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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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 22.82% solids in water

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/JHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: 21.7 - 24.0 grams
- Housing: solid bottom cages with appropriate bedding and nestlets toys as enrichment. During quarantine, animals were housed in groups of 5 or fewer. Animals were single housed for approximately 2 hours following each application of the vehicle control, test substance, or positive control to allow additional time for drying and/or absorption. Following the 2-hour single-housing period, animals were returned to their group housing status.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC
- Humidity (%): 30-70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
0% (vehicle control), 5%, 25%, 50%, 100%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Evaluated; findings not reported

MAIN STUDY
Study Parameter            Frequency
Body Weight              Test days 0 and 5
Dosing                 Test days 0-2
Days of Rest              Test days 3-4
Injection of Radioactivity          Test day 5
Removal of Lymph Nodes          At sacrifice (test day 5)
Disintegrations per minute (dpm) data    Test day 6

ANIMAL ASSIGNMENT AND TREATMENT
- Assignment to Groups: Prior to study start using a randomly generated, computer-based algorithm such that individual pretest body weights did not vary more than 20% of the group mean.
- Daily Animal Health Observations: At least once daily to detect moribundity and mortality.
- Careful Clinical Observations: Prior to administration of each dose and prior to sacrifice

TREATMENT PREPARATION AND ADMINISTRATION:
Twenty-five μL of vehicle control, test substance, or positive control were administered topically to the dorsum of each mouse ear for 3 consecutive days (test days 0-2). Test days 3-4 were days of rest followed by intravenous injection of 20 μCi of ³H-thymidine in PBS per mouse on test day 5. One mouse in the 100% test substance group was excluded due to incomplete IV injection, and the lymph nodes for this mouse were not analysed.
Approximately 5 hours after the injection, animals were sacrificed by isoflurane anaesthesia followed by carbon dioxide asphyxiation, draining auricular lymph nodes were removed, and single cell suspensions were prepared. The single cell suspensions were incubated at 2-8ºC overnight. On test day 6, the single cell suspensions were counted on a beta counter and reported as disintegrations per minute (dpm).

CRITERIA USED TO CONSIDER A POSITIVE RESPONSE: A stimulation index (SI) was derived for each experimental group by dividing the mean dpm of each experimental group by the mean dpm of the vehicle control group. A stimulation index of greater than or equal to 3.0 together with consideration of dose response and, where appropriate, statistical significance were used in the determination of a positive response.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Significance was judged at p < 0.01. Lymph node dpm data were transformed to Log to obtain normality or homogenous variances. See Table 1 below.

Results and discussion

Positive control results:
A 25% concentration of the positive control, HCA, produced a dermal sensitisation response in mice.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
No statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at any test concentration. SIs of less than 3.0 were observed at all test concentrations of the test substance. Therefore, the EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI = 3) for the test substance under the conditions of this study was not calculable. A 25% concentration of the positive control, HCA, produced a dermal sensitisation response in mice.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See Table 2.

Any other information on results incl. tables

No test substance-related changes in body weights were observed at any test concentration. One mouse in the vehicle control group had a 6% weight loss at test day 5, when compared to the test day 0 body weight. No clinical signs of toxicity were observed in the study.

Table 2 Stimulation Index Data

GROUP

MATERIAL TESTED

n

MEAN

(dpm)

S.D.

(dpm)

SI

1

0% Vehicle Control

5

412.25

209.62

N/A

2

5%

5

429.85

131.77

1.04

3

25%

5

471.65

355.00

1.14

4

50%

5

325.85

76.27

0.79

5

100%

4b

329.25

179.05

0.80

6

25% Positive Controla

5

1924.65

241.24

4.67

a Data were not included in the statistical analysis of the test substance groups.

b One mouse was not injected with the appropriate amount of radioactive material and the lymph nodes for this mouse were not analysed.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Not a dermal sensitiser in mice.
Executive summary:

The objective of this study was to evaluate the potential of the test substance to produce a dermal sensitisation response in mice using the local lymph node assay (LLNA). Five groups of 5 female CBA/JHsd mice were dosed for 3 consecutive days with 0% (vehicle control), 5%, 25%, 50%, or 100% test substance on both ears. Propylene glycol (PG) was used as the diluting vehicle. One group of 5 female mice was dosed for 3 consecutive days with 25% hexylcinnamaldehyde (HCA) in PG as a positive control. On test day 5 of the assay, mice received ³H-thymidine by tail vein injection and were sacrificed approximately 5 hours later. The cell proliferation in the draining auricular lymph nodes of the ears from the test substance groups was then evaluated and compared to the vehicle control group. No test substance-related changes in body weights were observed at any test concentration. One mouse in the vehicle control group had a 6% weight loss at test day 5, when compared to the test day 0 body weight. No clinical signs of toxicity were observed in the study. No statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at any test concentration. Stimulation indices (SIs) of less than 3.0 were observed at all test concentrations of the test substance. Therefore, the EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI = 3) for the test substance under the conditions of this study was not calculable. A 25% concentration of the positive control, HCA, produced a dermal sensitisation response in mice. Therefore, the LLNA test system was valid for this study. Under the conditions of this study the test substance did not produce a dermal sensitisation response in mice. Based on these data, the test substance is not a dermal sensitiser in mice.