Reaction mass of 2,6-Bis[(dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)phenol and 4-(1-{3,5-Bis[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)-2,6-bis[(dimethylamino)methyl]phenol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2015 (first response) and August 2015 (second response)

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:

- 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, 69-79 (2005)

- 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, 290-298 (2008).

- Vohr, H.-W.: Conformation of the Function of a Local Lymph Node Assay in Mice (LLNA/IMDS, Secondary Response) with 1-chloro-2,4-dinitrobenzene. Bayer HealthCare AG, PH-38331, December 02, 2014.

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:

4-(1-{3,5-Bis[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)-2,6-bis[(dimethylamino)methyl]phenol

Molecular formula:

C27H44N4O2

IUPAC Name:

4-(1-{3,5-Bis[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)-2,6-bis[(dimethylamino)methyl]phenol

Constituent 2

Reference substance name:

2,6-Bis[(dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}- 1-methylethyl)phenol

Molecular formula:

C24H37N3O2

IUPAC Name:

2,6-Bis[(dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}- 1-methylethyl)phenol

Constituent 3

Reference substance name:

2-[(Dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)phenol

Molecular formula:

C21H30N2O2

IUPAC Name:

2-[(Dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)phenol

impurity 1

Reference substance name:

Unknown impurities

IUPAC Name:

Unknown impurities

impurity 2

Reference substance name:

Xylene

EC Number:

215-535-7

EC Name:

Xylene

Cas Number:

1330-20-7

Molecular formula:

C8H10

IUPAC Name:

Benzene, dimethyl-

impurity 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

impurity 4

Reference substance name:

Sulphated ash

IUPAC Name:

Sulphated ash

Test material form:

solid: bulk

Details on test material:

- Name of test material (as cited in study report): HPP 12879-1

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 4 days.
- Treatment of test material prior to testing: The test item was formulated at day 1 or 2 of the study, respectively, or all applications.
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:

NMRI

Remarks:

Strain and breeder of the animals were changed for the challenge experiment study, due to availabilities at the suppliers.

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: first response: HsdWin: NMRI; second response: Crl: NMRI BR
- Source:first response: Harlan Nederland, 5960 AD Horst, The Netherlands; second response: Charles River, Sandhofer Weg 7; 97633 Sulzfeld, Germany
- Age at study initiation: first response: 9 weeks; second response: 8 weeks
- Weight at study initiation: first response: 29-37 g; second response: 29 -33 g
- Housing: During the study period the animals were single-housed in Makrolon type II cages.
- Diet and water: ad libitum
- Acclimation period: first response: at least 4 days; second response: 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:

acetone/olive oil (4:1 v/v)

Concentration:

First response:
0% (vehicle control), 2, 10, 50 % (based on the experience with this test system and expected solubility of the test item)

Second response: 0% (vehicle control) and 10% for the induction; 0% (vehicle control) and 2% for the challenge
10% were chosen, because this is the lowest concentration that showed a significant increase in the stimulation indices of cell counts and ear swelling in the first response-study. 2 % was chosen for the challenge, because this is the highest concentration that had no effect on the these parameters.

No. of animals per dose:

six (first and second response treatment)

Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:
First response:
The test item (2% and 50%) was formulated at day 1 of the study for all applications. The 10% test item formulation was formulated at day 1 (for the first application) and day 2 (for the second and third application) of the study. 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 and day. 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.

Second response:
The test item was formulated at day 1 of the study for all applications.
- Induction: 50 µl/flank and day epicutaneously onto the pre-shaven left flank of the animals on three consecutive days (d1, d2 and d3).
- Challenge: The dorsal part ofboth ears of the mice was treated two weeks later with 25 µl/ear and day. This treatment was repeated on three consecutive days (d15,d16 and d17).

The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4 or d18, respectivily). 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 by this 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.
A similar method reliability check for the secondary response was performed using 1-Chloro-2,4-dinitrobenzene (DNCB). The reliability test with the concentrations of 1 % for the induction and 0.3 %for the challenge clearly showed the sensitizing potential of the test item by immune memory induction.

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 in the evaluation of the biological relevance. All individual data were recorded and archived also in the cases where individual data were not reported, e.g. ear swelling and ear weight. The means of the weights of the lymph nodes were not reported as well.

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

Results 1

Parameter:

SI

Value:

1.29

Test group / Remarks:

low dose (2%)

Remarks on result:

other: first response study: "positiv" level of 1.4 (cut-off value for skin sensitisation) for the cell count index was not exceeded

Results 2

Parameter:

SI

Value:

1.84

Test group / Remarks:

mid dose (10 %)

Remarks on result:

other: first response study: "positiv" level of 1.4 (cut-off value for skin sensitisation) for the cell count index was exceeded

Results 3

Parameter:

SI

Value:

2.88

Test group / Remarks:

high dose (50%)

Remarks on result:

other: first response study: "positiv" level of 1.4 (cut-off value for skin sensitisation) for the cell count index was exceeded

Results 4

Parameter:

SI

Value:

1.12

Test group / Remarks:

induction with the vehicle and challenge with the test item

Remarks on result:

other: second response study: "positiv" level of 1.4 (cut-off value for skin sensitisation) for the cell count index was not exceeded

Results 5

Parameter:

SI

Value:

