1,3,5-Triazine-2,4-diamine, N2,N2,N4,N4-tetrabutyl-6-chloro-

Endpoint summary

Description of key information

OECD 429, GLP, mice, 5%, 10%, and 25% (w/w) in methyl ethyl ketone (MEK), sensitizing (BASF SE, 2019)

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:

Jul 20108 - Feb 2019

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:

Jul 2010

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen, Germany
- Batch No.of test material: GF02 (791)
- Purity: 97.2%
- Content: 99.1 g/100 g
- Expiry date: 21 Mar 2023
- Physical state / color: liquid / colorless, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Storage stability: guaranteed
- Homogeneity: homogeneous by visual inspection

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test-substance preparation was produced on a weight per weight (w/w) basis shortly before application. After stirring with a magnetic stirrer, the test substance was solved in the vehicle.

FORM AS APPLIED IN THE TEST: solution

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd/SPF

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS GmbH, NL-5800 AN Venray
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 19.1 g - 21.3 g (pre-test); 17.8 g - 21.5 g (main test)
- Housing: single housed
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12, illumination period: 6 am to 6 pm
- IN-LIFE DATES: From: 10 Jul 2018 To: 23 Jul 2018

Vehicle:

methyl ethyl ketone

Concentration:

5%, 10%, and 25% (w/w)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS: Two mice were treated with test-substance concentrations of 1%, 10% and 50% each on three consecutive days.
- Compound solubility: performed according to the recommendations given by OECD 429
- Irritation: performed (day 1, 2 and 5)
- Systemic toxicity: performed, no signs have been observed
- Ear thickness measurements: performed
- Erythema scores:

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- Criteria used to consider a positive response: A test item is regarded as sensitizer in the LLNA if exposure to at least one concentration of the test item results in an incorporation of 3H-thymidine at least 3-fold or greater than that determined in control mice as indicated by the stimulation index (SI ≥ 3.0). However, the biological relevance of the obtained stimulation indices is considered in conjunction with the other assessed end points (i.e. lymph node cell counts, lymph node weights, ear weights). Hereby, the thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of ≥ 1.5 and ≥ 1.25, respectively.

TREATMENT PREPARATION AND ADMINISTRATION:
The study comprised four test groups. Each group consisted of 5 mice. Mice were randomized. Obvious signs of systemic toxicity and/or local inflammation at the application sites were noted for each animal. A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
- epicutaneous application
- 25 µl per ear
- dorsal surface of both ears
- Three consecutive applications (day 0 - day 2) to the same application site

Positive control substance(s):

other: not included in this study

Statistics:

3H-thymidine incorporation, cell count, lymph node weight and ear weight: WILCOXON-Test

Positive control results:

Studies using the positive control substance Alpha-Hexylcinnamaldehyde, techn. 85%, are performed twice a year in the laboratory to show that the test system is able to detect sensitizing compounds under the test conditions chosen (reliability check).

Parameter:

SI

Value:

1.21

Test group / Remarks:

5% in MEK

Parameter:

SI

Value:

1.88

Test group / Remarks:

10% in MEK

Parameter:

SI

Value:

3.88

Test group / Remarks:

25% in MEK

Parameter:

EC3

Value:

18.4

Test group / Remarks:

Threshold concentration >10%<25%

Remarks on result:

other: Value given in percentage

Cellular proliferation data / Observations:

CELL COUNT:
The 25% test-substance preparation induced a biologically relevant (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) and statistically significant response in the auricular lymph node cell counts. The SI of the 10% test-substance preparation lies below the border of biological relevance and was statistically significant.

LYMPH NODE WEIGHTS:
Statistically significant increases in lymph node weights were noted at the 25% and 10% concentration.

EAR WEIGHTS:
The test-substance concentrations did not cause increases (SI ≥ 1.25) in ear weights demonstrating the absence of excessive ear skin irritation. However, a statistically significant increase in ear weight was noted at the 25% concentration.

CLINICAL OBSERVATIONS:
No signs of systemic toxicity were noticed in all animals during general observation.

BODY WEIGHTS:
No relevant influence on the mean body weights was observed during the study.

LOCAL FINDINGS:
Slight swelling of the ear skin on study days 1 and 2 and slight scaling on study day 5 were observed in all animals of the 25% concentration. In addition, application of the 25% concentration caused ear skin lesions in 2 animals on study day 5.

