Reaction products of N6,N6'-hexane-1,6-diylbis[N2,N4-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4,6-triamine] and allylbromide, subsequently reacted with ethaneperoxoic acid, hydrogenated

Endpoint summary

Description of key information

The substance was sensitizing in the Local lymph node assay (LLNA), the EC3 value was calculated at 3%.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 July 2021 - 23 Sep 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 22, 2010

Deviations:

yes

Remarks:

study procedure was extended to include additional measures of lymph node response including lymph node cell count, lymph node weight and ear weight as an indicator of skin irritation

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Remarks:

CaOlaHsd, SPF

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS GmbH Kreuzelweg 53 NL-5961 NM Horst
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 16.1 - 20.4 g
- Housing: single housing
- Diet (e.g. ad libitum): Mouse and rat maintenance diet “GLP”, Granovit AG, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: To: 06 July 2021 - 19 July 2021

Vehicle:

methyl ethyl ketone

Remarks:

MEK was used as vehicle because good solubility was achieved.

Concentration:

1%, 2.5% and 5%
The doses were determined in a pretest, in which application of 10% up to 50% (highest technically achieved concentration) caused slight to severe compound residues on the ears and slight to severe bald spots on the head during the observation period. After application of 10% concentration, the animals still showed severe increases (≥ 25%) in ear thickness measurements as indication of excessive ear skin irritation. Therefore, concentrations over 5% were considered not appropriate for testing.

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: up to 50%
- Irritation: irritation was observed at doses of 10% or higher
- Systemic toxicity: no signs of systemic toxicity were observed.
- Ear thickness measurements: ear thickness measurements showed a severe increase of ear thickness (≥ 25%) as indication of excessive ear skin irritation at doses of 10% and higher

MAIN STUDY
Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation
or the vehicle alone to the dorsal surfaces of both ears on three consecutive days.
Three days after the last application, ca. 20 μCi 3H-thymidine in 250 μL sterile saline were
injected into the tail vein of the mice. About 5 hours after the 3H-thymidine injection, the mice
were sacrificed, and the auricular lymph nodes were removed. Lymph node response was
evaluated measuring 3H-thymidine incorporation (indicator of cell proliferation). The cell count
and weight of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm
diameter sample was punched out of the apical part of each ear, and for each animal, the
weight of the pooled punches was determined to obtain an indication of possible skin irritation.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Mean values and standard deviations of the measured parameters were calculated per test
group from the individual values. The stimulation indices of 3H-thymidine incorporation, cell
count, lymph node weight and ear weight measurements were calculated by dividing the mean
values per test group and/or single animal values by the mean of the vehicle treated group. Statistical significance of 3H-thymidine incorporation, cell count, lymph node weight and ear weight were determined using the Wilcoxon-Test.

Parameter:

SI

Value:

1.92

Test group / Remarks:

1% test substance in MEK

Parameter:

SI

Value:

2.65

Test group / Remarks:

2.5% test substance in MEK

Key result

Parameter:

SI

Value:

4.29

Test group / Remarks:

5% test substance in MEK

Parameter:

EC3

Remarks:

[%]

Value:

3

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: see table in "any other information on results [...]"

EC3 CALCULATION: was calculated by linear regression to be 3%

CLINICAL OBSERVATIONS: No signs of systemic toxicity were noticed in all animals during general observation. No local findings were observed during the observation period.

BODY WEIGHTS: The expected body weight gain was generally observed during the study.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness).

³H-thymidine incorporation, cell count and lymph node weight: test group mean value, standard deviation and stimulation index

 

Test Group	Treatment	³H-thymidine incorporation [DPM/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	vehicle MEK	839.0	222.2	1.00
2	1% in MEK	1,614.9	817.5	1.92
3	2.5% in MEK	2,220.7	266.4	2.65 ##
4	5% in MEK	3,602.2	443.0	4.29 ##

 

Test Group	Treatment	Cell Counts [Counts/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	vehicle MEK	10,229,227	2,416,099	1.00
2	1% in MEK	12,854,400	3,030,382	1.26
3	2.5% in MEK	14,925,600	3,494,065	1.46 #
4	5% in MEK	18,229,333	2,185,078	1.78 ##

 

Test Group	Treatment	Lymph Node Weight [mg/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	vehicle MEK	4.8	0.5	1.00
2	1% in MEK	5.7	0.5	1.18 #
3	2.5% in MEK	7.6	1.0	1.58 ##
4	5% in MEK	7.9	0.6	1.65 ##

 

 

Table 13: Ear weight: test group mean value, standard deviation and stimulation index

 

Test Group	Treatment	Ear Weight [mg/animal]
		Mean	S.D.	Stimulation Index^1
1	vehicle MEK	29.0	1.4	1.00
2	1% in MEK	30.0	1.2	1.03
3	2.5% in MEK	30.1	2.0	1.04
4	5% in MEK	30.6	1.2	1.05

 

¹ vs. mean of test group 1 (vehicle control)

# = statistically significant for the value p ≤ 0.05

## = statistically significant for the value p ≤ 0.01

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitizing potential of the test material was assessed using the radioactive Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears. Groups of 5 female mice each were treated with 1%, 2.5% and 5% (w/w)
preparations of the test substance in MEK or with the vehicle alone. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation or the vehicle alone to the dorsal surfaces of both ears on three consecutive days. Three days after the last application, 3H-thymidine was injected and about 5 hours after the 3H-thymidine injection, the mice were sacrificed, and the auricular lymph nodes were removed. Lymph node response was evaluated measuring 3H-thymidine incorporation (indicator of cell proliferation). The cell count and weight of each animal’s pooled lymph nodes were also determined. In addition, an ear punch was taken for each animal and the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed in all animals during general observation. The mean stimulation indices (expressed as multiples of the vehicle control) for 3H-thymidine incorporation were 1.92, 2.65 and 4.29 for 1%, 2.5% and 5% treated animals, respectively. The EC 3 for 3H-thymidine incorporation was calculated by linear regression from the results of these concentrations to be 3.0%.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

 

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The EC3 derived in the LLNA with the registrered substance was 3%. As a result classification for skin sensitization as category 1B under Regulation (EC) No. 1272/2008 is warranted.