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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Remarks:
LLNA:BrdU
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-11-04 to 2017-03-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Version / remarks:
adopted 22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Method B.51 of the Council Regulation n° 640/2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 23 October 2015
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Constituent 1
Test material form:
liquid: viscous

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Remarks:
CBA/J (CBA/JRj)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage Janvier Labs - 53941 Le Genest saint Isle - France
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: 19.1 - 25.0 g
- Housing: Full barrier in an air-conditioned room; The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding (lot no. 291109, preliminary test; 101109, main test)
- Diet (ad libitum): Altromin 1324 maintenance diet for rats and mice (lot no. 1130)
- Water (ad libitum): Tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal resdue control, microbiological controls at regular intervals).
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3°C
- Relative humidity: 55 +/- 10 %
- Air changes: At least 10 X / hour
- Photoperiod (hrs dark / hrs light): 12/12

No further information on the test animals was stated.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25 %, 50 % and 100 %
No. of animals per dose:
4 female mice per test group
4 female mice for negative control (vehicle)
Details on study design:
PRELIMINARY STUDY:
As no information was available regarding irritant potential or systemic toxicity of the test item in the mouse, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test undiluted (100%) to the dorsal surface of each ear for three consecutive days (days 1, 2, 3). The mouse was observed daily from day 1. Any signs of toxicity or excessive irritation noted during this period were recorded. Ear thickness was recorded on day 1, day 3 and on day 6. The bodyweight of the mouse was recorded on day 1 (prior to dosing) and day 6.

MAIN STUDY:
Test item administration:
Groups of four mice were treated with the test item undiluted (100%) and diluted at 50% and 25% in Acetone/olive oil (4:1, v/v). 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 was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.
An additional mouse was treated in each group in case of problem which may occur during the study, in particular during tthe excision of lymph nodes. As no problem occured in groups during the collecting of lymph nodes, the lymph nodes of the additional mouse were not collected and the data concerning this animal was not used in the study.
Clinical observations:
- Clinical observations and mortality: All animals were observed daily on days 1, 2, 3, 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Bodyweights: The bodyweight of each mouse was recorderd on day 1 (prior to dosing) and on day 6 (prior to termination).
- Ear thickness measurements and recording of local reactions: Ear thicknesss measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses. On day 1 and on day 3 (before applicaiton) as well as on day 6 (after sacrifice) of each experiment, the thickness of the righ ear of each animal of the vehicle control and treated groups was measured by a micrometer. Furthermore, on day 6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighed in order to assess the irritation potential of the test item and the two lymph nodes per mouse were weighed.
- Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (dryness, presence of residual test item...) was noted.

BrdU INJECTION:
- Day 5
- 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route.
- The BrdU solution was prepared by weighing 201.9 mg of 5-bromo-2'-deoxyuridine (SIGMA - batch n° HMBF3943V) in a glass sample bottle and adding 20.19 mL of physiological saline. Then, the preparation was magnetically stirred and by Vortex, just before the treatement.

TERMINAL PROCEDURES:
- Termination: On day 6 (end of the test), the animals were anaesthetised with sodium pentobarbital and administration continued to fatal levels. The draining auricular lymph nodes from the four mice were excised.
-Preparation of Single Cell Suspension: From each mouse, a single-cell suspension of lymph node cells (LNC) excised bilaterally was prepared by gentle mechanical disaggregation through a disposable plastic pestle to crush the lymph nodes followed by passage through a #70 nylon mesh in 15 mL of DPBS (Ca 2+/mg 2+ - free) into a well of a multi-well 6. The optimised volume was based on achieving a mean absorbance of the negative control group within 0.1 - 0.2.

STIMULATION INDEX DETERMINATION:
BrdU was measured by ELISA using a commercial kit (e.g. Roche Applied Science, Mannheim, Germany, Catalogue Numbr 11 647 229 001 - batch n° 17267000). Briefly, 100 µL of the LNC suspension was added to the wells of a flat-bottom microplate at least in triplicate. After fixation and denaturation of the LNC, anti-BrdU antibody was removed by washing and the substrate solution was then added and allowed to produce chromogen. After 5 to 30 min, 30 µL of 1M H2SO4 was added in each well, then shaken for one minute. Absorbance at 450 nm with a reference wavelength of 690 nm was then measured.
The BrdU labelling index was defined as:
BrdU labelling index = (ABSem -ABSblankem) - (ABSref - ABSblankref)
em = emission wavelength and ref = reference wavelength
The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 16 compared to control values.
However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result (i.e. SI value between 1.6 and 1.9 ) is declared positive.
Any test item failing to produce a SI > 1.6 will be classified as a 'non-sensitiser'.