1.95

Test group / Remarks:

induction and challenge with the test item

Remarks on result:

other: second response study: "positiv" level of 1.4 (cut-off value for skin sensitisation) for the cell count index was exceeded

Cellular proliferation data / Observations:

BODY WEIGHTS: The body weights of the animals were not affected by any of the treatment (first and second response study)

Any other information on results incl. tables

Table 1: First Response Study - 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	1.09	1.67*	2.57*
Stimulation index: cell count in draining lymph nodes	1.00	1.29	1.84*	2.88*
Ear swelling in 0.01 mm on day 4 (index)	18.08 (1.00)	18.92 (1.05)	20.33* (1.24)	44.00* (2.43)
Ear weight in mg / 8 mm diameter punch on day 4 (index)	13.43 (1.00)	13.40 (1.00)	17.87* (1.33)	34.33* (2.56)

* statistically significant increase (p <=0.05)

Test item- sensitizing potential: The NMRI mice showed clear increases in the weights of the draining lymph nodes and in the stimulation indices of cell counts compared to control animals after application of the test item in the mid and high dose group, which are of statistical significance. The "positive level", which is 1.4 for cell count indices, has been exceeded in these groups.

Test item - irritation potential: The "positive level" of ear swelling, which is 2x10-2 mm increase, i.e. about 10 % of the control values, was exceeded in the mid and high dose group. These increases are of statistical significance. A significant increase of ear weights in the mid and high dose group compared to the vehicle treated animals was detected as well. The ears of the animals in the high dose group were strong reddened and hardened.

In summary, the results of the Local Lymph Node Assay with HPP 12879-1 (T101736-8, first response) showed an irritating response, which could be combined with a skin sensitizing potential of a test compound. Without consideration of the irritating potential the EC 1.4 value calculated is 3.6 % for this test item.

Table 2: Second Response Study - Summary of the LLNA/IMDS results (means of 6 animals per group)

Parameter investigated	Vehicle control	Dose 2% - induction with vehicle	Dose 2% - induction with 10% test item
Stimulation index: weight of draining lymph nodes	1.00	1.13	1.81*
Stimulation index: cell count in draining lymph nodes	1.00	1.12	1.95*
Ear swelling in 0.01 mm on day 18 (index)	17.92 (1.00)	17.33 (0.97)	18.25 (1.02)
Ear weight in mg / 8 mm diameter punch on day 18 (index)	12.83 (1.00)	13.19 (1.03)	13.66 (1.06)

* statistically significant increase (p <= 0.05)

Test item- sensitizing potential: The NMRI mice showed significant increases in stimulation indices of cell counts and for weights of the draining lymph nodes compared to control animals after induction and challenge exposure to the test item. In addition, the "positive level", which is 1.4 for cell count indices, has been exceeded in these mice.

Test item- irritation potential: The "positive level" of ear swelling, which is 2 x 10-2 mm increase, i.e. about 10 % of the control values, was not exceeded in tets item treated group. No test item specific effects were determined for ear weights.

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: sensitising

Executive summary:

Two mouse local lymph node assays (LLNA/IMDS; T101736-8 first response and T102171-2 secondary response) were carried out with the test item HPP 12879-1. The first LLNA/IMDS (T101736-8) was carried out in female NMRI mice after epicutaneous application of a formulation containing 0, 2, 10 or 50% of the test item in acetone/olive oil (4:1 v/v) for 3 consecutive days onto both ears of the animals. The study results did point to a non-specific (irritant) and to a specific immunostimulating (sensitizing) potential after application of the 10% and 50% test item preparation The "positive level", which is 1.4 for cell count index, has been exceeded in these mice (1.84 and 2.88). A sensitizing potential can be assumed from the increases in cell proliferation in the draining lymph nodes when the cell count index is increased by 0.4 (i.e. index 1.4), which is the defined threshold. The calculated Differentiation indices (DI) which describe the relationship between local lymph node activation (% of maximal increase in lymph node cell count index) and skin inflammation(% of maximal ear swelling) were < 1 for the mid and high concentration, i.e. 0.88 and 0.33. These DI values do point to an irritating potential of the test item. Nevertheless a sensitizing potential could not fully be excluded by the data available so far.

To clarify whether a specific stimulation is covered by the acute inflammatory reaction (irritation), caused by the test item in the 10% and 50% groups, a LLNA/IMDS, secondary response (T102171-2) was conducted. After a topical induction with a 10 % preparation of the test item on the flanks of female NMRI mice the challenge responses were determined following application of a 2 % preparation on the dorsal sides of the ears. There were increases compared to control animals regarding the stimulation indices for cell counts and for weights of the draining lymph nodes. There were no increases for ear swelling and ear weights. In addition, the "positive level", which is 1.4 for cell count index, has been exceeded in these mice (1.95). The Differentiation index (DI) for this study was> 1, i.e. 3,96 also indicating an allergic reaction pattern. These results show that there is an indication for a skin sensitizing effect after administration in this test system using 10% HPP 12879-1 for induction and 2% HPP 12879-1 as a challenge concentration.

The overall result of both studies showed an irritating response, which could be combined with a skin sensitizing potential of the test compound. Without consideration of the irritating potential the EC 1.4 value calculated is 3.6 % for this test item. In accordance with the classification proposed in the Technical Report No. 78 of the ECETOC this value corresponds to a moderate skin sensitizer.