Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-05-10 to 2004-05-13
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study well documented, meets generally accepted scientific principles, acceptable for assessment Deviations from the OECD 429 guideline: - animals were not individually housed - according to the guideline OECD 429 (2002), positive controls are used to demonstrate appropriate performance of the assay and competency of the laboratory to successfully conduct the assay. The positive control should produce a positive LLNA response at an exposure level expected to give an increase in the stimulation index (SI) > 3 over the negative control group. Furthermore, testing with the positive controls may be appropriate at intervals of no greater than 6 months. In this study only a differentiation index was given and no stimulation index. For this reason, the performance of this assay can not be assessed. In addition, the stated reliability check was 8 months old. Lastly, it was not stated if a vehicle was used and which concentrations were applied during the reliability check. - a range finding study for the selection of the test concentration was missing, but the test concetration were selected according to a skin irritation test (please refer to "Cross-reference to other study" below). Other guideline: - the individual LN cell count was done manually using Trypan blue exclusion using a NEUBAUER-chamber. Normally, the LN cell count is done by conductometry.
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 2004-04-24
Deviations:
yes
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Principles of method if other than guideline:
The test was performed in accordance with the the following method according to:
- Vohr, H._W. et al. (2000): An intra-laboratory validation of the integrated Model for Differentiation of Skin Reactions (IMDS): discrimination between (Photo)allergic and (photo)irritant skin reactions in mice. Arch Toxicol 73: 501 - 509.
-Homey, B. von Schilling, C., Blümel, J., Schuppe, H.-C., Ruzicka T., Ahr, H.J., Lehmann, P., Vohr, H.-W. (1998): An Integrated Model for the Differentiaiton of Chemical-Induced Allergic and Irritant Skin Reactions. Toxicology and Applied Pharmacology 153: 83 - 94.
- Vohr, H.-W., Homey, B., Schuppe, H.-C., Kind, P. (1994). Detection of photoreactivity demonstrated in a modified local lymph node assay in mice. Photodermatol Photoimmunol Photomed 10: 57 - 64.

The average LN cell count as feature for the activation of the immune system (local lymph nodes) is used for the calculation of a proliferation stimulation index compared with the negative control:
Index = LN cell count (test group)/LN cell count (negative control)
A lymph node index of 1.25 was determined as positive threshold value for the mouse strain NMRI by Vohr et al. The index is to fix at 1.00 if this threshold value is not come up or passed.
The ear swelling being the difference between the average ear thickness of the test and control animals on day 3 is calculated as feature for the skin inflammation at the site of topical treatment:
ear swelling = ear thickness on day 3 (test group) - ear thickness on day 3 (negative control)
An ear thickness increase of at least 0.02 mm was determined as positive threshold value by VOHR et al. The ear swelling is to fix at 0.01 mm if this threshold value is not come up or passed.
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2003-06-04
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
480-070-0
EC Name:
-
Cas Number:
85-27-8
Molecular formula:
C14H14O2
IUPAC Name:
4-(1-phenylethyl)benzene-1,3-diol
Details on test material:
- Name of test material: BIO 377
- Chemical name: 1,3-Benzenediol, 4-(1-phenylethyl)-
- Physical state: clear, viscous to solid
- Storage condition of test material: cool, dry, dark

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS - SPF albino mice of the strain Shoe:NMRI
- Source: DIMED Schönewalde GmbH, D-16352 Schönwalde
- Age at study initiation: about 9 weeks old
- Weight at study initiation: body weight ranging from 29 g to 42 g
- Housing: the mice were kept in groups in transparent polycarbonate cages (macrolone type III, floor area 810 cm^2) with five animals in each cage. The cages were cleaned and the bedding changed at least twice a week. Bedding was pinewood sawdust "Lignocel-Fasern" from Altromin, D-32791 lage, Lippe.
- Diet (ad libitum): a pelleted complete rodent diet "Altromin 1314" from Altromin GmbH, D-32791 Lage, Lippe
- Water (ad libitum): domestic quality drinking water acidified with hydrochloric acid to pH 2.5 in order to prevent microbial growth
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS - animal room provided with filtered air
- Temperature: 21 °C +/- 3°C
- Relative humidity: 55 % +/- 15 %
- Air changes: 10 times/hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1%, 2.5 % and 5 % (w/w)
No. of animals per dose:
5 female mice
(Note: in the negative control group 5 female mice were used, but means were later calculated for 4 animals because one mouse showed a markedly loss of weight during the study and was excluded from the evaluation)
Details on study design:
TESTING PROCEDURE:
On day 0 the animals were weighed, the thickness of ears was measured by using a micrometer (Oditest S0247) and an amount of 25 µL of the test item or vehicle respectively was applied topically on the dorsal side of the ears.
The open exposure procedure was repeated on day 1 and 2 with the different concentrations of the test item or with the vehicle.
On day 3 the mice were weighed again and the thickness of ears was measured. Subsequently the animals were killed by inhalation of CO2 and a dissection with removal of the auricular lymph node was carried out. Furthermore circular tissue pieces with a diameter of 7 mm were punched from the ears of the animals in order to determine their weight. The lymph node pairs were also weighed and LN cell suspensions were prepared by mechanical tissue disruption.
The cell counts per millilitre of these suspensions were determined manually by Trypan blue exclusion using a NEUBAUER-chamber.

