Myrac Aldehyde

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 June 2009 - 05 August 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

24th April 2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

EC Number:

915-650-9

Molecular formula:

C13H20O

IUPAC Name:

Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

Test material form:

liquid

Details on test material:

Batch No. VE00032417
Purity: 99.9% (sum of the two main constituents)
Physical state: colourless-pale yellow liquid
Storage Conditions: room temperature and protected from light
Expiry Date: 10 January 2010

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source (breeder): Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 9 weeks old
- Weight at study initiation: 20.4-24.7 g
- Housing: The animals were housed individually in disposable crystal polystyrene cages (22 cm x 8.5 cm x 8 cm), containing autoclaved sawdust.
- Diet: SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum.
- Water: drinking water was filtered by a FG Millipore membrane (0.22 micron) and provided ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00)
- IN-LIFE DATES: From: 01 July 2009 To: 06 July 2009

Study design: in vivo (LLNA)

Vehicle:

other: ethanol/diethylphthalate (1:3, v/v)

Concentration:

0, 2.5, 5, 10, 25 and 50%

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The test item was soluble in ethanol/diethylphthalate (1:3; v/v). A solution was obtained with this vehicle at the maximum tested concentration of 75%. The test item was prepared at the chosen concentrations in the vehicle. The dosage form
preparations were homogenized by vortex. The positive control was dissolved in the vehicle at the concentration of 25% (v/v). All dosage form preparations were made freshly on the morning of administration.
- Concentrations (preliminary test): 25, 50, 75 and 100%
- Ear thickness measurements: each day before treatment and 72 hours after the last application, using a micrometer.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: auricular lymph node cell proliferation assay
- Criteria used to consider a positive response: see table 1

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. 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. On day 6 of the main test, all animals of all groups received a single intravenous injection of 250 μL of 0.9% NaCl containing 20 μCi of 3H-TdR (specific activity of 25 Ci/mmol) via the tail vein. Approximately 5 hours later, the animals were sacrificed by cervical dislocation and the auricular lymph nodes were excised. For each animal, both auricular lymph nodes were pooled together and processed separately from other animals. Then, a cell suspension of both auricular lymph nodes was prepared for each animal by mechanical disaggregation in Petri dishes with the plunger of a syringe. Cell suspensions were washed with 4 mL of 0.9% NaCl and pellets obtained were re-suspended in
0.9% NaCl for numeration of lymphocytes (cellularity) and determination of their viability by exclusion of trypan blue. Each cell suspension was then centrifuged and pellets were precipitated with 1 mL of 5% (w/v) trichloroacetic acid in purified water at +4°C overnight. After a last centrifugation, the pellets were precipitated with 0.25 mL of 5% TCA. Three mL of Ultima GoldxR scintillation
fluid (Packard) were added in order to measure incorporation of 3H-TdR using β-scintillation counting.
The simulation index (SI) and the cellularity index (CI) were calculated.

OBSERVATIONS
- Clinical signs, morbidity and mortality: at least once a day
- Body weight: individually, on the first day (day 1) and on the day of sacrifice (day 6)
- Ear thickness measurements: left ears, on days 1, 2 and 3 (before each cutaneous application) and on day 6 (immediately after sacrifice), using a micrometer.
- Irritation reaction: erythema and edema, parallel with ear thickness measurements
- other: coloration, presence of residual test item

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Statistical analysis was performed using the software SAS Enterprise Guide Version 2.05.89 (SAS Release 8.02 TS Level 02M0, SAS Institute Inc).
The results of the ear thickness and the simulation index data were expressed as group mean values ± standard deviation.

Assessment of a dose-response relationship was performed using a Kendall test.

