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Description of key information

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:
21 February - 06 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
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: individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet: ad libitum 2014C Teklad Global
Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK
- Water: ad libitum mains drinking water
- Acclimation period: 5 days minimum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 15/h minimum
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 21/02/2013-06/03/2013
Vehicle:
other: 1% pluronic L92 in distilled water
Concentration:
Screening test: 72.5% w/w (equivalent to 50% w/w active ingredient)
Main test: 72.5%, 36% or 14.5% w/w (equivalent to 50%, 25% and 10% w/w active ingredient)
No. of animals per dose:
Screening test: 1 animal
Main test: 4 animals/dose
Details on study design:
RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item at a concentration of 72.5% w/w (equivalent to 50% w/w active ingredient) in 1% pluronic L92 in distilled water, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay in the Mouse. The assay has undergone extensive inter-laboratory validation and has been shown to reliably detect test materials that are moderate to strong sensitisers.
- 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 ration of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test substance is regarded as a sensitiser if at least one concentration of the test substance results in a threefold or greater increase in 3HTdR incorporation compared to control values.

TREATMENT PREPARATION AND ADMINISTRATION: For the purpose of the study, the test item was freshly prepared as a suspension 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. 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. A further group of four mice received the vehicle alone in the same manner.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
S.I. = 8.31, i.e. the positive control is considered to be a sensitiser under the conditions of the test.
Parameter:
SI
Remarks on result:
other: vehicle: na 14.5 %w/w (equivalent to 10 %w/w a.i.): 1.58 36 %w/w (equivalent to 25 %w/w a.i.): 1.66 72.5 %w/w (equivalent to 50 %w/w a.i.): 1.39
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: vehicle: 8295.77 dpm 14.5 %w/w (equivalent to 10 %w/w a.i.): 13082.50 dpm 36 %w/w (equivalent to 25 %w/w a.i.): 13812.15 dpm 72.5 %w/w (equivalent to 50 %w/w a.i.): 11501.18 dpm

Preliminary screenting test: Slight brown coloured residual test item on the ears was noted post dose on Days 2 and 3. No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Based on this information the dose levels selected for the main test were 72.5%, 36% or 14.5% w/w in 1% pluronic L92 in distilled water (equivalent to 50%, 25% and 10% w/w active ingredient, respectively).

Main test: 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 were comparable to those observed in the corresponding control group animals over the same period.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the results of this LLNA, SFL is not sensitising
Executive summary:

A study was performed according to OECD Guideline 429 to assess the skin sensitisation potential of Sugar Factor Lime (SFL) in the CBA mouse following topical application to the dorsal surface of the ear. Three groups, each of four animals, were treated with 50 µl (25 µl per ear) of the test substance as a suspension in 1% pluronic L92 in distilled water at concentrations of 72.5%, 36% or 14.5% w/w (equivalent to 50%, 25% and 10% w/w a.i., respectively). A further group of four 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 of the vehicle control group was 1.58, 1.66 and 1.39 at concentrations of 10, 25 and 50 %w/w a.i., respectively. SFL is considered to be a non sensitiser under the conditions of the test.

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

In a study performed according to OECD Guideline 429 under GLP [Harlan Laboratories (2013f)] the skin sensitisation potential of Sugar Factor Lime (SFL) in the CBA mouse was assessed following topical application to the dorsal surface of the ear. Three groups, each of four animals, were treated with 50 µl (25 µl per ear) of SFL as a suspension in 1% pluronic L92 in distilled water at concentrations of 72.5%, 36% or 14.5% w/w (equivalent to 50%, 25% and 10% w/w a.i., respectively). A further group of four 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 of the vehicle control group was 1.58, 1.66 and 1.39 at concentrations of 10, 25 and 50 %w/w a.i., respectively. SFL is considered to be a non sensitiser under the conditions of the test. 

This study is supported by a study performed using calcium carbonate, the main constituent of SFL. In this study, performed according to OECD Guideline 429 under GLP, calcium carbonate (nano) was applied topically to the dorsal surface of the ears of mice (CBA/Ca) [Bradshaw (2010b)]. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test substance as a suspension in dimethyl formamide at concentrations of 5%, 10% or 25%w/w. A further group of four animals was treated with dimethyl formamide alone. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group was 1.74, 1.13 and 1.19 at concentrations of 5, 10 and 25%w/w, respectively. The test material was considered to be a non sensitiser under the conditions of the test.


Migrated from Short description of key information:
SFL is non-sensitising based on the results of a LLNA study.

Justification for selection of skin sensitisation endpoint:
Study performed using SFL

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In a study performed according to OECD Guideline 429 under GLP SFL produced a Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group of 1.58, 1.66 and 1.39 at concentrations of 10, 25 and 50 %w/w a.i., respectively. SFL is considered to be a non sensitiser under the conditions of the test and therefore does not require classification.