Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-01-10 to 2012-02-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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 lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

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 (preliminary test and main study: lot no. 0815)
- Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at re gular intervals)
- The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding (preliminary test and main study: lot no. 11082011)
- 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:
acetone/olive oil (4:1 v/v)
Concentration:
Based on the results observed in the preliminary test the following test item concentrations were selected for the main study:
6.25%, 12.5% and 25% (v/v)
The preparations were made immediately prior to each dosing.
AOO was used as vehicle and served as negative control.
No. of animals per dose:
5 animals/dose group, 5 animals/control group
Details on study design:
Before the initiation of the preliminary test, a solubility test was performed to define the vehicle and the maximum concentration which is technically applicable to the animals.
The maximum technically applicable concentration of the test item was found to be 25% in AOO (4:1 (v/v) acetone / olive oil; see 11.2).
In order to determine the highest tolerated and non-irritant test concentration a preliminary test was performed.
For this purpose, two animals were treated by topical application with the test item on three consecutive days at a concentration of 25% (suspended in AOO) to the entire dorsal surface of each ear.
One further animal was treated with 100% AOO and served as negative control.
Immediately before the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of both ears of all animals was measured.
During this period also all clinical signs were recorded.
Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).
It could be detected no signs of systemic toxicity at the application site in any animal. In animals 1 and 2 there was observed very slight erythema (barely perceptible) at the application site.
All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the duration of the preliminary test.

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.
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 following risk phrases may be assigned 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, third revised edition, July 2009:
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 December 2011. The raw data of this study are kept in the BSL archives (BSL Project ID 114612 I).
Positive-control substance: P-phenylenediamine (CAS 106-50-3, Sigma,
purity > 98%; Lot 060M0186V6) 1%
Vehicle: AOO (4:1 (v/v) acetone/olive oil)
Species/strain: healthy CBA/CaOlaHsd mice
Source: Harlan Winkelmann GmbH, 33178 Borchen, Germany
Concentrations: 1% on three consecutive days
The Stimulation Index was calculated to be 7.7 (SD=2.2)

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Each of the three tested concentrations of the test item exceeded the stimulation index of 3. The stimulation index at a concentration of 6.25% was 6.6 The stimulation index at a concentration of 12.5% was 5.8 The stimulation index at a concentration of 25% was 5.3
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Group Dose-Level Animal # DPM _____________________________________________________________ Negative 16 1071.0 Control 17 968.0 18 1460.0 19 2772.0 20 1337.0 ______________________________________________________________ FAT 40858/A 6.25 1 6064.0 TE 2 13579.0 in AOO 3 8273.0 4 8321.0 5 9079.0 ________________________________________________________________ FAT 40858/A 12.5 6 7422.0 TE 7 6125.0 in AOO 8 6887.0 9 5315.0 10 8773.0 __________________________________________________________ FAT 40858/A 25 11 5626.0 TE 12 7908.0 in AOO 13 5126.0 14 4779.0 15 8630.0 ______________________________________________________________ Background 13.0 Szinti and 12.0 TCA 13.0 13.0 11.0

Any other information on results incl. tables

Table Ear Thickness – Preliminary Test (mm)

  Animal No. Measurement of Ear Thickness (mm)
Concentration Day 1 Day 3 Day 6
  left right left right left right
FAT 40858/A TE
25% in AOO
1 0.18 0.19 0.19 0.19 0.19 0.19
         
FAT 40858/A TE
25% in AOO
2 0.17 0.18 0.20 0.20 0.20 0.20
         
Negative Control
100% AOO
3 0.17 0.18 0.19 0.18 0.19 0.19

Table Absolute Body Weights – Preliminary Test (g)

Concentration Animal
No.
Start
of Study
End
of Study
Weight
Gain
FAT 40858/A TE
25% in AOO
1 22 22 0
         
FAT 40858/A TE
25% in AOO
2 23 24 1
         
Negative Control
100% AOO
3 20 21 1

Table Radioactive Determination of the Positive-Control Group of the Recent Study

