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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:
key study
Study period:
2014-03-13 to 2014-06-03
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)
Version / remarks:
(adopted: July 22, 2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
EPA 712-C-03-197, March 2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Specific details on test material used for the study:
Identification : FAT 40045/Z TE

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Test System:
Species/strain: healthy CBA/CaOlaHsD mice
Source: Harlan Laboratories GmbH, 5800 AN Venray, The Netherlands
Sex: female (nulliparous and non-pregnant)
Age at the beginning of the study: 8-9 weeks
Number of animals: 5 mice / group, 3 mice / prescreen test
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals are bred for experimental purposes.

Housing and Feeding 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. 131113)
- 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
Concentration:
6.25, 12.5 and 25 % (w/v)
No. of animals per dose:
5
Details on study design:
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 Regime
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.
In certain situations where the dose response does not incorporate a data point lying below the SI value of three, provided the data are of good quality (relatively close to an SI of three and evidence of a dose response), an EC3 value may be estimated by using the two doses closest to the SI value of three. The EC3 value is estimated by log-linear interpolation between these two points on a plane where the x-axis represents the dose level and the y-axis represents the SI. The point with the higher SI is denoted (a,b) and the point with the lower SI is denoted (c,d). The formula for the EC3 estimate is as follows: EC3=2^{(log2(c)+(3-d)/(b-d)*[(log2(a)-log2(c)]}, by log-transforming the doses, EC3 estimates will never fall below zero.
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:
Reliability Check
The recent reliability check was performed in March 2014. The raw data of this study are kept in the BSL archives (BSL Project ID 134702 U).
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 8.4.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
ca. 3.7
Test group / Remarks:
Conc. 6.25%
Remarks on result:
other: Stimulation index > 3
Key result
Parameter:
SI
Value:
ca. 5.6
Test group / Remarks:
Conc. 12.5%
Remarks on result:
other: stimulation index > 3.
Key result
Parameter:
SI
Value:
ca. 6.2
Test group / Remarks:
Conc. 25%
Remarks on result:
other: stimulation index > 3.

Any other information on results incl. tables

Radioactive Determination of the Test Substance Groups

POS  CPM  Test Item Conc.[%] Animal number  DPM DPMmean background DPM/ Node Stimulation Index
16 875 Negative Control 100 16 2291 2272.6 1136.3  
17 1959 17 5162 n.d.  n.d.   
18 889 18 2331 2312.6 1156.3  
19 1108 19 2916 2897.6 1448.8  
20 842 20 2220 2201.6 1100.8  
MV 928.5 MV 2439.5 2421.1 1210.6 1
SD 105 SD 278 278 139  
1 1633 FAT 40045/Z
TE in DMSO
6.25 1 4269 4250.6 2125.3 1.8
2 3177 2 8348 8329.6 4164.8 3.4
3 3805 3 9910 9891 6  4945.8 4.1
4 5975 4 15730 15711.6 7855.8 6.5
5 2585 5 6762 6743.6 3371 8  2.8
MV 3435 MV 9003.8 8985.4 4492.7 3.7
SD 1457.7 SD 3845.4 3845.4 1922.7 1.6
6 3928 FAT 40045/Z
TE in DMSO
12.5 6 10338 10319.6 5159.8 4.3
7 5850 7 15393 15374.6 7687.3 6.4
8 4299 8 11179 11160.6 5580.3 4.6
9 7441 9 19617 19598.6 9799.3 8.1
10 4153 10 10858 10839.6 5419.8 4.5
MV  5134.2 MV  13477 13458.6 6729.3 5.6
SD  1337.8 SD  3559.9 3559.9 1779.9 1.5
11 2046 FAT 40045/Z
TE in DMSO
25 11 5375 5356.6 2678.3 2.2
12 5846 12 15415 15396.6 7698.3 6.4
13 6134 13 16242 16223.6 8111.8 6.7
14 5302 14 13886 13867.6 6933.8 5.7
15 9035 15 23725 23706.6 11853.3 9.8
MV  5672.6 MV  14928.6 14910.2 7455.1 6.2
SD  2228.8 SD  5860 5860 2930 2.4
36 8 Background
Szinti and
TCA
    20      
37 7   19      
38 6   15      
39 10   27      
40 4   11      
MV  7 MV  18.4 0 0 0
SD  2 SD  5.4      

Clinical Observation

Time of Observation  Systemic Effects  Local Effects
Group 1, animals no. 1-5 / test item at a concentration of 6.25% in DMSO
Day 1  nsf  nsf
Day 2 nsf  R
Day 3  nsf  R
Day 4 nsf  R
Day 5 nsf  R
Day 6 nsf  R
Group 2, animals no. 6-10 / test item at a concentration of 12.5% in DMSO
Day 1  nsf  nsf
Day 2 nsf  R
Day 3  nsf  R
Day 4 nsf  R
Day 5 nsf  R
Day 6 nsf  R
Group 3, animals no. 11-15 / test item at a concentration of 25% in DMSO
Day 1  nsf  nsf
Day 2 nsf  R
Day 3  nsf  R, D
Day 4 nsf  R
Day 5 nsf  R
Day 6 nsf  R
Group 4, animals no. 16-20 / negative control DMSO
Day 1  nsf  nsf 
Day 2 nsf  nsf 
Day 3  nsf  nsf 
Day 4 nsf  nsf 
Day 5 nsf  nsf 
Day 6 nsf  nsf 

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
FAT 40045/Z should be considered to be a skin sensitiser and is classified into Category 1B.

Executive summary:

The skin sensitisation potential of FAT 40045/Z is assessed in a LLNA study conducted according to OECD Guideline 429. Based on the results of the prescreen test the test item was assessed for sensitising properties at concentrations of 6.25, 12.5 and 25 % (w/v), each diluted with DMSO. 5 mice per concentration were used.

Each of the three tested concentrations exceeded the stimulation index of 3.

The stimulation index at a concentration of 6.25 % was 3.7

The stimulation index at a concentration of 12.5 % was 5.6

The stimulation index at a concentration of 25 % was 6.2

The EC3 value (estimated by linear interpolation) was calculated to be at a test item concentration of 4.79 %. Consequently, according to OECD 429 solutions or preparations containing more than 4.79 % FAT 40045/Z TE are expected to have a stimulation index of >3 and are therefore considered to be dermal sensitisers. According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test item FAT 40045/Z TE has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.