Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

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

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
modifications: 1. non-radioactive alternative, measuring lymph node cell counts; 2. in addition, measurement of ear swelling and ear weight to discriminate the irritating potential from the sensitizing potential of the test substance
Principles of method if other than guideline:
Modified LLNA (IMDS: Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised in the OECD TG 429. Information on validation of IMDS and scientific justification is given in: Vohr HW et al. Arch. Toxicol., 73, 501-509, (2000): Ehling G et al., Toxicology, 212, 60-68 and 69-75 (2005); Gamer AO et al., Regul. Toxicol. Pharmacol., 52, 290-298 (2008).
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
17 alpha-Cyanomethyl-17 ß-hydroxy-5(10)-estren-3-one
IUPAC Name:
17 alpha-Cyanomethyl-17 ß-hydroxy-5(10)-estren-3-one
Test material form:
solid: crystalline

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Strain: Crl:NMRI BR
- Age at study initiation: 8 weeks
- Housing: 1 animal/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40-70
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0, 2, 10, 40 %
No. of animals per dose:
6
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation or the vehicle were applied epicutaneously onto the dorsal part of both ears of the animals. This  treatment was repeated on three consecutive days (d1, d2 and d3). The volume administered was 25 µL/ear/day. The used concentrations were based on the experiences with the test system and the toxic properties of the test substance.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4). The lymphatic organs (the auricular lymph nodes) were then removed and transferred into physiological saline (PBS).

Investigations:
- weight of draining lymph nodes (given as weight index compared to vehicle controls)
- cell counts in draining lymph nodes (given as cell count index compared to vehicle controls)
Stimulation indices were calculated by dividing the absolute weight or number of cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling (given in 0.01 mm and as index)
- ear weight (given in mg / 8 mm diameter piece and as index)
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The individual values from actively treated groups were compared with those from the control group. A pre-testing was carried out by a Cochran test. Furthermore, depending on the statistical result, a Bonferroni-Holm test (Mann-Whitney test included) or a Dunnett test significance test was conducted (significance levels of 5 %; two-tailed).

In this method of statistical processing of measurements a large number of comparisons are made, and as a result of the multiple tests the overall probability of error is considerably greater than the p values suggest (increased number of false-positive results). On the other hand, the known methods of adjusting p values lead to an excessive increase in the number of false negatives. In view of these problems the biological and toxicological relevance is also taken into consideration in the evaluation of statistical significance.

For this reason, in the case of indices only the standard deviations between groups and difference analysis of the mean values were used for the final evaluation of the biological relevance.

Results and discussion

Positive control results:
Alpha hexyl cinnamic aldehyde showed a clear sensitizing potential in the local lymph node assay (IMDS).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Remarks:
cell count index (%)
Value:
1.35
Test group / Remarks:
2 % test item
Key result
Parameter:
SI
Remarks:
cell count index (%)
Value:
1.07
Test group / Remarks:
10 % test item
Key result
Parameter:
SI
Remarks:
cell count index (%)
Value:
1.31
Test group / Remarks:
40 % test item

Any other information on results incl. tables

Table 1: Summary of the LLNA results (means of 6 animals per group)

Parameter investigated

 Vehicle control

  Test item 2 %

 Test item 10 %

Test item 40 %

 Positive control

Stimulation index:

weight of draining lymph nodes

1.00 

1.19 

1.13 

1..03 

1.49 * 

Stimulation index:

cell count in draining lymph nodes

1.00 

1.35

1.07 

1.31 

2.21 * 

Ear swelling in 0.01 mm on day 4 (index)

18.00 (1.00) 

18.08 (1.00) 

 17.83 (0.99)

18.17 (1.01) 

24.17 * (1.34)

Ear weight in mg / 8 mm diameter punch on day 4 (index)

13.15 (1.00)

12.91 (0.98) 

12.27 (0.93) 

11.88 ** (0.90)

18.23 * (1.39) 

* statistically significant increase (p ≤ 0.05)

** statistically significant decrease (p ≤ 0.05)

The mice did not show increases in stimulation indices for cell counts or for weights of the draining lymph nodes after application of the test item. The “positive level” indicating sensitizing potential, which is 1.4 for the cell count index, was never reached or exceeded in any dose group.

The “positive level” of ear swelling indicating irritating potential, which is 2 x 10-2 mm increase, i.e. about 10 % of the control values, was not reached or exceeded in any dose group. No substance specific effects were determined for ear weights either. In the highest dose group there was a slight statistical significant decrease for ear weights. This change is, however, within the normal range of variance for this parameter, which means that it is only of statistical significance because the mathematical conditions were favorable.

The validity of the assay was demonstrated by the positive results of the positive control group (alpha hexyl cinnamic aldehyde).

The body weights of the animals were not affected by any treatment.

Applicant's summary and conclusion

Executive summary:
5 -Dehydrocyanomethylketon was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429. Concentrations of 0 (vehicle control), 2, 10 and 40 % formulated in dimethylformamide were tested. The results show that the test item has neither an irritating nor a sensitizing potential in mice after dermal application.