POS CPM     Test Item Conc. [%] Animal number DPM DPM- mean back- ground DPM/ Node Stimu-lation Index
25 616.0     Negative   21 1240.0 1222.0 611.0  
26 751.0      Control   22 1525.0 1507.0 753.5  
27 1204.0         23 2441.0 2423.0 1211.5  
28 587.0         24 1175.0 1157.0 578.5  
29 946.0         25 1894.0 1876.0 938.0  
MV 820.8         MV 1655.0 1637.0 818.5 1.0
SD 229.8         SD 467.9 467.9 233.9  
30 3578.0     Phenylene- 1 26 7249.0 7231.0 3615.5 4.4
31 5201.0     diamine   27 10507.0 10489.0 5244.5 6.4
32 6133.0         28 12692.0 12674.0 6337.0 7.7
33 8339.0         29 16865.0 16847.0 8423.5 10.3
34 7782.0         30 15917.0 15899.0 7949.5 9.7
MV 6206.6         MV 12646.0 12628.0 6314.0 7.7
SD 1729.5         SD 3527.2 3527.2 1763.6 2.2
102 7.0     Background     15.0      
103 9.0     Szinti and     19.0      
104 8.0     TCA     15.0      
105 13.0           27.0      
106 7.0           14.0      
MV 8.8         MV 18.0 0.0 0.0 0.0
SD 2.2         SD 4.8      

Table Radioactive Determination of the Test Substance Groups

POS CPM Test Item Conc. [%] Animal number DPM DPM- mean back- ground DPM/ Node Stimu-lation Index
30 527.0 Negative   16 1071.0
1058.6
529.3  
31 476.0  Control   17 968.0 955.6 477.8  
32 722.0     18 1460.0 1447.6 723.8  
33 1332.0*     19 2772.0* n.d. n.d.  
34 657.0     20 1337.0 1324.6 662.3  
MV 595.5     MV 1209.0 1196.6 598.3 1.0
SD 98.4     SD 197.8 197.8 98.9  
78 2982.0 FAT 40858/A 6.25 1 6064.0 6051.6 3025.8 5.1
79 6607.0** TE   2 13579.0** n.d. n.d. n.d.
80 4005.0 in AOO   3 8273.0 8260.6 4130.3 6.9
81 4061.0     4 8321.0 8308.6 4154.3 6.9
82 4451.0     5 9079.0 9066.6 4533.3 7.6
MV 3874.8     MV 7934.3 7921.9 3960.9 6.6
SD 543.3     SD 1126.1 1126.1 563.1 0.9
85 3654.0 FAT 40858/A 12.5 6 7422.0 7409.6 3704.8 6.2
86 3016.0 TE   7 6125.0 6112.6 3056.3 5.1
87 3348.0 in AOO   8 6887.0 6874.6 3437.3 5.7
88 2608.0     9 5315.0 5302.6 2651.3 4.4
89 4291.0     10 8773.0 8760.6 4380.3 7.3
MV 3383.4     MV 6904.4 6892.0 3446.0 5.8
SD 571.7     SD 1174.2 1174.2 587.1 1.0
90 2728.0 FAT 40858/A 25 11 5626.0 5613.6 2806.8 4.7
91 3831.0 TE   12 7908.0 7895.6 3947.8 6.6
92 2523.0 in AOO   13 5126.0 5113.6 2556.8 4.3
93 2331.0     14 4779.0 4766.6 2383.3 4.0
94 4195.0     15 8630.0 8617.6 4308.8 7.2
MV 3121.6     MV 6413.8 6401.4 3200.7 5.3
SD 747.5     SD 1555.4 1555.4 777.7 1.3
114 7.0 Background     13.0      
115 6.0 Szinti and     12.0      
116 6.0 TCA     13.0      
117 6.0       13.0      
118 6.0       11.0      
MV 6.2     MV 12.4 0.0 0.0 0.0
SD 0.4     SD 0.8      
* = outlier, failed Grubbs, Nalimov and Dixon; ** = outlier, failed Nalimov; n.d. = not determined