RELIABILITY CHECK
Control tests with positive reference substances are performed in order to verify the experimental technique.
The last study was carried out between September 2nd, 2003 and September 5th, 2003.

ASSESSMENT
A Differentiation Index (DI) is calculated from the different parameters of investigation which describes the relation between the activation of the local skin-draining lymph nodes and the skin inflammation at the site of topical treatment as well as serves to distinguish between inflammatory (non-specific) and allergic (specific) reaction:
0 < DI < 1 inflammation
DI > 1 allergic reaction
- The average LN cell count as feature for the activation of the immune system (local lymph nodes) is used for the calculation of a proliferation stimulation index compared with the negative control:
Index = LN cell count (test group)/LN cell count (negative control)
A lymph node index of 1.25 was determined as positive threshold value for the mouse strain NMRI by Vohr et al. The index is to fix at 1.00 if this threshold value is not come up or passed.
Based on the fixing of a maximal LN cell counts index being 5 it is possible to calculate a percentage of the dfined maximal index:
% max. LN cell counts index = (stimulation index X 100 %)/5
- The ear swelling being the difference between the average ear thickness of the test and control animals on day 3 is calculated as feature for the skin inflammation at the site of topical treatment:
ear swelling = ear thickness on day 3 (test group) - ear thickness on day 3 (negative control)
An ear thickness oncrease of at least 0.02 mm was determined as positive threshold value by VOHR et al. The ear swelling is to fix at 0.01 mm if this threshold value is not come up or passed.
Based on the fixing of a maximal ear swelling being 0.15 mm it is possible to calculate a percentage of the defined maximal ear swelling:
% max. ear swelling = (ear swelling X 100 %)/0.15 mm
- The quotient from the two percentages comes to the Differentiation Index (DI):
DI = % max. LN cell count index/ % max.ear swelling
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Please refer to "details on study design"

Results and discussion

Positive control results:
A Differentiation Index of 2.28 was calculated and a specific (sensitizing) stimulation potential was demonstrated for the undiluted test item α-HEXYLCINNAMALDEHYDE, Lot 13102MO.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The average LN cell count as feature for the activation of the immune system (local lymph nodes) is used for the calculation of a proliferation stimulation index compared with the negative control: Index = LN cell count (test group)/LN cell count (negative control) A lymph node index of 1.25 was determined as positive threshold value for the mouse strain NMRI by Vohr et al. The index is to fix at 1.00 if this threshold value is not come up or passed. The ear swelling being the difference between the average ear thickness of the test and control animals on day 3 is calculated as feature for the skin inflammation at the site of topical treatment: ear swelling = ear thickness on day 3 (test group) - ear thickness on day 3 (negative control) An ear thickness increase of at least 0.02 mm was determined as positive threshold value by VOHR et al. The ear swelling is to fix at 0.01 mm if this threshold value is not come up or passed. Lymph node index: 5 % w/w: SI: 0.95 (LN cell count; 2 pieces) 2.5 % w/w: SI: 1.11 (LN cell count; 2 pieces) 1 % w/w: SI: 0.97 (LN cell count; 2 pieces) Ear swelling - mean(Day 3) [x 0.01]mm: 5 % w/w: 20.4 mm 2.5 % w/w: 21.1 mm 1 % w/w: 19.6 Negative control: 19.8 mm (mean was calculated for 4 animals because one mouse showed a markedly loss of weight during the study and was excluded from the evaluation)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Not applicable

Any other information on results incl. tables

During the study a remarkable loss of weight was revealed in one animal of the negative control group and caused the exclusion of this animal from the study evaluation of its data.

The remaining nineteen animals showed a normal body weight gain with and without an increase in weight. Only the two test groups with the highest test concentrations showed an ear swelling compared with the negative control but did not exceed the positive threshold value. On day 3 a slight decrease of ear thickness was observed at the test group with the lowest test concentration. The weight determination of ear tissue showed a slight increase at the test groups in comparison to the negative control. This trend towards an increase at the test groups was confirmed for the weights of lymph node pairs. The lymph node cell count of the middle test group was a little above the negative control level. The other two test groups showed lower cell counts compared to the negative control.

Since in all cases the treatment with the test item did not lead to an exceeding of the positive threshold values for both the ear swelling and the lymph node cell count a specific or non-specific stimulation potential can be excluded for the tested concentrations of the test item.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is not a skin sensitizer.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is is not a skin sensitizer.