trans-crotonic acid

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

trans-crotonic acid

EC Number:

203-533-9

EC Name:

trans-crotonic acid

Cas Number:

107-93-7

Molecular formula:

C4H6O2

IUPAC Name:

but-2-enoic acid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DEBR 012553

In vivo test system

Test animals

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.

Study design: in vivo (LLNA)

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).

Results and discussion

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

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

5 %

Results 2

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

10 %

Results 3

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

25 %

Results 4

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

50 %

Results 5

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

AOO (Negative control for the positive control)

Results 6

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

DMF (negative control for the test item)

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.