Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-11-02 tot 2011-11-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

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)
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:
Species/strain: healthy CBA/CaOlaHsD mice
Source: Harlan Winkelmann, 33178 Borchen, Germany
Sex: female (nulliparous and non-pregnant)
Age at the
beginning of the study: 8 – 9 weeks
Number of animals: 5 mice / group
3 mice / preliminary 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 (lot no. 1956)
- 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 (lot no. 190711)
- 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% (w/v)
The preparations were made immediately prior to each dosing.
No. of animals per dose:
Number of animals: 5 mice / group
3 mice / preliminary test
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.
Positive control substance(s):
other: P-Phenylenediamine (CAS No.: 106-50-3)

Results and discussion

Positive control results:
Stimulation Index of 1% P-Phenylenediamine in AOO: 7.8

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The stimulation index at a concentration of 6.25% was 8.2 The stimulation index at a concentration of 12.5% was 9.8 The stimulation index at a concentration of 25% was 16.9
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Negative Control: 1105.0 1601 1044.0 1009.0 1005.0 6.25%: 8560.0 5803.0 8082.0 11804.0 7872.0 12.5%: 4802.0 10861.0 16255.0 6754.0 11556.0 25%: 18607.0 14773.0 15143.0 18537.0 19850.0

Any other information on results incl. tables

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 (Acetone, Merck, lot no.K41154114, expiry date: 06/2015, olive oil highly refined, Sigma, lot no. BCBD1085, expiry date: 10/2011).

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 sacrificingthe thickness of both ears of all animals was measured:
  Animal No. Measurement of Ear Thickness (mm)
Concentration Day 1 Day 3 Day 6
  left right left right left right
FAT 40853/A TE
25% in AOO
1 0.18 0.19 0.18 0.19 0.19 0.19
         
FAT 40853/A TE
25% in AOO
2 0.17 0.18 0.17 0.18 0.17 0.18
         
Negative Control
100% AOO
3 0.18 0.19 0.18 0.19 0.18 0.19

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).

Neither signs of systemic toxicity nor signs of irritation at the application site could be detected in any animal.

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

Concentration Animal
No.
Start
of Study
End
of Study
Weight
Gain
FAT 40853/A TE
25% in AOO
1 22 23 1
         
FAT 40853/A TE
25% in AOO
2 20 23 3
         
Negative Control
100% AOO
3 21 23 2

Radioactive Determination of the Test Substance Groups

POS CPM     Test Item Conc. [%] Animal number DPM DPM- mean back- ground DPM/ Node Stimu-lation Index
18 536.0     Negative   16 1105.0 1090.2 545.1  
19 778*      Control   17 1601* n.d. n.d.  
20 502.0         18 1044.0 1029.2 514.6  
21 484.0         19 1009.0 994.2 497.1  
22 483.0         20 1005.0 990.2 495.1  
MV 501.3         MV 1040.8 1026.0 513.0 1.0
SD 21.4         SD 40.1 40.1 20.0  
30 4138.0     FAT 40853/A TE 6.25 1 8560.0 8545.2 4272.6 8.3
31 2805.0         2 5803.0 5788.2 2894.1 5.6
32 3862.0         3 8082.0 8067.2 4033.6 7.9
33 5643.0         4 11804.0 11789.2 5894.6 11.5
34 3778.0         5 7872.0 7857.2 3928.6 7.7
MV 4045.2         MV 8424.2 8409.4 4204.7 8.2
SD 917.0         SD 1935.7 1935.7 967.8 1.9
37 2315.0     FAT 40853/A TE 12.5 6 4802.0 4787.2 2393.6 4.7
38 5210.0         7 10861.0 10846.2 5423.1 10.6
39 7792.0         8 16255.0 16240.2 8120.1 15.8
40 3234.0         9 6754.0 6739.2 3369.6 6.6
41 5388.0         10 11556.0 11541.2 5770.6 11.2
MV 4787.8         MV 10045.6 10030.8 5015.4 9.8
SD 1902.2         SD 3995.8 3995.8 1997.9 3.9
42 8968.0     FAT 40853/A TE 25 11 18607.0 18592.2 9296.1 18.1
43 7147.0         12 14773.0 14758.2 7379.1 14.4
44 7240.0         13 15143.0 15128.2 7564.1 14.7
45 8691.0         14 18537.0 18522.2 9261.1 18.1
46 9575.0         15 19850.0 19835.2 9917.6 19.3
MV 8324.2         MV 17382.0 17367.2 8683.6 16.9
SD 966.9         SD 2036.9 2036.9 1018.5 2.0
66 7.0     Background     14.0      
67 6.0     Szinti and     12.0      
68 7.0     TCA     15.0      
69 7.0           15.0      
70 9.0           18.0      
MV 7.2         MV 14.8 0.0 0.0 0.0
SD 1.0         SD 1.9      
*outlier, failed in Grubbs, Nalimov, Dixon            
n.d. = not determined            

Body Weight Development:

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

Concentration Animal
No.
Start
of Study
End
of Study
Weight
Gain
FAT 40853/A TE 1 21 22 1
  2 21 22 1
  3 21 24 3
  4 19 20 1
6.25% in AOO 5 20 21 1
         
FAT 40853/A TE 6 19 20 1
  7 18 19 1
  8 18 20 2
  9 19 21 2
12.5% in AOO 10 17 18 1
         
FAT 40853/A TE 11 21 21 0
  12 18 19 1
  13 19 20 1
  14 19 21 2
25% in AOO 15 22 23 1
         
  16 21 22 1
Negative 17 18 20 2
Control 18 22 24 2
100% AOO 19 19 20 1
  20 20 21 1

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) was calculated to be at a test item concentration of 0.64%.
Consequently, according to OECD 429 solutions or preparations containing more than 0.64% FAT 40853/A 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 40853/A TE has obligatory labelling requirement for skin sensitisation and is classified into Category 1A.
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 sub-category 1A

X

 

 

classified into sub-category 1B

 

 

 

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 sub-category 1A

X

 

 

classified into sub-category 1B

 

 

 

unclassified

 

Based on the results of the preliminary test the test item was assessed for sensitising properties at concentrations of 6.25%, 12.5% 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:

All of the three tested concentrations of the test item reached the stimulation index of3.

The stimulation index at a concentration          of       6.25% was     8.2

The stimulation index at a concentration          of       12.5% was     9.8

The stimulation index at a concentration          of       25%    was     16.9