Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 January 2015 - 26 January 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Performed in a GLP laboratory in accordance with OECD and EU test guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
not specified
Details on test material:
Physical state/ Appearance: off white solid blocks.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Age at study initiation: 8 to 12 weeks old.
- Weight at study initiation: 15 to 23 g
- Housing: animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): Free access to food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains tap water.
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours of night and 12 hours of continuous light.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
10, 5 and 2.5% (w/w) of the test substance in the vehicle (dimenthyl formamide). A further group of animals received the vehicle alone.
No. of animals per dose:
Four mice per dose group.
Details on study design:
PRELIMINARY SCREENING TEST:
The mice were treated by daily application of 25 µL of the test item at concentrations of 50%, 25% or 10% w/w in dimethyl formamide, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily.

MAIN TEST:
Groups of four mice were treated with the test item at concentrations of 10%, 5% or 2.5% w/w in dimethyl formamide. 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.

Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.

Terminal Procedures:
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". 3HTdR incorporation was measured by β-scintillation counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The concentration of α-Hexylcinnamaldehyde, tech., 85% expected to cause a 3 fold increase in 3HTdR incorporation (EC value) was calculated to be 6%.

Conclusion

α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
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: CONCENTRATION (% W/W) IN DIMETHYL FORMAMIDE: 2.5 STIMULATION INDEX: 8.21 RESULT: Positive CONCENTRATION (% W/W) IN DIMETHYL FORMAMIDE: 5 STIMULATION INDEX: 13.03 RESULT: Positive CONCENTRATION (% W/W) IN DIMETHYL FORMAMIDE: 10 STIMULATION INDEX: 11.18 RESULT: Positive
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 4.

Any other information on results incl. tables

Table 4: Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index.

Concentration
(% w/w) in
dimethyl formamide
dpm dpm/Node Stimulation
Index
Result
Vehicle 9753.09 1219.14 na na
2.5 80106.12 10013.27 8.21 Positive
5 127067.3 15883.41 13.03 Positive
10 109008.2 13626.03 11.18 Positive

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Category 1, sub-category 1A Criteria used for interpretation of results: EU
Conclusions:
The test item was considered to be a sensitizer under the conditions of the test.

The test item was classified as a contact sensitizer (Category 1, sub-category 1A) according to the Globally Harmonized System of Classification and Labelling of Chemicals.
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 10% 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 10%, 5% or 2.5% 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:

Concentration (% w/w) in dimethyl formamide

Stimulation Index

Result

2.5

8.21

Positive

5

13.03

Positive

10

11.18

Positive

 

The test item was considered to be a sensitizer under the conditions of the test.

The test item was classified as a contact sensitizer (Category 1, sub-category 1A) according to the Globally Harmonized System of Classification and Labelling of Chemicals.