Registration Dossier

Administrative data

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:
Nov. 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Gouvernment of the United Kingdom
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
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: eight to twelve weeks
- Weight at study initiation: 15 to 23 g
- Housing: individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): 2014C Teklad Global Rodent diet ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06.00 to 18.00) and twelve hours darkness
Vehicle:
dimethylformamide
Concentration:
50%, 25% or 10% w/w in dimethyl formamide
No. of animals per dose:
4
Details on study design:
The test item was freshly prepared as a solution in dimethyl formamide. 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 exception is considered not to affect the purpose or integrity of the study.
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.
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 methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse. Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. 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."
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
A group of five animals was treated with 50 µL (25 µL per ear) of a Hexylcinnamaldehyde, tech., 85% as a solution in dimethyl formamide at a concentration of 15% v/v. A further control group of five animals was treated with dimethyl formamide alone. 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 5.34. a Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: 10% w/w resulted in SI of 1.14 25% w/w resulted in SI of 1.22 50% w/w resulted in SI of 1.15
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM: control: 15297.24 10% w/w: 17383.45 25% w/w: 18652.32 50% w/w: 17570.61

There were no deaths. No signs of systemic toxicity were noted in the test or control animalsduring 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.

Measurement of Ear Thickness and Mean Ear Thickness Changes - Preliminary Screening Test

Concentration
(%
w/w) in
dimethyl formamide

Animal Number

Ear Thickness Measurement (mm)

Day 1

Day 3

Day 6

pre-dose

post dose

left

right

left

right

left

right

50

S-1

0.21

0.21

0.20

0.20

0.21

0.21

overall mean (mm)

0.21

0.20

0.21

overall mean ear thickness change (%)

na

-4.76

0.00

na=       Not applicable

Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration
(%
w/w) in
dimethyl formamide

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

15297.24

1912.16

na

na

10

17383.45

2172.93

1.14

Negative

25

18652.32

2331.54

1.22

Negative

50

17570.61

2196.33

1.15

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

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: other: EU-GHS
Conclusions:
The study was performed according to the OECD TG 429 without deviations and according to the good laboratory practice principles, it is considered to be of the highest quality (reliability Klimisch 1). The criteria of validity of the test system are fulfilled. The test item was considered to be a non-sensitizer under the conditions of the test.
Executive summary:

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. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% 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 solution in dimethyl formamide at concentrations of 50%, 25% or 10% 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 are as follows: 1.14 for 10% w/w, 1.22 for 25% w/w and 1.15 for 50% w/w. The test item was considered to be a non-sensitizer under the conditions of the test.

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

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. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% 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 solution in dimethyl formamide at concentrations of 50%, 25% or 10% 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 are as follows: 1.14 for 10% w/w, 1.22 for 25% w/w and 1.15 for 50% w/w. The test item was considered to be a non-sensitizer under the conditions of the test.


Migrated from Short description of key information:
LLNA (2015), GLP, OECD 429, mouse, solution in dimethyl formamide of 50%, 25% or 10% w/w, non-sensitizer

Justification for selection of skin sensitisation endpoint:
well documented study with GLP certificate

Justification for classification or non-classification