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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
end on 16-JUL-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: test performed according to OECD guideline and GLP

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Potassium (R)-(4-hydroxyphenyl)[(3-methoxy-1-methyl-3-oxoprop-1-enyl)amino]acetate
EC Number:
273-992-8
EC Name:
Potassium (R)-(4-hydroxyphenyl)[(3-methoxy-1-methyl-3-oxoprop-1-enyl)amino]acetate
Cas Number:
69416-61-1
Molecular formula:
C13H15NO5.K
IUPAC Name:
potassium (4-hydroxyphenyl)[(3-methoxy-1-methyl-3-oxoprop-1-en-1-yl)amino]acetate
Details on test material:
- Name of test material (as cited in study report): D(-)-p-HydroxyphenylglycineDaneSalt (K,Methyl), Code Name: HPG-DS
- Substance type: monoconstituent substance
- Physical state: data not available
- Stability under test conditions: not indicated by the sponsor
- Storage condition of test material: at room temperature, light and moisture protected

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V. (The Netherlands)
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.8 to 23.4 g
- Housing: single
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days prior to the start of dosing under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2°C
- Humidity: 45-65%
- Air changes: data not available
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m

IN-LIFE DATES: data not available

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
5, 10, and 25%
No. of animals per dose:
4 females per group
Details on study design:
RANGE FINDING TESTS:
- Compound solubility:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which ccould be technically achieved was a 50 % suspension in dimethylsulfoxide. At a test item concentration of 25% and below, a solution could be achieved in dimethylsulfoxide.
- Irritation:
To determine the highest non-irritant test concentration, two pre-tests were performed. Three mice were treated with test item concentrations of 10 and 25% or 50% each once daily on three consecutive days.
In the pre-tests clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. At all tested concentrations the animals did not show any signs of irritation or systemic toxicity. However, during the pre-test using the 50% test item suspension, it became apparentthat a reliable application of the mentioned suspension was not feasible.
- Lymph node proliferation response: not determined

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
> First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
> Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.


TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 5, 10, and 25% (w/v) in dimethylsulfoxide. The application volume, 25 µl, was spread over the entire dorsal surface (diameter ca. 8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application, all mice were administered with 250 µl of 80.9 µCi/ml 3HTdR (corresponds to 20.2 µCi 3HTdR per mouse) by intravenous injection via a tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanised.
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group).
The level of 3HTdR incorporation was then measured on a beta-scintillation counter.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
not applicable

Results and discussion

Positive control results:
EC3 = 12.9 % (positive)

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: %5: 1.42 10%: 1.42 25%: 1.76
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Control group: 4776 5%: 6794 10%: 6771 25%: 8402

Any other information on results incl. tables

No deaths occurred during the study period.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

The body weight of the animals, recorded prior to the first application and prior to treatment with3HTdR, was within the range commonly recorded for animals of this strain and age.

Table 1: results:

Vehicle: dimethylsulfoxide

Test item concentration % (w/v)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

13

---

---

---

---

---

BG II

12

---

---

---

---

---

1

4788

4776

8

596.9

 

5

2

6806

6794

8

849.2

1.42

10

3

6783

6771

8

846.3

1.42

25

4

8414

8402

8

1050.2

1.76

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

The EC3 value could not be calculated, since all S.I.´s are below 3.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item D(-)-p-HydroxyphenylglycineDaneSalt (K,Methyl) P-Hydroxy-phenyl hydantoin, Code Name: HPG-DS was not a skin sensitiser under the described conditions.
Executive summary:

In the study the test item D(-)-p-HydroxyphenylglycineDaneSalt (K,Methyl), Code Name: HPG-DS diluted in dimethylsulfoxide was assessed for its possible contact allergenic potential (OECD 429, GLP).

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 1.42, 1.42, and 1.76 were determined with the test item at concentrations of 5, 10, and 25% in dimethylsulfoxide, respectively.

The test item D(-)-p-HydroxyphenylglycineDaneSalt (K,Methyl), Code Name: HPG-DS was not a skin sensitiser under the test conditions of this study.