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Description of key information

OECD TG 429 (Sanders, 2011): Not sensitizing 


 


The in vivo skin sensitizing study conducted was conclusive for classification. The need to conduct an in vitro skin sensitization test was waived due to results from an in vivo test. The results from the in vivo OECD TG 429 was sufficient to classify the test item as not sensitising to the skin according to CLP Regulation (EC) No 1272/2008. 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 January 2011 and 22 February 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was conducted in accordance with OECD Guideline 429 "Skin Sensitisation: Local Lymph Node Assay". This study was also performed in accordance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
No statistical analysis was performed
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)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Batch Number: 101009
- Expiration date of the lot/batch: Not stated
- Purity test date: Not stated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store at room temperature in the dark
- Stability under storage conditions: Not stated
- Stability under test conditions: Not stated
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Not stated
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): Not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test material was prepared as a solution in acetone/olive oil 4:1.
- Preliminary purification step (if any): N/A
- Preparation of a nanomaterial dispersion (incl. dilution): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Females- nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Not stated
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation:15 to 23g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet:Ad libitum access to food (2014 Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK)
- Water: Ad libitum access to mains tap water
- Acclimation period: of at least five days
- Indication of any skin lesions: Not stated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 degC
- Humidity (%): 30 to 70 %
- Air changes (per hr): 15 changes per hour
- Photoperiod (hrs dark / hrs light):12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
50 %, 25 % and 10 %
No. of animals per dose:
4 animals per dose.
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Not stated
- Irritation: The preliminary screening test suggested that the test item would not produce excessive local irritation at a concentration of 50 % (v/v) in acetone/olive oil 4:1.
- Systemic toxicity: The preliminary screening test suggested that the test item would not produce systemic toxicity at a concentration of 50 % (v/v) in acetone/olive oil 4:1.
- Ear thickness measurements: No signs of irritation indicated by a ≥ 25% increase in mean eat thickness was noted in animals treated with the test item at concentrations of 50%, 25% or 10% v/v in acetone
- Erythema scores: Not stated

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD TG No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010)
- Criteria used to consider a positive response: A positive result is that where the Stimulating Index (SI) ≥ 3.

TREATMENT PREPARATION AND ADMINISTRATION:

the test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1 at concentrations of 50%, 25% and 10% v/v. The mice were treated by daily application of 25 uL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 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. the control group 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 uL of phosphate buffer saline (PBS) containing 3H-methyl thymidine (3HTdR: 8uCi/mL, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 uCi to each mouse.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The stimulation index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
Concentration (% v/v) in acetone/olive oil 4:1: 25
Stimulation Index: 7.25
Therefore it is considered to be positive - a skin sensitiser
Parameter:
SI
Value:
0.41
Test group / Remarks:
10%
Parameter:
SI
Value:
0.52
Test group / Remarks:
25%
Parameter:
SI
Value:
0.79
Test group / Remarks:
50%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA

DETAILS ON STIMULATION INDEX CALCULATION: Outlined in Table 1

CLINICAL OBSERVATIONS: There were no deaths, no signs of systemic toxicity were noted in the test or control animals during the test. Clinical observations are outlined in Table 2.

BODY WEIGHTS: Body weight changes if the test item treated animals between day 1 and day 6 are outlined in Table 3 they were comparable to those observed in the corresponding control group animals over the same period.

Table 1. Disintegrations per minute disintegrations per minute/node and stimulation index








































 Concentration (% v/v) in acetone/olive oil 4:1 DPM DPM/Node * Stimulation Index ** Result
 Vehicle 10,609.10 1,326.14 NA NA
 10 4,385.87 548.23 0.41 Negative
 25 5,530.79 691.35 0.52 Negative
 50 8,419.83 1,052.48 0.79 Negative

DPM = Disintegrations per minute


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


** = Stimulation index of 3.0 or greater indicates a positive result


NA = Not applicable


 


 


Table 2: Individual Clinical Observations and Mortality Data 




























































































































































































































Concentration (% v/v) in acetone/olive oil 4:1



Animal number



Day 1



Day 2



Day 3



Day 4



Day 5



Day 6



Pre- Dose



Post- Dose



Pre-Dose



Post -Dose



Pre -Dose



Post Dose



Vehicle



1-1



0



0



0



0



0



0



0



0



0



1-2



0



0



0



0



0



0



0



0



0



1-3



0



0



0



0



0



0



0



0



0



1-4



0



0



0



0



0



0



0



0



0



10



2-1



0



0



0



0



0



0



0



0



0



2-2



0



0



0



0



0



0



0



0



0



2-3



0



0



0



0



0



0



0



0



0



2-4



0



0



0



0



0



0



0



0



0



25



3-1



0



0



0



0



0



0



0



0



0



3-2



0



0



0



0



0



0



0



0



0



3-3



0



0



0



0



0



0



0



0



0



3-4



0



0



0



0



0



0



0



0



0



50



4-1



0



0



0



0



0



0



0



0



0



4-2



0



0



0



0



0



0



0



0



0



4-3



0



0



0



0



0



0



0



0



0



4-4



0



0



0



0



0



0



0



0



0



0 = No signs of systemic toxicity 


 


 


 


Table 3: Individual bodyweights and body weight change




















































































































Concentration (% v/v) in acetone/olive oil 4:1



Animal number



Bodyweight (g)



Body weight changes



Day 1



Day 6



Vehicle



1-1



18



20



2



1-2



19



19



0



1-3



20



19



-1



1-4



20



19



-1



10



2-1



21



22



1



2-2



19



17



-2



2-3



19



20



1



2-4



18



19



1



25



3-1



18



20



2



3-2



19



19



0



3-3



19



19



0



3-4



18



19



1



50



4-1



18



19



1



4-2



19



19



0



4-3



18



19



1



4-4



19



20



1



 


All other raw data can be found in the study report.

Interpretation of results:
other: No sensitising potential according to OECD TG 429
Remarks:
The SI was less than 3 for all three concentrations evaluated
Conclusions:
Under the conditions of the LLNA in the mouse the test item did not show any skin sensitising properties. The SI for all concentrations evaluated were less than 1. The available data on skin sensitising of the test substance do not meet the criteria for classification according to the CLP Regulation (EC) No 1272/2008, and therefore conclusive but not sufficient for classification.
Executive summary:

The study was performed to OECD TG 429 under GLP to assess the sensitization potential of the test material to the skin following a single application to the CBA/Ca mouse. A volume of 25ul pf the test material at concentrations of 50%, 25% or 10% in acetone/olive oil 4:1 was applied to the dorsal surface of each ear for three consecutive days( days 1, 2 and 3). There were no deaths and no signs of systemic toxicity in the test or control animals during the testing period. under the conditions of this study, the test item was considered not to be sensitizing to the skin. 

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

Justification for classification or non-classification

Results from the key studies OECD TG 429 (Sanders, 2011) were sufficient for a no sensitising classification according to CLP Regulation (EC) No 1272/2008.