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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 November 2011-22 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Scientifically well-performed study; Guideline study; GLP study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, 80538 München, Germany
Type of study:
mouse local lymph node assay (LLNA)
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
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: lot no. 1956, 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 regular 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: lot no. 19072011, 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
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100%, undiluted test item
50% and 25%, diluted with acetone/olive oil
No. of animals per dose:
5,
3 animals for the 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 results:
Phenylenediamine: Stimulation Index 9.0
Parameter:
SI
Remarks on result:
other: Negative Control: 1.0 Test Item 25%: 1.6 Test Item 50%: 1.8 Test Item 100%: 2.6
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Test Item Conc. [%] Animal number DPM Negative 16 1043.0 Control 17 1721.0 18 1437.0 19 1153.0 20 1211.0 MV 1313.0 SD 241.1 Polyglykol 25 1 1430.0 BB 300 2 2436.0 in AOO 3 3705* 4 2101.0 5 2268.0 MV 2058.8 SD 381.8 Polyglykol 50 6 2320.0 BB 300 7 1982.0 in AOO 8 2690.0 9 2235.0 10 3640* MV 2306.8 SD 253.8 Polyglykol 100 11 2830.0 BB 300 12 2418.0 13 4107.0 14 4124.0 15 3305.0 MV 3356.8 SD 680.1 Background 16.0 Szinti and 13.0 TCA 10.0 10.0 13.0 MV 12.4 SD 2.2

Absolute Body Weights in g:

Concentration Animal
No.
Start
of Study
End
of Study
Weight
Gain
  1 21 22 1
  2 20 20 0
Polyglykol BB 300 3 19 20 1
25% in AOO 4 17 18 1
  5 18 18 0
         
  6 19 21 2
  7 20 22 2
Polyglykol BB 300 8 22 21 -1
50% in AOO 9 18 19 1
  10 19 21 2
         
  11 22 22 0
  12 20 21 1
Polyglykol BB 300 13 19 20 1
100% 14 18 20 2
  15 19 19 0
         
  16 21 21 0
Negative 17 21 21 0
Control 18 18 18 0
100% AOO 19 23 22 -1
  20 19 20 1

Clinical Observation

Time of Observation

Systemic Effects

Local Effects

Group 1, animals no. 1 – 5 / 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 2, animals no. 6 – 10 / test item at a concentration of 50% 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 100%

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

Ear Thickness - Preliminary Test

  Animal No. Measurement of Ear Thickness (mm)
Concentration Day 1 Day 3 Day 6
  left right left right left right
Polyglykol BB 300
50% in AOO
1 0.20 0.19 0.22 0.21 0.20 0.21
         
Polyglykol BB 300
100%
2 0.20 0.20 0.22 0.21 0.22 0.22
         
Negative Control
100% AOO
3 0.20 0.20 0.19 0.20 0.20 0.21

Absolute Body Weights - Preliminary Test in g

Concentration Animal
No.
Start
of Study
End
of Study
Weight
Gain
Polyglykol BB 300
50% in AOO
1 25 25 0
         
Polyglykol BB 300
100%
2 23 23 0
         
Negative Control
100% AOO
3 23 24 1
Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
No significant skin sensitization potential was found for the registration substance. No classification is to be assigned.
Executive summary:

The skin sensitization potential of the registration substance was investigated according to the OECD 429. Mice were treated topically on the ear with 0.1ml at concentration of 0, 25, 50 and 100% in AOO (4:1 (v/v) acetone/olive oil). The obtained SI values ranged 1.6, 1.8 and 2.6 at 25, 50 and 100% respectively. Based on the results obtained the registration substance is considered as a non-skin sensitizer.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:
Migrated from Short description of key information:
The skin sensitization property of the registration substance was investigated according to the OECD Guideline 429.
The registration substance is a non-skin sensitizer according to the results obtained in this study.

Justification for selection of skin sensitisation endpoint:
study on registration substance; scientifically well performed; Guideline study; GLP study

Justification for classification or non-classification

The skin sensitization property of the registration substance was investigated according to the OECD Guideline 429. No significant skin sensitization potential was found. No classification is to be assigned for the registration substance.