Two separate statistical comparisons were performed:
- negative control group versus positive control group (first comparison),
- test item-treated groups versus negative control group (second comparison).
For each of these statistical comparisons, the normality and homogeneity of variances were tested using Kolmogorov Smirnov and Bartlett tests:
- if normality and homogeneity of variances are demonstrated, a Student’s t-test (for the first comparison) or a one-way analysis of variance followed, if necessary, by a Dunnett tests (for the second comparison) is implemented,
- if normality and homogeneity of variances are not demonstrated, a Mann & Withney test (for the first comparison) or a Kruskall Wallis test followed, if necessary, by Dunn tests (for the second comparison) is conducted.

Results and discussion

Positive control results:

Simulation Index: 2.99

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Remarks:

%

Value:

24.04

Remarks on result:

other: 5.6 µg/cm^2

Results 2

Parameter:

SI

Value:

1.03

Remarks on result:

other: 2.5% test group

Results 3

Parameter:

SI

Value:

0.88

Remarks on result:

other: 5% test group

Results 4

Parameter:

SI

Value:

2.56

Remarks on result:

other: 10% test group

Results 5

Parameter:

SI

Value:

3.03

Remarks on result:

other: 25% test group

Results 6

Parameter:

SI

Value:

4.82

Remarks on result:

other: 50% test group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: A significant lymphoproliferation was noted at the concentrations of 25 and 50%.

DETAILS ON STIMULATION INDEX CALCULATION: according to the following formula:
SI= Mean disintegration per minute per node of treated group/mean disintegration per minute per node of vehicle group

EC3 CALCULATION:
EC3 = c+[(3-d)/(b-d)] x (a-c)
a = the lowest concentration giving stimulation > 3
b = the actual stimulation index caused by a
c = the highest concentration failing to produce a stimulation index of 3
d = the actual stimulation index caused by c

SOLUBILITY: The test item was soluble in ethanol/diethylphthalate (1:3; v/v). A solution was obtained with this vehicle at the maximum tested concentration of 75%.

CLINICAL OBSERVATIONS: Neither mortality nor clinical signs were observed during the study.

BODY WEIGHTS: The body weight change of treated animals was similar to that of control animals.

OTHER: A dryness of the skin was noted on day 6 in 2/5 females treated at the concentration of 50%. No excessive increase in ear thickness was observed in the animals of the treated groups.

According to the absence of local irritation, a statistically significant lymphoproliferative response at the concentration of 50%, and a statistically significant dose-response relationship, the test item is considered as a skin sensitizer. The EC3 value (24.04%) is equal to 5.60 μg/cm².

ACCEPTABILITY: The incorporation of 3H-TdR in the vehicle control group was at least 2-fold higher than that of control blank, the mean cell viability in the vehicle group was higher than 70%.
The threshold positive value of 3 for the SI was not reached in the positive control group (2.99). However, because a postive repsonse was observed after treatment with the test item, this has no effect on the outcome of the study.
The study was therefore considered to be valid.

Any other information on results incl. tables

Table 2 Results

Treatment	Concentration (%)	Irritation level	Stimulation index (SI)
Test item	2.5	Non-irritant	1.03
Test item	5	Non-irritant	0.88
Test item	10	Non-irritant	2.56
Test item	25	Non-irritant	3.03
Test item	50	Slightly irritant	4.82
HCA	25	-	2.99

Applicant's summary and conclusion

Interpretation of results:

other: Skin sensitiser

Remarks:

according Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

The substance could elicit a SI ≥ 3. An EC3 value of 24.04% v/v was calculated, which is equal to 5.60 μg/cm².

Executive summary:

In a Local Lymph Node Assay, the skin sensitisation potential of the substance has been tested according to OECD TG 429 test guideline and GLP principles. Per concentration, 5 female mice were treated. Skin sensitization potential was evaluated by ear thickness measurements. At 2.5, 5, 10, 25 and 50% the substance showed SI values of 1.03, 0.88, 2.56, 3.03 and 4.82, respectively. Negative and positive controls were included and all acceptability criteria were met. These results show that the substance could elicit a SI ≥ 3. An EC3 value of 24.04% was calculated, which is equal to 5.60 μg/cm². Therefore, the substance has to be classified as a skin sensitiser according to CLP regulation (EC) n° 1272/2008.