Reaction mass of 2-(3-{6-[3-(2-Hydroxyethyl)-3-methylureido]hexyl}-1-methylureido)ethanol and 6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 to 22 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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). By comparing the specific immune reaction induced by the test item in the draining lymph nodes (cell counts / lymph node weights) with the immediate nonspecific acute skin reaction (ear swelling / ear weight), it is possible to differentiate the irritant potential from the sensitizing potential of the compound tested .Modifications are authorized in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Detailed Information on validation of IMDS, scientific justification, method and assessment of results are given in:

- Homey, B., von Schilling, C., Blümel, J., Schuppe, H.-C., Ruzicka, T., Ahr, H.-J., Lehmann, P. and Vohr, H.-W.: An integrated Model for the Differentiation of Chemical-Induced Allergic and Irritant Skin Reactions (IMDS). Toxicol. and Appl. Pharmacol. 153, 83-94 (1998).

- Vohr, H.-W., Blümel, J., Blotz, A., Homey, B. and Ahr, H.J. An intra-laboratory validation of IMDS: Discrimination between (Photo) Allergic and (Photo) Irritant Skin Reactions in Mice. Arch. Toxicol., 73, 501-509 (2000).

- ECETOC Technical Report No. 87, Brussels (2003)

- Ehling G., Hecht M., Heusener A., Huesler J., Gamer A.O., v. Loveren H., Maurer Th., Riecke K., Ullmann L., Ulrich P., Vandebriel R., Vohr H.-W. An European Inter-Laboratory Validation of Alternative Endpoints of the Murine Local Lymph Node Assay. 2nd ROUND.Toxicology 212 (2005), 69-79

- Gamer A.O., Nies E., Vohr H.-W.: Local Lymph Node Assay (LLNA): Comparison of different protocols by testing skin-sensitizing epoxy resin system components. Reg. Tox. Pharmacol. 52 (2008), 290-298.

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:

N,N''-hexane-1,6-diylbis[N'-(2-hydroxyethyl)-N'-methylurea]

EC Number:

253-281-9

EC Name:

N,N''-hexane-1,6-diylbis[N'-(2-hydroxyethyl)-N'-methylurea]

Cas Number:

36938-15-5

Molecular formula:

C14H30N4O4

IUPAC Name:

2-(3-{6-[3-(2-Hydroxyethyl)-3-methylureido]hexyl}-1-methylureido)ethanol

Constituent 2

Reference substance name:

6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

Molecular formula:

C14H30N4O4

IUPAC Name:

6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

Test material form:

solid: bulk

Details on test material:

- Batch no.: EGGE 2806-1
- Currenta sample no.: 2187

Specific details on test material used for the study:

- Solubility and stability of the test substance in the solvent/vehicle: The stability of the test item in the vehicle was analytically verified for up to 2 hours. The test item formulations in the vehicle were visually described as solutions.SOURCE OF TES
- Treatment of test material prior to testing:The test item was melted for approx. 3 hours in a hot cabinet (110°C) before weighing. The test item was formulated in DMF by mixing immediately before each application.

FORM AS APPLIED IN THE TEST (if different from that of starting material): The formulations were visually described as solutions.

In vivo test system

Test animals

Species:

mouse

Strain:

other: HsdWin: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: 9 weeks
- Weight at study initiation: 28-36 g
- Housing: During the study period the animals were single-housed in Makrolon type II cages.
- Diet and water: ad libitum
- Acclimation period: at least 6 days
- Indication of any skin lesions: no


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 % (based on the experience with this test system and expected solubility of the test item)

No. of animals per dose:

six

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 expected solubility of the test item. 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 phosphate buffered solution (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

- LLN weight and cell count determinations: The weight and cell count determinations were carried out by appropriate laboratory procedures. The weights of the lymph nodes were determined on a Mettler automatic balanceand manually recorded. LN cell suspensions were generated by crushing the lymph nodes through a sieve into 2 ml medium, the cell counts per ml were then determined using a Multisizer 3® from Coulter Electronics. These data were directly collected and processed by computer (Multisizer 3 software and Excel). Means, stimulation indices and standard deviations were calculated by an Excel data sheet. The so-called stimulation (or LLN-) index is calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones. Thus, in case of no stimulating effect the index is about 1.00 (±relative standard deviation), and the indices of vehicle treated animals are set to 1.00 (±relative standard deviation).

