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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:
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.
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)
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.
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)
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.
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.

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
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A single study has been conducted in accordance with GLP and the OECD 429 guideline. The results of the study indicate that the substance is a potent skin sensitiser in mice with a measured EC3 value of <2.5% w/w in dimethylformamide. An extrapolated EC3 value of 1.2% was calculated in accordance with the methodology set out in Ryan et al 2007 Cutan Ocul Toxicol 26(2)135-45. This is converted to a dose of 300µg/cm2 in accordance with ECHA guidance document Chapter R8, in Appendix R.8-10.; this point of departure is considered to be a LOAEL.


Migrated from Short description of key information:
Skin sensitisation: Local Lymph Node Assay: mouse (CBA/Ca) female (OECD 429, GLP): the EC3 for the substance is <2.5% w/w in dimethylformamide. The calculated EC3 is 1.2%.

Justification for selection of skin sensitisation endpoint:
The test substance was analysed in accordance with OECD 429 and EU method B42. The substance was determined to be a sensitizer (Category 1, sub-category 1A).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance has been tested in the mouse local lymph node assay in accordance with OECD 429. Application of the classification criteria set out in CLP Regulation Annex I Section 3.4.2.2.3 results in a classification of Skin sensitiser Category 1A for the calculated EC3 value of 1.2% w/v in dimethylformamide.