Registration Dossier

Administrative data

Endpoint:
skin sensitisation
Remarks:
in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 November 2000 - 19 December 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Principles of method if other than guideline:
OECD guideline 406 is given in the report, which is probably an error and it is assumed that guideline 429 was followed.
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
It cannot be entirely excluded that the test material used was the mono potassium salt rather than the di potassium salt.
Specific details on test material used for the study:
It cannot be entirely excluded that the test material used was the mono potassium salt rather than the di potassium salt. However, based on the very similar properties of these compounds, the toxicological data presented is considered valid and suitable to describe the toxicological property of the di potassium salt even if generated with the mono salt.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca/Ola/Hsd
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK, Blackthorne, Bicester, Oxon, UK
- Age at study initiation: Young adults, approx. 13 weeks old
- Weight at study initiation:
- Housing: A maximum of 4 mice was housed per cage, in cages suitable for animais of this strain and weight range
- Diet: RM 1, supplied by Special Diets Services Limnited, Withamn, Essex, UK, available ad libitum.
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): A minimum of 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
other: propylene glycol (test substance) and acetone (positive control)
Concentration:
3%, 10% or 30% w/v
No. of animals per dose:
4
Details on study design:
MAIN STUDY
- Criteria used to consider a positive response: The criterion for a positive response is that one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.

TREATMENT PREPARATION AND ADMINISTRATION:
Approximately 25 µl of a 3%, 10% or 30% w/v preparation of the test substance in propylene glycol was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using propylene glycol alone. The procedure was repeated daily for 3 consecutive days. A concurrent naive control group was not treated with the test substance or the vehicle.
Three days after the third application, all the animals were injected, via the tail vein, with approximately 250 µl of phosphate buffered saline (PBS) containing approximately 20 µCi of a 2.0 Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.
A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10ml of PBS. Approximately 3 ml of 5% w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4°C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1 ml of TCA.
The lymph node suspensions were transferred to scintillation vials and 10 ml of scintillant (Optiphase) was added prior to ß-scintillation counting using a Packard Tri-Carb 2500TR Liquid Scintillation Counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
other:

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.57
Test group / Remarks:
3%
Parameter:
SI
Value:
1.55
Test group / Remarks:
10%
Parameter:
SI
Value:
0.75
Test group / Remarks:
30%

Any other information on results incl. tables

SKIN SENSITISATION POTENTIAL - TEST SUBSTANCE

Test item concentration number of lymph nodes Disintegrations per minute (dpm) DPM/lymph node (x10-2) Test control ratio
Naive control 8 2233 2.79 N/A
vehicle control 8 1729 2.16 N/A
3 % (w/v) 8 981 1.23 0.57
10 % (w/v) 8 2670 3.34 1.55
30 % (w/v) 8 1296 1.62 0.75

SKIN SENSITISATION POTENTIAL OF THE POSITIVE CONTROL SUBSTANCE

concentration of hexyleinnamaldehyde number of lymph nodes Disintegrations per minute (dpm) DPM/lymph node (x10-2) Test control ratio
Naive control 8 3672 4.59 N/A
vehicle control 8 2064 2.58 N/A
1 % (w/v) 8 6839 8.55 3.31
3 % (w/v) 8 12164 15.21 5.90
10 % (w/v) 8 23776 29.72 11.52

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test article is unlikely to be a moderate or strong skin sensitiser under the conditions of the test.
Executive summary:

The test article was assessed for its skin sensitisation potential using the mouse Local Lymph Node Assay. The test substance was applied as 3%, 10% or 30% w/v preparations in propylene glyco1. The test substance did not have the capacity to cause skin sensitisation when applied as 3%, 10% or 30% w/v preparations in propylene glycol. In a positive control study, hexylcinnamaldehyde was shown to have the capacity to cause skin sensitisation when applied as 3% or 10% w/v preparations in acetone, confirming the validity of the protocol used for this study. In conclusion, the test substance is unlikely to be a moderate or strong skin sensitiser under the conditions of this test.