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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 guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(2010)
Deviations:
yes
Remarks:
- modified LLNA (IMDS): Measurement of cell counts instead of radioactive labeling. In addition, measurements of ear swelling and ear weights were done 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 authorized in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). 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 69-79 (2005).
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Reaction mass of 2,2'-iminodiethanol and 2-{[bis(2-hydroxyethyl)amino]methyl}-4-[2-(4-hydroxyphenyl)propan-2-yl]phenol and 2,2-bis(4-hydroxyphenyl)propane and 2-{[bis(2-hydroxyethyl)amino]methyl}-4-[2-(3-{[bis(2-hydroxyethyl)amino]methyl}-4-hydroxyphenyl)propan-2-yl]phenol
- Appearance: viscous, clear, yellowish liquid

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: HsdWin: NMRI (SPF-bred)
- Source: Harlan Nederland, 5960 AD Horst, The Netherlands
- Age at study initiation: 10 weeks
- Weight at study initiation: 30-38 g
- Housing: During the study period the animals were single-housed in type II cages.
- Diet and 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 (vehicle control), 2, 10, 50 % (Due to the viscosity of the test item it was not possible to pipette the test item pure)
No. of animals per dose:
6
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item was formulated once before application in the vehicle. The formulation was 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. The concentrations used were based on the experiences with the test system and the toxic properties of the test substance. For negative control a dose group treated only with the vehicle in the above described manner was used.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4). The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline (PBS).
Investigations:
- weight of lymph nodes
- cell counts in lymph nodes
- stimulation index is calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling
- ear weight
- body weights

The methodological reliability is checked in regular intervals using alpha cinnamic aldehyde as positive control. The last reliability test using alpha hexyl cinnamic aldehyde formulated in acetone/olive oil (4:1) at concentrations of 2.5 %, 10 % and 40 % clearly showed the sensitizing potential of the test item.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
When it was statistically reasonable, the values from treated groups were compared with those from the control group by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to a homogeneity testing like Cochran's test. Alternatively, if the variances are considered to be heterogeneous (p<=0.05), a non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) at significance levels of 5 %. Two sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. Outlying values in the LN weights were eliminated at a probability level of 99 % by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Scheffels method, which according to Sachs can be used for both equal and unequal sample sizes.

Results and discussion

Positive control results:
After treatment with Alpha Hexyl Cinnamic Aldehyde mice showed clear increases in the weights of the draining lymph nodes and in the stimulation indices for cell counts compared to control animals, which are of statistical significance.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The “positive level”, which is 1.4 for cell count indices, was never reached or exceeded in any dose group. Cell count index (test item concentration): 1.00 (0 %) / 0.88 (2 %) / 0.88 (10 %) / 1.27 (50 %).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: modified LLNA; measurement of cell counts instead of radioactive labelling

Any other information on results incl. tables

It has to be clarified that the “positive level” mentioned above is exclusively defined for the NMRI outbreed mice used for this study. Such positive limits have to be calculated for each strain of mice individually.

The mice did not show increases for weights of the draining lymph nodes after application of the test item (weight index: 1.0 (0 %), 0.98 (2 %), 1.01 (10 %), 1.35 (50 %)).

The positive level of ear swelling, which is 2 x 10EXP-2, i.e. about 10 % of the control values, has not been reached or exceeded in any dose group (ear swelling: day 1 = 18.67 (0 %) / 17.92 (2 %) / 17.75 (10 %) / 17.92 (50 %); day 4 = 19.17 (0 %) / 18.67 (2 %) / 19.00 (10 %) / 18.83 (50 %); Index day 4 = 1.00 (0 %) / 0.97 (2 %) / 0.99 (10 %) / 0.98 (50 %).

No substance specific effects were determined for ear weights either (day 4 = 13.77 (0 %) / 12.98 (2 %) / 13.36 (10 %) / 13.30 (50 %); Index day 4 = 1.00 (0 %) / 0.94 (2 %) / 0.97 (10 %) / 0.97 (50 %)).

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

Analyses during the study verified that the test item content agreed with the target concentrations.

Applicant's summary and conclusion

Executive summary:

A modified LLNA (IMDS; OECD TG 429) was performed on 6 female NMRI mice per dose group using test substance of 0 % (vehicle control), 2 %, 10 % and 50 %. Due to the viscosity of the test item it was not possible to pipette the test item pure, which limited the maximum dose to 50 %.

Compared to vehicle treated animals, none of the parameters measured in the substance treated groups, i.e. cell counts and weights of the draining lymph nodes, ear swelling and ear weights, reached or exceeded the "positive levels" defined for this assay. Therefore, no indication for a skin sensitizing effect after administration of a concentration up to and including 50 % was seen.

No indication for a non‑specific (irritant) activation was detected either. Overall, the concentration of 50 % turned out to be the NOEL for the parameters investigated in this study.