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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to current guidelines 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:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
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:
- Name of test material (as cited in study report): p-Cresolmethylether
- Physical state: liquid, colorless, clear
- Analytical purity: 98.6%
- Batch No.: 107450 09T0
- Stability under test conditions: stability analytically verified
- Storage condition of test material: room temperature, protection from light

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 Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 19.4 - 22.2 g
- Housing: single, Makrolon Type II, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst / Netherlands)
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 20 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
10, 25 and 50% (w/v) test item
No. of animals per dose:
4
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 can be technically used was 100% of the undiluted test item. Dilutions were formulated in methyl ethyl ketone.
- Irritation: To determine the highest test item concentration that does not induce local signs of skin irritation or systemic toxicity, a pre-test was performed in two animals. Two mice were treated with test item concentrations of 50% and 100% each on three consecutive days and irritating potential was measured by ear thickness measurements (via micrometer and weighing of punch biopsies). The maximum concentration of test item to be investigated in the LLNA was the dose that could be uniformly applied to the dorsal surface of the ears of the mice and which did at the same time not cause excessive skin irritation or systemic toxicity., i.e. 10, 25 and 50% (w/v).


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT

- Criteria used to consider a positive response:
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.
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:

Topical Application:
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 10, 25 and 50% (w/v) in methyl ethyl ketone. The application volume, 25 µl, was spread over the entire dorsal surface (8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the vehicle alone (control animals).

Administration of 3H-Methyl Thymidine:
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.

For the determination of lymph node weights, after excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.

For the determination of incorporated 3HTdR, lymph nodes were rapidly excised and pooled per group (8 nodes per group), single cell suspensions were prepared by gentle mechanical disaggregation through stainless steel gauze and the level of 3HTdR incorporation was measured on a scintillation counter.
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 on the ear and lymph node weights to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. However, the primary point of consideration is the biological relevance of the results.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: S.I.: 10% test item: 1.56 25% test item: 2.08 50% test item: 2.39
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM 0 % test item: 2150 10% test item: 3363 25% test item: 4473 50% test item: 5142

Any other information on results incl. tables

Pretest:

Ear swelling was observed on day 6 before sacrifice in the animal treated with 100% test item concentration and the ear thickness measurement on day 6 showed an ear swelling of approximately 28% in comparison to the measurement on day 1 prior to

treatment.

Main test:

No deaths occurred during the study period. No symptoms of local irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period. The body weight of the animals, recordedprior to the first application and prior to treatment with3HTdR, was within the range commonly recorded for animals of this strain and age.

A statistically significant increase in lymph node weights was not observed in any of the test item treated groups in comparison to the vehicle control group. A statistically significant increase in ear weights was not observed in in any of the test item treated groups in comparison to the vehicle control group.

Test item concentration % (w/v)

Group

Measurement DPM

Calculation

Result

DPM-BGa

number of lymph nodes

DPM per lymph nodeb

S.I.

---

BG I

15

---

---

---

---

---

BG II

20

---

---

---

---

0

1

2167

2150

8

268.7

 

10

2

3380

3363

8

420.3

1.56

25

3

4490

4473

8

559.1

2.08

50

4

5159

5142

8

642.7

2.39

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 none of the tested concentrations induced an S.I. greater than 3.

Applicant's summary and conclusion