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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 September 2010 to 6 October 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted in accordance with current test guidelines and the principles of GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
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
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Glycidol
IUPAC Name:
Glycidol
Constituent 2
Chemical structure
Reference substance name:
2,3-epoxypropan-1-ol
EC Number:
209-128-3
EC Name:
2,3-epoxypropan-1-ol
Cas Number:
556-52-5
Molecular formula:
C3H6O2
IUPAC Name:
oxiran-2-ylmethanol
Details on test material:
- Name of test material (as cited in study report): Glycidol

- Physical state: colourless liquid
- Analytical purity: 97.5%
- Purity test date: 20 August 2010
- Lot/batch No.: GD-AH-0.13
- Expiration date of the lot/batch: 22 November 2010
-Storage condition of test material:in a sealed container, at 2 to 8°C 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: CBA/CaCrl strain obtained from Charles River UK Margate, England
- Age at study initiation: 8-9 weeks old
- Weight at study initiation: 16-20g on day prior to initiation
- Housing: group housed during acclimatisation and singly housed for remainder of study period in standard laboratory cages
- Diet (e.g. ad libitum): SQC(E) rat and Mouse Maintenance Diet No 1 from SDS Ltd ad libitum
- Water (e.g. ad libitum): mains potable tap water via cage bottles ad libitum
- Acclimation period: 15 - 22 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65% RH
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 September 2010 To: 6 October 2010

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
the first in the approved list of vehicles that produced a solution at 50% concentration
Concentration:
100% (undiluted); 50% v/v and 25% v/v selected on basis of preliminary screening test results.
The humane termination of the screening animal after two dosing occasions (convulsions on removal from cage immediately prior to the third dose administration were attributed to a sporadic background effect of handling) was not attributed to a treatment related effect and the undiluted test material did not elicit irritation.

While there were three deaths in the study (one screening animal and one mouse from each of the high and intermediate dose group), there were no indications that a treatment relations had been established in causing death. In the opinion of the study director, the convulsive episodes and clinical signs were indicative of spontaneously occuring sporadic epileptic episodes known to occur in mice when they are handled. There were no corroborative necropsy findings to indicate any neurotoxic or treatment related effects.

Since death is not one ofthe criterai for assessing sensitisation potential and the lymph node response is based on pooled results for each group, the deaths of these individuals is not considered to have adversely affected the integrity or validity of the response determined in this study.
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: dimethylformamide was the first of the recommended vehicles to allow a 50% v/v solution to be prepared. In the screening test for aural irritation the test material was only applied as supplied - undiluted (100%).
- Irritation: undiluted gylcidol did not elicit significant irritation
- Lymph node proliferation response: not recorded in screening test

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: local lymph node assay
- Criteria used to consider a positive response:
The test result is not valid for those groups producing an SI value of 3.0 or more when the sites of application have shown excessive irritation and for those groups that have shown indications of systemic toxicosis.
The test article is regarded as a sensitiser when the maximum value of the SI is 3.0 or above.
The test article is classified as a non sensitiser when the maximum value of the SI is less than 3.0. (This result is unchanged by observations of irritation at sites of application of the test formulation).

TREATMENT PREPARATION AND ADMINISTRATION:
Formulations were freshly prepared as required using dimethylformamide on Days 1, 2 and 3.

Groups of four female mice were assigned to four groups. Doses were selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1.0%, 0.5% based on theresults ofthe screening test. Three consecutive concentrations were selected - 100%, 50%, 25%, - so that the highest concentration maximised exposure whilst avoiding systemic toxicity and excessive local irritation.

Each mouse was manually restrained with both auditory pinnae left free. The outer aspect of both pinnae of each mouse was treated by direct application of the appropriate test or control formulation (0.025 mL/pinna) dispensed from an automatic micro pipette. Treatment was applied on Days 1-3.

On Day 6 the mice were placed in a thermacage in order to dilate the peripheral blood vasculature and thus facilitate intravenous dosing. Each mouse was transferred to a cylindrical restrainer. A plastic syringe and fine gauge hypodermic needle were used to administer 0.25 mL phosphate buffered saline incorporating 20 μCi of 3HTdR into a tail vein of each mouse by slow bolus injection. Approximately five hours after intravenous injection of the 3HTdR, all mice were killed and auricular lymph nodes excised. The interval between death and the recovery of the auricular lymph nodes was restricted to no more than fifteen minutes

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
mercaptobenzothiazole (CAS No 149-30-4)
Statistics:
Incorporation of 3HTdR is measured by ß-scintillation counting as disintegrations per minute (DPM) over a ten-minute period. This value was corrected to account for the background containing 5% w/v aqueous trichloroacetic acid and scintillation fluid.
The scintillation counter printed data including the DPM value (disintegrations per minute during a ten minute period). The DPM value was transformed into a DLM value (disintegrations per minute per lymph node) by dividing the number of sites yielding lymph nodes. The DLM value for each test group was divided by the DLM for the control group to provide the Stimulation Index (SI) value for each test group

Results and discussion

Positive control results:
Results of nine positive control studies were all positive for sensitisation giving SI values in excees of 3.0 and a linear dose response, confirming the validity of the assay methods

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 0.5; 1.2 and 1.6 for 25%, 50% and 100%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Sample identity Number of sites Disintegrations Disintegrations Stimulation yielding per minute per minute Index (SI) lymph nodes (DPM) per node (DLM) Scintillation fluid with 5% w/v trichloroacetic acid -- 61 -- -- Vehicle control 8 808 101 -- Test article, 25% v 8 401 50 0.5 Test article, 50% v 6 751 125 1.2 Test article, 100% v/v 6 947 158 1.6

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The Local Lymph Node Assay demonstrated that Glycidol does not have the potential to cause skin sensitisation.
The test article did not meet the criteria for classification as a sensitiser according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
Executive summary:

A local lymph node assay was conducted to assess the potential of Glycidol to cause skin sensitisation in the mouse.

Following a preliminary screening test using the undiluted test article, the main study was conducted using the undiluted test article and concentrations of 25 and 50% v/v in dimethylformamide. Groups of four female CBA / CaCrl mice were subjected to topical applications of vehicle or of one of the test formulations to the outer aspect of the auditory pinnae once daily on Days 1, 2 and 3.

On Day 6 a 20 μCi dose of tritiated3H-methyl thymidine was injected intravenously into each mouse. Five hours later the auricular lymph nodes were recovered from each animal. The nodes from mice subjected to the same treatment were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter.

Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation count per group obtained from the test groups relative to the corresponding mean scintillation count from controls. The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0. The Local Lymph Node Assay demonstrated that Glycidol does not have the potential to cause skin sensitisation.The test article did not meet the criteria for classification as a sensitiser according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).