Registration Dossier

Administrative data

Description of key information

Crotonic acid was not sensitizing in the LLNA.

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
Study period:
2012-07-04 to 2012-07-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DEBR 012553
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: 12 weeks
- Weight at study initiation: 17.7-20.7 g
- Housing: grouped caging in small groups (5 animals/cage)
- Diet: Pellet standard diet
- Water: Tap water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3°C
- Humidity: 30-70%
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Vehicle:
dimethylformamide
Concentration:
5, 10, 25, 50 % (w/v)
No. of animals per dose:
5 females per dose
Details on study design:
RANGE FINDING TESTS: pre-test was performed in three groups of 2 animals
- Treatment: 10, 25, 50% (w/v) in DMF once daily on three consecutive days

TREATMENT PREPARATION AND ADMINISTRATION:
Each mouse was topically treated (dorsal surface of each ear) once a day for three consecutive days (Days 1, 2 and 3) with 25 µL of the appropriate formulations of the test item, of the positive control substance (positive control group) or of the vehicles (negative control groups). On Day 6 each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS containing 20 µCi of 3H-methyl-thymidine. Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation and lymph nodes were removed. The lymph nodes of individual mice were collected separately. A single cell suspension (SCS) of lymph node cells (LNCs) of each individual animal was prepared and washed. After wash, the pellets were gently agitated before suspending the LNCs in 3 mL of 5 % trichloracetic acid (TCA) at 2-8°C overnight (approx. 18 hrs) for precipitation of the macromolecules. After incubation pellets were re-suspended in 1 mL of 5 % TCA and dispersed. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and measured via a beta-scintillation counter. The beta-counter expresses the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % TCA. Ear punch weights and ear thickness were measured.

CRITERIA USED TO CONSIDER A POSITIVE RESPONSE
- Exposure to at least one concentration of the test item results in an incorporation of 3HTdR at least 3-fold or greater than 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The heterogeneity of variance between groups was checked by Bartlett's test for the measured DPM values corrected with the mean background value. Since no significant heterogeneity was detected, a one-way analysis of variance was carried out. Since the result was positive Duncan's Multiple Range test was used to assess the significance of inter-group differences. Significance of the positive control response was evaluated by T-test versus control (AOO).
Positive control results:
Significant larger lymph nodes than the control was observed in the positive control group:
Mean DPM: 6570.6 +/- 2281.8 %
S.I.: 10.2
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
5 %
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
10 %
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
25 %
Key result
Parameter:
SI
Value:
1.3
Test group / Remarks:
50 %
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
AOO (Negative control for the positive control)
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
DMF (negative control for the test item)

Table 1. Disintegrations per minute (DPM). No statistically significant lymphoproliferation was observed in any group treated with Crotonic acid

Crotonic acid in DMF

DPM

5 %

762.8±270.2

10 %

548.4± 192.5

25 %

479.6±250.0

50 %

678.32± 440.6

AOO (negative control for the positive substance)

642.4± 362.1

DMF (negative control for the test item)

517.2±332.5

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present Local Lymph Node Assay, Crotonic acid tested at the maximum attainable concentration of 50 % and at concentrations of 25%, 10 % and 5 % as solution in a suitable vehicle was shown to have no sensitization potential.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In a Local Lymph Node Assay according to OECD 429 and GLP guideline (Toxi-Coop Zrt. (c), 2012) mice (5/concentration) were treated with the test item at concentrations of 5, 10, 25 and 50 % (w/v) and with an equivalent volume of the relevant vehicle (N,N-Dimethylformamide, DMF) alone. Two further groups of mice were treated with the positive control (25 % alpha-Hexylcinnamaldehyde) and with the related vehicle control (Acetone: Olive oil 4:1 (v/v) mixture (AOO)). Each substance was applied on the external surface of each ear (25 µL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6 animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR), than sacrificed approximately 5 hours after the injection. Auricural lymph nodes were removed and processed (individual approach). The cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the obtained values were used to calculate stimulation indices (SI). Ear punch weight and ear thickness were measured at sacrifice.

No mortality was observed during the test. No signs of systemic toxicity and no test item related effect on body weight gain occured. No significant local effects at the application sites (ears) were observed in any treatment group. No visible erythema was observed during the test. The measured ear thickness values did not increase by more than 25 % in any treatment group. The measured ear punch weight values did not indicate any irritation effect of the test item in any dose group. Larger lymph nodes than the control were observed in the positive control group only. No statistically significant lymphoproliferation was observed in any group treated with Crotonic acid. The SI values were 1.3, 0.9, 1.1 and 1.5 at concentrations of 50 %, 25 %, 10 % and 5 %, respectively. No dose-related response was observed. The positive control item induced the appropriate stimulation (SI = 10.2), thus confirming the validity of the assay. Under the conditions of the present Local Lymph Node Assay, Crotonic acid tested at the maximum attainable concentration of 50 % and at concentrations of 25 %, 10 % and 5 % as solution in a suitable vehicle was shown to have no sensitization potential.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitization, the test item does not need to be classified as skin sensitizing according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.