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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/JHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 9 weeks old
- Weight at study initiation: between 19.9 and 23.8 grams
- Housing: In stainless steel, wire-mesh cages suspended above cage boards. In pairs during quarantine. Singly after assignment to groups, and during the dosing and resting phases of the study. After final weighing (test day 5) until sacrifice, animals were housed one group per plastic shoebox cage with appropriate bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum of 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26ºC
- Humidity (%): 30-70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): an approximate 12-hour light/dark cycle
Vehicle:
dimethylformamide
Concentration:
0% (vehicle control), 5%, 25%, 50%, or 100%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Evaluated, findings not reported
- Irritation: Test substance did not appear to have severe skin-irritating capability (pH 10)

MAIN STUDY         
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.
- Clinical Observations: Prior to administartion of each dose and prior to sacrifice

TREATMENT PREPARATION AND ADMINISTRATION:
Twenty-five μL of vehicle control, test substance, or positive control (25% in vehicle 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 per mouse on test day 5.
Approximately 5 hours after the injection, animals were sacrificed 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 disintegrations per minute (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 < 0.05 except for dpm data that were judged at p < 0.01. Lymph node dpm data were transformed to Log to obtain normality or homogenous variances. See Table 1 below.
Positive control results:
A 25% concentration of the positive control, HCA, produced a dermal sensitization response in mice.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See Table 2 for data.
Parameter:
SI
Value:
1.55
Test group / Remarks:
25%
Remarks on result:
other: see Remark
Remarks:
Statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at the 50% and 100% test concentrations. 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.
Parameter:
SI
Value:
2.33
Test group / Remarks:
50%
Remarks on result:
other: see remarks
Remarks:
Statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at the 50% and 100% test concentrations. 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.
Parameter:
SI
Value:
2.29
Test group / Remarks:
100%
Remarks on result:
other: see remarks
Remarks:
Statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at the 50% and 100% test concentrations. 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.

No statistically significant differences in mean body weights and body weight compared to the vehicle control group were observed at any test concentration. A statistically significant increase in mean body weight gains compared to the vehicle control group was observed at the 25% test concentration. No clinical signs of toxicity were observed in the study. One mouse in the 100% test concentration group was found dead on test day 3.

Table 2

GROUP

MATERIAL TESTED

n

MEAN

(dpm)

S.D.

(dpm)

SI

1

0% Vehicle Control

5

685.65

165.43

N/A

2

5%

5

653.85

248.09

0.95

3

25%

5

1064.25

386.08

1.55

4

50%

5

1599.05#

399.13

2.33

5

100%

4b

1569.50#

592.08

2.29

6

25% Positive Controla

5

6154.25

747.70

8.98

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

b one mouse was found dead on test day 3.

# - Statistically significant increase in dpm data from vehicle control at p < 0.01 by Jonckheere-Terpstra trend test.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance is not a dermal sensitizer in mice.
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
Executive summary:

The objective of this study was to evaluate the potential of the test substance to produce a dermal sensitization 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% of test substance on both ears. Dimethylformamide (DMF) was used as the diluting vehicle. One group of 5 female mice was dosed for 3 consecutive days with 25% hexylcinnamaldehyde (HCA) in DMF 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 statistically significant differences in mean body weights and body weight compared to the vehicle control group were observed at any test concentration. A statistically significant increase in mean body weight gains compared to the vehicle control group was observed at the 25% test concentration. No clinical signs of toxicity were observed in the study. One mouse in the 100% test concentration group was found dead on test day 3. Although, statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at the 50% and 100% test concentrations, 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 sensitization response in mice. Therefore, the LLNA test system was valid for this study with the test substance.

Under the conditions of this study, the test substance did not produce a dermal sensitization response in mice. Based on these data, the test substance is not a dermal sensitizer in mice.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A mouse local lymph node assay was conducted at 0% (vehicle control), 5%, 25%, 50%, or 100% of test substance. Although, statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at the 50% and 100% test concentrations, 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 sensitization response in mice. Therefore, the LLNA test system was valid for this study with the test substance. Under the conditions of this study, the test substance did not produce a dermal sensitization response in mice.


Migrated from Short description of key information:
OECD 429; no sensitization was observed in mice. Reliability = 2

Justification for selection of skin sensitisation endpoint:
OECD Guideline, GLP study

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

There was no evidence of respiratory sensitization during inhalation exposures.


Migrated from Short description of key information:
There was no evidence of respiratory sensitization during inhalation exposures.

Justification for classification or non-classification

There was no evidence of dermal sensitization in a mouse local lymph node assay or respiratory sensitization during inhalation exposures. The substance does not need to be classified for sensitization according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.