Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-02-02 to 2009-02-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study with minor deviation to the guideline without effects on results: - animals were individually housed - 15 % concentration of hexyl cinnamic aldehyde was used as positive control - preliminary study: erythema was not scored according to the scale stated in the guideline - preliminary study: ear thickness was not taken - identification data of vehicle missing (purity, concentration) - mean and standard deviation of body weight was not calculated

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
, adopted 2002-04-24
Deviations:
yes
Remarks:
, identification data (purity; volume used) of the vehicle missing
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
, 2008
Deviations:
yes
Remarks:
, identification data (purity; volume used) of the vehicle missing
GLP compliance:
yes (incl. certificate)
Remarks:
date of inspection 2008-08-19
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Physical state: solid, whitish to white crystals
- Storage condition of test material: room temperature 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:
Harlan Laboratories UK Limited, Bicester, Oxon, UK (CBA/Ca (CBA/CaOlaHsd) strain mice)
B & K Universal Ltd. Hull, UK (CBA/CaBkl strain mice)
- Age at study initiation: eight to twelve weeks old
- Weight at study initiation: preliminary screening test: 18 g; main study: 17 to 21 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (ad libitum): 2014 Teklad Global Rodent diet
- Water (ad libitum): mains tap water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Relative humidity: 30 to 70 %
- Air changes: approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
2.5 % w/w, 5 % w/w and 10 % w/w in the vehicle
No. of animals per dose:
4 female mice
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: vehicle was chosen as it produced the highest concentration that was suitable for dosing.

A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test material at a concentration of 10 % w/w in vehicle, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.

Results:
No signs of systemic toxicity were noted.
The dose levels selected for the main test were 10 %, 5 % and 2.5 % w/w in dimethyl formamide.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- groups of four mice were treated with the test material at the three concentrations in the vehicle.
- mice were treated by daily application of 25 µL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).
- a further group of four mice received the vehicle alone in the same manner.
- five days following the first topical application of the test material (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) giving a total of 20 µCi to each mouse.
- five hours following the administration of 3HTdR all mice were killed.
- draining auricular lymph nodes from the four mice were excised and pooled for each experimental group.
- 1 mL of PBS was added to the pooled lymph nodes.
- single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze.
- lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish.
- lymph node cell suspension was transferred to a centrifuge tube.
- petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube.
- pooled lymph node cells were pelleted at 1400 rpm for ten minutes.
- pellet was resuspended in 10 mL of PBS and re-pelleted.
- pellet was resuspended in 3 mL of 5 % Trichloroacetic acid (TCA).
- after approximately eighteen hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid.
- 3HTdR incorporation was measured by β-scintillation counting.
- vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes.
- after approximately twenty minutes, the vials were shaken.
- number of radioactive disintegrations per minute was then measured using a scintillation system.

- Criteria used to consider a positive response: the test material was regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation was classified as a "non-sensitiser".

OBSERVATIONS:
- 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.
- Bodyweights: bodyweight of each mouse was recorded on Day 1 (prior to dosing ) and Day 6 (prior to termination).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegration 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).

Results and discussion

Positive control results:
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: SI = 4.55
The positive control was considered to be a sensitiser.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: A stimulation index of less than 3 was recorded for all concentrations. 2.5 % w/w: SI = 1.12 5 % w/w: SI = 1.22 10 % w/w: SI = 1.24
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Vehicle: 3564.63 (dpm/group); 445.58 (dpm/node) 2.5 % w/w: 4003.20 (dpm/group); 500.40 (dpm/node) 5 % w/w: 4350.42 (dpm/group); 543.80 (dpm/node) 10 % w/w: 4429.53 (dpm per group); 553.69 (dpm/node) dpm/node obtained by dividing the dpm value by 8 (total number of lymph nodes)

Any other information on results incl. tables

OBSERVATIONS:

- clinical observations: there were no deaths and no signs of systemic toxicity.

- bodyweight: changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The substance is not a skin sensitiser. According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classification as skin sensitiser.
Executive summary:

The skin sensitisation potential of the substance were investigated in female CBA mice using the Local Lymph Node Assay (LLNA) (OECD 429). Based on the results of preliminary testing, concentrations of 2.5, 5, and 10 % of the test item in dimethyl formamide were used in the main test. Groups of four animals were treated with 25 µL per ear of the test item concentrations. A further group of four animals was treated with the vehicle. Historical data were used for the positive control substance (15% α- hexyl cinnamic aldehyde). Treatment was conducted on three consecutive days followed by 3H-methyl thymidine administration on day 6. Five hours following the administration of 3H-methyl thymidine animals were killed and draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. The number of radioactive disintegrations per minute (dpm) was measured using a scintillation system and the Stimulation index was determined for each treatment group.

Overall, any sign of systemic toxicity or mortality was not observed. The body weight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals.

Based on the pooled data of cellular proliferation, stimulation indices of 1.12, 1.22, and 1.24 were determined at substance concentrations of 2.5 %, 5 %, and 10 %, respectively. With respect to the derived stimulation index, which is lower than 3, the substance tested not positive for skin sensitisation. The results of the positive control with 15 % α-HCA confirmed the validity of the assay.

According to the Regulation (EC) No 1272/2008 and subsequent adaptations, the test substance is not classified as skin sensitiser.