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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:
Aug 03-18,2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed in compliance with the “OECD Principles of Good Laboratory Practice“, as revised in 1997 [C(97)186/Final]. This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: OECD Guidelines for Testing of Chemicals, Updated Guideline 429: Skin Sensitisation: Local Lymph Node Assay (adopted 24 April 2002). "European Union Method B.42, Skin Sensitisation: Local Lymph Node Assay - Commission regulation (EC) No 440/2008, Official Journal of the European Union (EN), dated May 30, 2008"

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity (HPLC): 99.9 % (w/w)

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS

Source: Harlan Netherlands B.V. Postbus 6174 NL - 5960 AD Horst / The Netherlands
Age at study initiation: 8 - 12 weeks (beginning of treatment)
Weight at study initiation: 22 +/- 1.5


Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Identification Single caging. The animals were distributed into the test groups at random and identified by cage number.



ENVIRONMENTAL CONDITIONS

Housing: single
Cage Type: Makrolon Type II, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)
Feed: pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst / Netherlands)
Water: tap water, ad libitum
Environment: temperature 22 + 2°C
relative humidity 45-65%
artificial light 6.00 a.m. - 6.00 p.m.


Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
5, 10, 25 %
No. of animals per dose:
Total 16 animals
3 test groups with 4 animals
1 control group with 4 animals
Details on study design:
according to guideline
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.
The ANOVA (Dunnett-test) was conducted to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. Statistical significance was at the five per cent level (p < 0.05). However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
Experiment performed in May 2010.
Positive control substance: -Hexylcinnamaldehyde
Vehicle: acetone:olive oil (4+1)

Test item concentration % (w/v) Group Measurement DPM Calculation Result
DPM-BG a) number of lymph nodes DPM per lymph node b) S.I.
--- BG I 13 --- --- --- ---
--- BG II 12 --- --- --- ---
0 1 4561 4549 8 568.6 1.00
5 2 9275 9263 8 1157.8 2.04
10 3 15507 15495 8 1936.8 3.41
25 4 27961 27949 8 3493.6 6.14


BG = Background (1 ml 5% trichloroacetic acid) in duplicate
1 = Control Group
2-4 = Test Group
S.I. = Stimulation Index
a) = The mean value was taken from the figures BG I and BG II
b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see DPMs
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Test item concentration % (w/w) Group Measurement DPM Calculation Result DPM-BGa) number of lymph nodes DPM per lymph nodeb) S.I. --- BG I 78 --- --- --- --- --- BG II 58 --- --- --- --- --- 1 2962 2894 8 361.8 1.00 5 2 3150 3082 8 385.3 1.06 10 3 4555 4487 8 560.9 1.55 25 4 3647 3579 8 447.4 1.24

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item Phenolphthalein was not a skin sensitiser in this assay.
Executive summary:

Purpose

The purpose of this Local Lymph Node assay was to identify the contact allergenic potential of Phenolphthalein when administered to the dorsum of both ear lobes of mice. This study provides a rational basis for risk assessment to the sensitising potential of the test item in man.

Study Design

In order to study a possible allergenic potential of Phenolphthalein, three groups each of four female mice were treated with different concentrations of the test item dissolved in DMF by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter.

Results

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. A statistically relevant increase in ear thickness was not observed.

In this study Stimulation Indices (S.I.) of 1.06, 1.55, and 1.24 were determined with the test item at concentrations of 5, 10, and 25% in dimethylformamide, respectively.

Conclusion

The test item Phenolphthalein was not a skin sensitiser in this assay.