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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 September 2016 - 04 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
d.d 24/9/2015
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sebacohydrazide
EC Number:
213-126-8
EC Name:
Sebacohydrazide
Cas Number:
925-83-7
Molecular formula:
C10H22N4O2
IUPAC Name:
sebacohydrazide
Test material form:
solid: particulate/powder
Details on test material:
Appearance: White powder
Storage conditions: Room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
- Source: Envige RMS B.V., Inc., Horst, The Netherlands.
- Age at study initiation: Young adult animals (approx. 8-12 weeks old)
- Weight at study initiation: 15 - 23 g
- Housing: Animals were group housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Free access to pelleted rodent diet (2014C Teklad Global Rodent diet, Envigo RMS (UK) Limited, Oxon, UK)).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set to achieve)
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 19 September 2016 - 04 October 2016

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5%, 10% and 25% (w/w)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
(tektst onder 4.8.2 Pre-screen test)
A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the test item at a concentration of 25%, the highest concentration that could technically be applied.
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. The animal was treated on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge (prior to dosing on Days 1 and 3, and on Day 6.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: For the purpose of the study, the test item was freshly prepared as a suspension in acetone/olive oil 4:1. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The test item was formulated within 2 hours of being applied to the test system.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Viability: Twice daily on days 1,2, and 3 and on a daily basis on days 4, 5 and 6.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Twice daily on days 1,2, and 3 and on a daily basis on days 4, 5 and 6.
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing) according to a numerical scoring system (see table 1 below). Furthermore, a description of all other (local) effects was recorded according to guidelines.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Individual and group mean DPM values were assessed for dose response relationships. Data was assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variance, then parametric one way analysis of variance (ANOVA) and Dunnett's multiple comparison procedure were used to determine statistical significance. If assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.

Results and discussion

Positive control results:
The routine reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at Envigo is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.98
Test group / Remarks:
5% test item
Key result
Parameter:
SI
Value:
1.35
Test group / Remarks:
10% test item
Key result
Parameter:
SI
Value:
1.93
Test group / Remarks:
25% test item
Cellular proliferation data / Observations:
The mean DPM/animal were 3479, 3423, 4696 and 6712 for the control, 5%, 10% and 25% test item groups, respectively.
White test item on the ears was noted in anmals treated with the test item at a concentration of 25%.

Any other information on results incl. tables

Results Pre-screen test:

White test item on the ears was noted post dose on day 2 and at all subsequent observations. No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Based on this information the dose levels selected for the main test were 25%, 10% and 5% w/w.

Other results - main study:

Systemic Toxicity and Body weight

There were no deaths. No signs of systemic toxicity were noted in the test or control animals. Body weight change of the test animals between day 1 and day 6 was comparable to that observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, Sebacohydrazide was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 25% (w/w).
Executive summary:

An LLNA skin sensitisation study was performed according to OECD/EC test guidelines and in accordance with GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 5%, 10% and 25% w/w. White test item remnants were present on the ears of the animals in the 25% group between Days 1 and 5. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 3423, 4696 and 6712 DPM, respectively. The mean DPM/animal value for the vehicle control group was 3479 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 0.98, 1.35 and 1.93, respectively. As the SI appeared not to be ≥ 3 when tested up to 25% w/w, Sebacohydrazide was considered not to be a skin sensitiser.