement to the study report
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
The substance has a low vapour pressure and is insoluble in water.
The substance is stable under test conditions.
Species:
mouse
Strain:
CBA
Remarks:
CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food was allowed throughout the study.
- Diet (e.g. ad libitum): 2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Vehicle:
propylene glycol
Concentration:
Test item at concentrations 10 %, 5 % and 2.5 % w/w in propylene glycol.
No. of animals per dose:
Four
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Non-soluble
- Irritation: Non-irritant. No signs of visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.
- Systemic toxicity: No signs of systemic toxicity were noted
- Lymph node proliferation response: overall mean ear thickness change (%): -2.33

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay in the Mouse
- Criteria used to consider a positive response:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/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).
The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer

TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study, the test item was freshly prepared as a suspension in propylene glycol. 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.

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Groups of four mice were treated with the test item at concentrations of 10%, 5% or 2.5% w/w in propylene glycol. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin 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. No patch was used.

A further group of four mice received the vehicle alone in the same manner.

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-methylthymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse.
Positive control substance(s):
other: Phenylacetaldehyde (>90%)
Statistics:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/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). The Stimulation Index is expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group.
Positive control results:
The positive control study was performed according to the OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010) and Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) 440/2008.

Three groups, each of five animals, were treated with 50 μL (25 μL per ear) of Phenylacetaldehyde (>90%) as a solution in propylene glycol at concentrations of 2.5%, 5% or 10% v/v. A further group of five animals was treated with propylene glycol alone.

Phenylacetaldehyde (>90%) was considered to be a sensitizer under the conditions of the test, based on the stimulation index results.

The laboratory has further historical data on positive controls.
Key result
Parameter:
SI
Value:
0.93
Test group / Remarks:
Concentration of test item: 10 % w/w in propylene glycol
Remarks on result:
other: Negative for skin sensitisation
Remarks:
Average SI value for the 10 % dose group
Parameter:
SI
Value:
1.36
Test group / Remarks:
Concentration of test item: 5 % w/w in propylene glycol
Remarks on result:
other: Negative for skin sensitisation
Remarks:
Average SI value for the 5 % dose group
Parameter:
SI
Value:
1.42
Test group / Remarks:
Concentration of test item: 2.5 % w/w in propylene glycol
Remarks on result:
other: Negative for skin sensitisation
Remarks:
Average SI value for the 2.5 % dose group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
For the radioactive disintegrations per minute per lymph node and the stimulation index, see Table 1, Any other information on results incl. tables.

The proliferation response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node).

DETAILS ON STIMULATION INDEX CALCULATION
The proliferation response of lymph node cells is also expressed as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer."

EC3 CALCULATION
For the positive control, the concentration of Phenylacetaldehyde (>90%) expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 4%.

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

BODY WEIGHTS
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period (see Table 2, Any other information on results incl. tables).

Table 1. Average Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index.

Concentration (% w/w)
in propylene glycol
dpm dpm/Node Stimulation
Index
Result
Vehicle 9858.23 1232.28 na na
2.5 14000.02 1750.00 1.42 Negative
5 13388.71 1673.59 1.36 Negative
10 9215.09 1151.89 0.93 Negative

dpm = Disintegrations per minute

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

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

na = Not applicable

Table 2. Individual Body Weights and Body Weight Change

Concentration (% w/w)
in propylene glycol
Animal
Number
Body Weight (g) Body Weight
Change (g)
Day 1 Day 6
Vehicle 1-1 17.9 19.1 1.2
1-2 23.1 23.0 -0.1
1-3 18.3 19.7 1.4
1-4 19.1 20.3 1.2
2.5 2-1 19.4 18.8 -0.6
2-2 15.6 16.3 0.7
2-3 20.8 19.9 -0.9
2-4 21.6 20.3 -1.3
5 3-1 19.7 18.5 -1.2
3-2 20.0 21.7 1.7
3-3 22.0 22.1 0.1
3-4 18.6 19.8 1.2
10 4-1 18.2 18.0 -0.2
4-2 18.5 18.8 0.3
4-3 18.5 19.1 0.6
4-4 23.1 22.6 -0.5
Interpretation of results:
GHS criteria not met
Remarks:
The test item was considered to be a non-sensitizer under the conditions of the test. Criteria used for interpretation of results: EU
Conclusions:
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Based on these observations and the Stimulation Index at different tested concentrations, the results were negative for sensitization. The test item was considered to be a non-sensitizer under the conditions of the test. It is therefore not classified as sensitizing according to the CLP Regulation.
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.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as a suspension in propylene glycol at concentrations of 10%, 5% or 2.5% w/w. A further group of four animals was treated with propylene glycol alone.

Results

The Stimulation Index was expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group. For all of the tested concentrations of the test item, the result according to the Stimulation Index was negative.

Conclusions

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

No adverse effects were observed in the study, and the results based on the average Simulation Index values were considered negative. The test item was therefore considered to be a non-sensitizer under the conditions of the test.

There are no data gaps in sensitisation. Even though there is no human data available on the hazards of the substance for sensitisation, there is no reason to believe that the observed results in the Local Lymph Node Assay in the Mouse (CBA/Ca strain) would not be relevant to humans. In vitro/in chemico tests for skin sensitisation do not need to be conducted, as an in vivo skin sensitisation study is available for the substance. The study allows the conclusion to be made that the substance is not a skin sensitiser and is not presumed to have the potential to produce significant sensitisation in humans. The substance is therefore not classified as a skin sensitiser according to the CLP Regulation. Therefore, a risk assessment on the skin sensitisation of the substance is not required.


Justification for selection of skin sensitisation endpoint:
Only one study available. In accordance with column 2 of REACH Annex VII, the in vitro/in chemico tests for skin sensitisation (required in section 8.3.1) do not need to be conducted, as an in vivo study according to point 8.3.2 is available for the substance.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Justification for selection of respiratory sensitisation endpoint:
Not relevant for the sensitisation since no testing proposal is needed to perform Annex VII or VIII studies.

Justification for classification or non-classification

The test item was considered to be a non-sensitizer under the conditions of the skin sensitisation test. It is therefore not classified as sensitising according to the CLP Regulation.

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