DETERMINATION OF THE EC1.6 VALUE:
The EC1.6 value (theoretical concentration resulting in a SI value of 1.6) was determined by linear interpolation of points on the dose-response curve, immediately above and below the 1.6-fold threshold. The equation used for calculation of EC1.6 was:
EC1.6 = c+[(1.6-d)/(b-d)]*(a-c) with
a = the lowest concentration giving stimulation index > 1.6
b = the actual stimulation index caused by a
c = the highest concentration failing to produce a stimulation index of 1.6
d= the actual stimulation index caused by c

INTERPRETATION OF THE RESULTS:
In accordance with the Regulation (EC) n° 1272/2008, the studies obtained for test item should be classified:
- If at least one concentration of the test item results in a SI ≥3, as sensitizer and characterised by the signal word 'Warning' with the hazard statement H317 'May cause an allergic skin reaction'.
The value 3 is based on the interpretation of [3H]-Thymidine incorporation in lymphocytes as descripted in the OECD test guideline n° 429.
According to the OECD test guideline n°442-B, the decision process regards a result as positive when SI ≥ 1.6. However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result (i.e. SI value between 1.6 and 1.9 ) is declared positive.
For a borderline positive response between a SI of 1.6 and 1.9, users may want to consider additional information such as dose-response relationship, evidence of systemic toxiicity or excessive irritation and where appropriate, statistical significance together with SI values to confirm that such results are positive. Consideration should also be given to known skin sensitizers, whether it causes excessive skin irritation in the mouse, and the nature of the dose-response observed.
In accordance with the Regulation (EC) n° 286/2011, the positive test item will be classified in sub-category 1A or 1B in accordance with the following:
- Sub-category 1A: EC value ≤ 2%
- Sub-category 1B: EC value > 2%
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
In the positive control group given Hexylcinnamaldehyde (CAS n° 101-86-0 - purity 95%), the Stimulation Index expressed as the BrdU labelling idnex for each treatement group divide by the mean BrdU labelling index of the vehicle control group are as follows:
- C = 5%: SI = 1.34
- C = 10%: SI = 1.63
- C = 25%: SI = 1.95
In conclusion, in view of the results, under these experimental conditions, the substance Hexylcinnamaldehyde in accordance with the Regulation (EC) n° 1272/2008 has to be calssified in category 1 'Skin sensitization'.
The study was therefore considered valid.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Remarks:
mean
Value:
1.14
Variability:
+/- 0.05
Test group / Remarks:
concentration = 25%
Key result
Parameter:
SI
Remarks:
mean
Value:
1.77
Variability:
+/- 0.07
Test group / Remarks:
concentration = 50%
Key result
Parameter:
SI
Remarks:
mean
Value:
1.56
Variability:
+/- 0.19
Test group / Remarks:
concentration = 100%
Key result
Parameter:
other: EC1.6
Value:
43.25
Remarks on result:
other: %
Cellular proliferation data / Observations:
One Stimulation index of more than 1.6 was recorded at the tested concentration of 50%.
The Stimulation Index (SI) calculated by individual approach was 1.14, 1.77 and 1.56 for the treated groups at 25%, 50% and 100% respectively.
The EC1.6 determined by linear regression was 43.25%.
The result obtained may be considered as a borderline result (i.e. positive response between an SI 1.6 and 1.9) and no dose response was noted.
The lowest SI value at the tested concentration 100% may be explained by a best bioavailability of the test item at lowest concentration.

Any other information on results incl. tables

PRELIMINARY SCREENING TEST:

No mortality and no signs of systematic toxicity were noted.

Blue coloration was noted on the treated area between days 2 and 6.

Therefore, 100% was chosen as the highest concentration for the main study.

MAIN TEST:

Clinical obsevations and mortality:

No mortality and no signs of systematic toxicity were noted in the test and control animals during the test. Residual test item was noted in all animals treated at 50% and 100% on day 2 to day 6.

Weight evolution:

Bodyweight changes of the test animals between day 1 and day 6 were comparable to those observed in the corresponding control group animals over the period.

Local irritation:

No increase in ear thickness and in ear weight was noted in animals treated at 25% and 50%. Therefore the test item has to be considered as not irritant at these concentrations.

An increase in ear thickness (+19.7%) and in ear weight (+ 34%) was noted in animals treated at 100%. Therefore, the test item has to be considered as slightly irritant at this concentration.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The results obtained in these experimental conditions, enable to conclude that the test item has to be classified as a sensitiser in Category 1 (sub-category 1B) in accordance with the Regulation (EC) n° 1272/2008 on classification, labelling and packaging of substances and mixtures. The signal word 'Warning' and hazard statement H317 'May cause an allergic skin reaction' are required.