Table Absolute Body Weights in g

Concentration Animal
No.
Start
of Study
End
of Study
Weight
Gain
  1 17 19 2
  2 17 18 1
FAT 40858/A TE 3 21 22 1
6.25% in AOO 4 18 19 1
  5 19 20 1
         
  6 21 24 3
  7 22 24 2
FAT 40858/A TE 8 20 21 1
12.5% in AOO 9 20 21 1
  10 17 17 0
         
  11 17 18 1
  12 20 21 1
FAT 40858/A TE 13 23 23 0
25% in AOO 14 19 20 1
  15 20 21 1
         
  16 22 22 0
Negative 17 19 20 1
Control 18 21 22 1
100% AOO 19 19 21 2
  20 18 20 2

Table Clinical Observation

Time of Observation

Systemic Effects

Local Effects

Group 1, animals no. 1 – 5 / test item at a concentration of 6.25% in AOO

Day 1

nsf

nsf

Day 2

nsf

nsf

Day 3

nsf

nsf

Day 4

nsf

nsf

Day 5

nsf

nsf

Day 6

nsf

nsf

Group 2, animals no. 6 – 10 / test item at a concentration of 12.5% in AOO

Day 1

nsf

nsf

Day 2

nsf

nsf

Day 3

nsf

nsf

Day 4

nsf

nsf

Day 5

nsf

nsf

Day 6

nsf

nsf

Group 3, animals no. 11 – 15 / test item at a concentration of 25% in AOO

Day 1

nsf

nsf

Day 2

nsf

nsf

Day 3

nsf

nsf

Day 4

nsf

nsf

Day 5

nsf

nsf

Day 6

nsf

nsf

Group 4, animals no. 16 – 20 / negative control AOO

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 1 (skin sensitising) based on GHS criteria
Remarks:
Migrated information
Conclusions:
The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were above 3.
Consequently, according to OECD 429 the test item FAT 40858/A TE as described in this report is expected to have sensitising properties and therefore should be regarded as a dermal sensitiser.
Since it was not possible to calculate the EC3 value, the test item cannot be classified into any of the sub-categories (category 1A or B) according to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System). Therefore, according to Commission Regulation (EU) No 286/2011 and OECD GHS the test item FAT 40858/A TE is classified into Category 1 and has obligatory labelling requirement for skin sensitisation.
For details of the classification criteria see Evaluation of Results.
Executive summary:

On the basis of the test results given below and in conformity with the criteria given inCommission Regulation (EU) No 286/2011 the substance should be:

classified into category 1

X

 

unclassified

 

On the basis of the test results given below and in conformity with the criteria given inGHS (Globally Harmonized Classification System) the substance should be:

classified into category 1

X

 

unclassified

 

Based on the results of the preliminary test the test item was assessed for sensitising properties at concentrations of 6.25%, 12.25% and 25% (w/v), each diluted with AOO 4:1 (v/v).

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

Species/strain:                     Mice, CBA/CaOlaHsd

Number of animals:            20/main test

Vehicle:                               AOO (4:1 (v/v) acetone/olive oil)

Summary Results:

Each of the three tested concentrations of the test item exceeded the stimulation index of3.

The stimulation index at a concentration          of       6.25% was     6.6

The stimulation index at a concentration          of       12.5% was     5.8

The stimulation index at a concentration          of       25%    was     5.3


Conclusion:

The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were above 3.Consequently, according to OECD 429[3]the test item FAT 40858/A TE as described in this report is expected to have sensitising properties and therefore should be regarded as a dermal sensitiser.

Since it was not possible to calculate the EC3 value, the test item cannot be classified into any of the sub-categories (category 1A or B) according to Commission Regulation (EU) No 286/2011[6]as well as GHS (Globally Harmonized Classification System)Therefore, according to Commission Regulation (EU) No 286/2011 and OECD‑GHS the test item FAT 40858/A TEis classified into Category 1 and has obligatory labelling requirement for skin sensitisation.

For details of the classification criteria seeEvaluation of Results.