Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 29 july 2003 to 30 jan 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD TG 429 compliant.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Relative humidity recorded in the animal room was sometimes outside of the target.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Species, strain and sex: CBA/J mouse, nulliparous and non-pregnant females.
- Source: Charles River Laboratories France, L'Arbresle, France.
- Age at study initiation: 8 to 10 weeks old
- Weight at study initiation: body weight ± standard deviation of 22.7 ± 1.7 g.
- Number: four females for the preliminary test, 28 females for the main test.
- Housing: the animals were housed individually in disposable crystal polystyrene cages (22 x 8.5 x 8 cm). Each cage contained autoclaved sawdust (SICSA, Alfortville, France). Sawdust is analysed by the supplier for composition and contaminants levels.
- Diet: free access to A04 C pelleted diet (SAFE, Villemoisson, Epinay-sur-Orge, France).
- Water): free access to tap water (filtered using a 0.22 micron filter) contained in bottles.
- Acclimation period: at least 5 days before the beginning of the study.


ENVIRONMENTAL CONDITIONS
• temperature: 22 ± 2°C
• relative humidity: 30 to 70%
• light/dark cycle: 12 h/12 h
• ventilation: approximately 12 cycles/hour of filtered, non-recycled air.


IN-LIFE DATES: From: 29 july 2003 To: 26 aug 2003

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
AOO
Concentration:
0, 5, 10, 25, 50 and 100%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was performed on a small number of animals, as follows:
• the test item was prepared at the concentrations of 10, 25, 50 and 100%,
• for three consecutive days, the animals received applications of 25 μL of the dosage form preparations to the external surface of both ears (one concentration per ear),
• measurement of the ear thickness (using a micrometer) was performed each day before treatment and 24 hours after the last application.
- Irritation: the test item was non-irritant in the preliminary test.
- Lymph node proliferation response: No cutaneous reactions and no noteworthy increase in ear thickness were observed, whatever the concentration.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The test item was considered as a skin sensitizer when the SI for a dose group is ≥ 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

TREATMENT PREPARATION AND ADMINISTRATION:
On days 1, 2 and 3, a dose-volume of 25 μL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration.
No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
In the positive control group given HCA at the concentration of 25%, a moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 15.23) were noted. The study was therefore considered valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: SI observed was between 10.42 (guetol at 5%) and 9.15 (guetol at 50%).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM was higher than control group (very significantly different), at all doses.

Any other information on results incl. tables

Table of results:

 

Group

Treatment and conc.

Cell count viable

Cell count dead

Viability (%)

Number of nodes per group

Dpm per group

Dpm per node

Stimulation index (SI)

EC3 value

1

AOO (4/1)

0

82

9

90.11

8

575.3

71.91

 

EC3<5%

2

Guetol 5%

359

62

85.27

8

5995.6

749.45

10.42

3

10%

442

33

93.05

8

5895.54

736.94

10.25

4

25%

265

11

96.01

8

6685.05

835.63

11.62

5

50%

198

22

90

8

5262.07

657.76

9.15

7

HCA 25%

272

56

82.93

8

8760.45

1095.06

15.23

 

Clinical signs and mortality:

All animals of the treated group 6 (undiluted test item), were found dead either on day 3 (females 51 and 53) or on day 4 (females 52 and 54). Hypoactivity piloerection and dyspnea were observed prior to death for animals 52 and 54.

Piloerection and dyspnea were recorded in all animals of the treated group 5 from day 3 up to day 6.

No either clinical signs were observed during the study.

Body weight:

A marked body weight loss was recorded in the animals of the treated groups 4 and 5.

The body weight gain of the other treated animals was similar to that of the control animals.

Local irritation:

No cutaneous reactions and no noteworthy increase in ear thickness were observed in the animals of the treated groups.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Guetol is sensitizing to the skin in LLNA.
IN EU GHS system (CLP 1272/2008), Guetol should be classified Skin Sens. Cat 1.
Executive summary:

In a murine Local Lymph Node Assay (LLNA) study (CIT, 2004), the potential of the test item Guetol to induce delayed contact hypersensitivity was evaluated. Evaluation of local irritation was also carried out in parallel.

Twenty-eight female CBA/J mice were allocated to seven groups:

           • five treated groups of four animals receiving the test item Guetol at the concentration of 5, 10, 25, 50 or 100%,

           • one negative control group of four animals receiving the vehicle (mixture acetone/olive oil (4/1)),

           • one positive control group of four animals receiving the reference item,α-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25%.

 

During the induction phase, the test item, vehicle or reference item was applied over the ears (25μL per ear) for three consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI).

 

All animals of the treated group 6 (undiluted test item) were found dead either on day 3 or on day 4. Hypoactivity, piloerection and dyspnea were observed prior to death. Piloerection and dyspnea were recorded in all animals of the treated group 5 (test item at the concentration of 50%) from day 3 up to day 6. No other clinical signs were noted during the study.

Positive lymphoproliferative responses (SI > 3) were noted at all tested concentrations but without clear evidence of a dose-response relationship.

 

No cutaneous reactions and no noteworthy increase in ear thickness were observed in the animals of the treated groups.

 

In the absence of local irritation, the observed lymphoproliferative responses were attributed to delayed contact hypersensitivity.

The EC3value for the test item Guetol is lower than 5%.

 

Based on these results, the test item Guetol is sensitizing in the murine Local Lymph Node Assay, and classified as Skin sens. 1 (H317) according to the EU GHS (CLP 1272/2008).