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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:
other: GLP compliant study conduct in accordance with international guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not applicable
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not applicable
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
not applicable
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N-dimethyl-3-oxobutyramide
EC Number:
218-059-8
EC Name:
N,N-dimethyl-3-oxobutyramide
Cas Number:
2044-64-6
Molecular formula:
C6H11NO2
IUPAC Name:
N,N-dimethyl-3-oxobutyramide
Constituent 2
Reference substance name:
N,N-Dimethylacetoacetamide
IUPAC Name:
N,N-Dimethylacetoacetamide
Test material form:
other: yellowish liquid
Details on test material:
Name of test material (as cited in study report): LZ705

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation:11-12 weeks (at start of the preliminary test and main test)
- Weight at study initiation: 17.6 to 21.1 g
- Housing: Grouped caging (5 animals/cage)
- Cage type: Type II. Polypropylene / polycarbonate
- Bedding: Laboratory bedding
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M-Z+H complete diet for rats and mice.
- Water: Tap water from municipal supply, as for human consumption, from a bottle ad libitum.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): not reported
- 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:
25%, 50% and 75% w/v in a final volume of 1 mL (main test)
No. of animals per dose:
12 animals/preliminary test (2 animals/treatment group)
40 animals/main test (5 animals/treatment group)
Details on study design:
RANGE FINDING TESTS:
Six groups of 2 CBA/Ca mice were treated with the undiluted test item (100 %) and formulations of 75 %, 50 %, 25 %, 10 % or 5 % (w/v) in the selected
vehicle (DMF) once daily for 3 consecutive days. All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site during the preliminary test. Body weights were recorded prior to the first treatment (Day 1) and prior to termination (Day 6, where applicable).
Both ears of each mouse were observed for erythema and scored. Measurement of ear thickness was taken using digital micrometer on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.

MAIN STUDY
The test item was administered at four different concentrations according to the results of the dose range finding test. The positive control substance (HCA) was tested at one concentration (see table under "Any other information on materials and methods incl. tables).

IN VIVO TREATMENT:
Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance (positive control group) or the vehicle (negative control group) using a pipette, on the dorsal surface of each ear. After the treatments animals returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not applicable

Results and discussion

Positive control results:
The positive control group animals were treated with 25 % (w/v) HCA solution (dissolved in AOO) concurrent to the test item groups. No mortality,
cutaneous reactions or signs of toxicity were observed in the positive control group.
The positive control substance induced the appropriate, statistically significant stimulation compared to the control (SI = 9.7; p < 0.01 Mann-Whitney U-test versus the relevant vehicle (AOO) control). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed sensitivity and validity of the assay.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 0.5 (undiluted) 0.7 (75%) 0.9 (50%) 1.3 (25%)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: LZ705 100% (undiluted) - 874.7 LZ705 75% in DMF - 1051.5 LZ705 50% in DMF - 1455.9 LZ705 25% in DMF - 2155.5

Any other information on results incl. tables

No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control was noted for LZ705 at the tested concentrations. The observed stimulation index values were 0.5, 0.7, 0.9 and 1.3 at test item concentrations of 100 %, 75 %, 50 % and 25 %, respectively. No dose-related response was observed. Significance of the lymphoproliferative response was statistically evaluated using the measured individual disintegration per minute (DPM) values corrected with the mean background value. No statistical significance was observed for the test item (Mann-Whitney U-test versus the relevant controls (untreated or DMF) was performed).

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
LZ705 tested at the maximum attainable concentration of 100% as the undiluted test item and at concentrations of 75%, 50 % and 25% as formulations in an appropriate vehicle (DMF) was shown to have no sensitization potential.
Executive summary:

A LLNA study was performed in July 2012 according to OECD Guideline 429 and GLP.

No signs of irritation (erythema on the ears) or any other local effect was observed in any of the treatment groups.

No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control was noted for LZ705 at the tested concentrations. The observed stimulation index values were 0.5, 0.7, 0.9 and 1.3 at test item concentrations of 100 %, 75 %, 50 % and 25 %respectively. LZ705 tested at the maximum attainable concentration of 100% as the undiluted test item and at concentrations of 75%, 50 % and 25% as formulations in an appropriate vehicle (DMF) was shown to have no sensitization potential.