The Local Lymph Node Assay Test methodology was checked for reliability in a test on female NMRI mice using alpha hexyl cinnamic aldehyde formulated in different vehicles (PEG 400, DAE 433, DMF, MEK, acetone/olive oil (4:1) and Cremophor ELl physiological saline solution 2% v/v) at concentrations of3 %, 10% and 30%. The sensitivity as well as the reliability of the experimental technique is thus confirmed bythis study.
A similar check is done in regular intervals using one of the above mentioned vehicles in order to confirm the reliability of the method. The last reliability test (March 2015) using alpha hexyl cinnamic aldehyde formulated in acetone/olive oil (4:1) at concentrations of2.5 %, 10% and 40% clearly confirmed 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 groups (vehicle) 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 heterogenous (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 (Mann-Whitney test included), respectively. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Scheffe's method, which can be used for both equal and unequal sample sizes. 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 offalse~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 in the evaluation of the biological relevance Individual data were recorded and archived, but in some cases e.g. ear swelling and ear weight are not reported.

Results and discussion

Positive control results:

The sensitivity as well as the reliability of the experimental technique is confirmed by the studies with Alpha Hexyl Cinnamic Aldehyde (see details on study design).

In vivo (LLNA)

Results

Parameter:

SI

Value:

0.94

Test group / Remarks:

high dose (50%)

Remarks on result:

other: cell count index of the high dose group (50%): "positiv" level of 1.4 (cut-off value for skin sensitisation) was not exceeded in any dose group

Cellular proliferation data / Observations:

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

Any other information on results incl. tables

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

Parameter investigated	Vehicle control	Dose 2%	Dose 10%	Dose 50%
Stimulation index: weight of draining lymph nodes	1.00	0.87	0.89	1.08
Stimulation index: cell count in draining lymph nodes	1.00	0.85	0.87	0.94
Ear swelling in 0.01 mm on day 4 (index)	19.67 (1)	19.00 (0.97)	19.0 (0.97)	19.5 (0.99)
Ear weight in mg / 8 mm diameter punch on day 4 (index)	14.39 (1)	13.71 (0.95)	13.98 (0.97)	13.53 (0.94)

Test item - sensitizing potential: The NMRI mice did not show an increase in stimulation indices for cell counts, which is of statistical significance, and for weights of the draining lymph nodes after application of the test item up to and including 50 %. In addition, the "positive level", which is 1.4 for cell count indices, was not exceeded in any dose group.

Test item - irritating potential: The "positive level" of ear swelling, indicating irritating potential, which is 2x10-2mm increase, i.e. about 10 % of the control values, was not exceeded in any dose group.

The validity of the assay was demonstrated by the positive results with Alpha Hexyl Cinnamic Aldehyde which has been demonstrated in regular intervals in separate studies.

Applicant's summary and conclusion

Interpretation of results:

other: not sensitising

Executive summary:

The test item was assessed for both its skin sensitizing and irritant potential in a modified Local Lymph Node Assay with the following concentrations: 0 % (vehicle control), 2 %, 10 % and 50 %. The test item was formulated in dimethylformamide (DMF). Compared to vehicle (DMF) treated animals there was no increase regarding the cell counts and the weights of the draining lymph nodes up to and including the highest dose group. Therefore there is no indication for a specific skin sensitizing effect after administration of a concentration of up to and including 50 % test item in this test system. The "positive level" of ear swelling which is 2 x 10-2mm increase, i.e. about 10 % of the control values, was not exceeded in any dose group. No substance specific effects were determined for ear weights either. Thus the study does not point to an irritant potential of the test item, too.The validity of the assay was demonstrated by the positive results with Alpha Hexyl Cinnamic Aldehyde which has been demonstrated in regular intervals in separate studies.

.