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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-07-11 to 2014-10-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

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
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Disperse Red 302

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (prescreen test and main study: lot no. 1526)
- Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control,
microbiological controls at regular intervals)
- The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
(prescreen test and main study: lot no. 290114)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days) under laboratory conditions

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Remarks:
Applichem, lot no. 2P003474, expiry date: 09/2014
Concentration:
The maximum technically applicable concentration of the test item in the vehicle was found to be 12.5 % in DMSO.
Based on the results observed in the preliminary test the following test item concentrations were selected for the main study:
3 %, 6.25 % and 12.5 % (w/v)
DMSO was used as vehicle and served as negative control.
No. of animals per dose:
5 mice / group; 3 mice / prescreen test
Details on study design:
Preparation of the Test Item
Based on the results observed in the prescreen test the following test item concentrations were selected for the main study:
3 %, 6.25 % and 12.5 % (w/v)
The preparations were made immediately prior to each dosing.

Preparation of the Animals
The animals were randomly selected. Identification was ensured by cage number and individual marking (tail).

Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity,
including dermal irritation at site of application.

Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

Dose Groups
3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.

Test Regimen

Topical Application
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days.

Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection
(tail vein) of 250 µL of 3H-methyl thymidine, diluted to a working concentration of 80 µCi/mL.

Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The
draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected
in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical
disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted
in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5 % TCA at approx. 4 °C for approximately 18 hours for
precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5 % TCA and 7 mL scintillation
fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.
Determination of Incorporated 3H -Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per
minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5 % TCA). Determination of radioactivity
was performed individually for each animal.

Evaluation of Results
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per
lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals
relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined,
background values were subtracted. EC3 values, calculated concentrations which induce stimulation indices of three, are
determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a c), between two points of the stimulation indices axis,
one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation
index of three, no EC3 value can be stated.

A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or
greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that
recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).
On the basis of the test results, the test substance may be classified into one of the following categories in conformity with
the criteria given in Commission Regulation (EU) No 286/2011 as well as in GHS - Globally Harmonised System of
Classification and Labelling of Chemicals, fifth revised edition, 2013:

Skin sensitiser
Category 1:
A substance is classified as a skin sensitiser
a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons, or
b) if there are positive results from an appropriate animal test.
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1A:
Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.
EC3 value ≤ 2 %
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1B:
Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.
EC3 value > 2 %
WARNING, exclamation mark. May cause an allergic skin reaction.

Positive control substance(s):
other: Phenylenediamine
Statistics:
Outlier tests according to Dixon, Grubbs and Nalimov were performed for the values measured for the number of disintegrations per minute (DPM).
If outliers were identified, these values were not included in the calculation of the stimulation indices. As at least four
values per group are required for the evaluation of the results, the outlier test was not repeated to detect further outliers.

Results and discussion

Positive control results:
The recent reliability check was performed in May 2014. The raw data of this study are kept in the BSL archives (BSL Project ID 142473 A).
Positive-control substance: P-Phenylenediamine (CAS 106-50-3, Sigma, purity > 98%; Lot No.: SLBC7171V) 1%
Vehicle: DMSO
Species/strain: healthy CBA/CaOlaHsd mice
Source: Harlan Laboratories GmbH, 5800 AN Venray, The Netherlands
Concentrations: 1% on three consecutive days

The Stimulation Index was 10.0

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Variability:
0.2
Test group / Remarks:
3%
Key result
Parameter:
SI
Value:
1.1
Variability:
0.1
Test group / Remarks:
6.25%
Key result
Parameter:
SI
Value:
1
Variability:
0.2
Test group / Remarks:
12.5%

Any other information on results incl. tables

All animals survived throughout the test period without showing any adverse clinical signs.

All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

Radioactive Determination of the Test Substance Groups

POS CPM Test Item Conc. [%] Animal number DPM DPM- mean back- ground DPM/ Node Stimulation Index
16 1382.0 Negative Control 100 16 3672.0 3645.8
1822.9
 
17 760.0 17 1993.0 1966.8 983.4  
18 1144.0 18 2979.0 2952.8 1476.4  
19 1690.0 19 4479.0 4452.8 2226.4  
20 1050.0 20 2783.0 2756.8 1378.4  
MV 1205.2 MV 3181.2 3155.0 1577.5 1.0
SD 313.8 SD 841.0 841.0 420.5  
41 891.0 FAT
93503/B TE
in DMSO
3 1 2363.0 2336.8 1168.4 0.7
42 1609.0 2 4227.0 4200.8 2100.4 1.3
43 1148.0 3 3018.0 2991.8 1495.9 0.9
44 1414.0 4 3763.0 3736.8 1868.4 1.2
45 1013.0 5 2663.0 2636.8 1318.4 0.8
MV 1215.0 MV 3206.8 3180.6 1590.3 1.0
SD 262.7 SD 691.9 691.9 345.9 0.2
46 1300.0 FAT
93503/B TE
in DMSO
6.25 6 3445.0 3418.8 1709.4 1.1
47 1268.0 7 3333.0 3306.8 1653.4 1.0
48 1486.0 8 3937.0 3910.8 1955.4 1.2
49 1299.0 9 3418.0 3391.8 1695.9 1.1
50 1971.0* 10 5231.0* n.d. n.d. n.d.
MV 1338.3 MV 3533.3 3507.1 1753.5 1.1
SD 86.3 SD 236.7 236.7 118.4 0.1
51 930.0 FAT 93503/B TE
in DMSO
12.5 11 2454.0 2427.8 1213.9 0.8
52 1052.0 12 2824.0 2797.8 1398.9 0.9
53 1436.0 13 3798.0 3771.8 1885.9 1.2
54 1393.0 14 3787.0 3760.8 1880.4 1.2
55 1159.0 15 3083.0 3056.8 1528.4 1.0
MV 1194.0 MV 3189.2 3163.0 1581.5 1.0
SD 194.6 SD 531.6 531.6 265.8 0.2
73 11.0 Background Szinti and TCA 28.0  
74 9.0 24.0  
75 10.0 25.0  
76 11.0 27.0  
77 11.0 27.0  
MV 10.4 MV 26.2 0.0 0.0 0.0
SD 0.8   SD 1.5      
* = outlier, failed Grubbs, Nalimov and Dixon;  n.d. = not determined

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
According to OECD 429 the test item is expected to have no sensitising properties and therefore should not be regarded as a dermal sensitiser.
According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test item has no obligatory labelling requirement for skin sensitisation and is unclassified.
Executive summary:

A local lymph node assay was carried out with the test substance in 5 female mice per test group according to OECD test guideline 429.

The maximum technically applicable concentration of the test item in the vehicle was found to be 12.5 % in DMSO.

Based on the results observed in the preliminary test the following test item concentrations were selected for the main study: 3 %, 6.25 % and 12.5 % (w/v). DMSO was used as vehicle and served as negative control.

At the daily clinical observation the animals did not show any visible clinical symptoms and no case of mortality was observed. Body weight development was regular.

None of the three tested concentrations of the test item reached the stimulation index of 3.

The stimulation index at concentrations of 3%, 6.25%, and 12.5% were 1, 1.1, and 1, respectively.      

The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3.

Consequently, according to OECD 429 the test item as described in this report is expected to have no sensitising properties and therefore should not be regarded as a dermal sensitiser.