Evaluation of results

In order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous application of several concentrations of the test substance to the skin of the dorsal surface of both ears is determined by measuring³H-thymidine incorporation into the lymph node cells. Additional parameters used to characterize the response are lymph node cell count and to a certain extent lymph node weight. Because irritation by the test substance may also induce lymph node responses the weights of ear punches taken from the area of test substance application are determined as an indicator for inflammatory ear swelling due to any irritant action of the test substance.

 

A test item is regarded as sensitizer in the LLNA if exposure to at least one concentration of the test item results in an incorporation of³H-thymidine at least 3-fold or greater than that determined in control mice as indicated by the stimulation index (SI ≥ 3.0). However, the biological relevance of the obtained stimulation indices is considered in conjunction with the other assessed end points (i.e. lymph node cell counts, lymph node weights, ear weights). Hereby, the thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of ≥ 1.5 and ≥ 1.25, respectively.

 

If applicable, the EC (estimated concentration) leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below and above the SI if possible or by using the two nearest points below or above the SI.

Table 1: Stimulation indices

Test group	Treatment	³H-thymidine incorporation Stimulation index^1	Cell count Stimulation index^1	Lymph node weight Stimulation index^1	Ear weight Stimulation index^1
1	Vehicle (MEK)	1.00	1.00	1.00	1.00
2	5% in MEK	1.21	1.05	1.10	1.02
3	10% in MEK	1.88 #	1.38 #	1.32 #	1.04
4	25% in MEK	3.88 ##	2.06 ##	1.98 ##	1.12 #

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

The statistical evaluations were performed using the WILCOXON-test (# for p ≤ 0.05, ## for p ≤ 0.01)

Interpretation of results:

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

Conclusions:

Thus, it is concluded that the test substance exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.
The threshold concentration for sensitization induction was >10% <25%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 18.4% and 12.6%, respectively.

Executive summary:

The skin sensitizing potential of the test substance was assessed using the radioactive Murine 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 CBA/CaOlaHsd mice each were treated with 5%, 10% and 25% (w/w) preparations of the test substance in MEK (methyl ethyl ketone) 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, applied to the dorsal surfaces of both ears on three consecutive days. Three days after the last application, 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). Cell counts, and weights of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter samplewas 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.

No signs of systemic toxicity were noticed in all animals during general observation. When applied as 25% preparation in MEK, the test substance induced a biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3) and statistically significant increase of ³H-thymidine incorporation into the cells from the auricular lymph nodes. The increase of the 10% test-substance preparation was statistically significant. Concomitantly, the 25% test-substance preparation induced a biologically relevant (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) and statistically significant response in the auricular lymph node cell counts. The SI of the 10% test-substance preparation lies below the border of biological relevance and was statistically significant. In addition, statistically significant increases in lymph node weights were noted at the 25% and 10% concentration. The test-substance concentrations did not cause increases (SI ≥ 1.25) in ear weights demonstrating the absence of excessive ear skin irritation. However, slight swelling of the ear skin on study days 1 and 2 and slight scaling on study day 5 were observed in all animals of the 25% concentration. In addition, application of the 25% concentration caused ear skin lesions in 2 animals on study day 5. Moreover, a statistically significant increase in ear weight was noted at this concentration. Nevertheless, the concentration of 25% has to be considered to indicate a skin sensitization potential because of lymph node responses exceeding the respective cutoff values in the absence of sufficiently strong ear skin irritation. The threshold concentration for sensitization induction was >10% <25%. The EC 3 (estimated concentration that leads to the SI of 3.0) for ³H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 18.4% and 12.6%, respectively. Thus, it was concluded that the test substance exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitizing potential of the test substance was assessed using the radioactive Murine Local Lymph Node Assay.

Groups of 5 female CBA/CaOlaHsd mice each were treated with 5%, 10% and 25% (w/w) preparations of the test substance in MEK (methyl ethyl ketone) 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, applied to the dorsal surfaces of both ears on three consecutive days. Each test animal was treated with 25 µL per ear of the appropriate test-substance preparation or the vehicle alone, applied to the dorsal surfaces of both ears on three consecutive days. Three days after the last application, 20 µCi³H-thymidine in 250 µL sterile saline were injected into the tail vein of the mice. About 5 hours after the³H-thymidine injection, the mice were sacrificed, and the auricular lymph nodes were removed. Lymph node response was evaluated measuring³H-thymidine incorporation (indicator of cell proliferation). Cell counts, and weights of each animal’s pooled lymph nodes were also determined. In addition,a 0.8cm diameter samplewas 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.

When applied as 25% preparation in MEK, the test substance induced a biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3) and statistically significant increase of³H-thymidine incorporation into the cells from the auricular lymph nodes. The increase of the 10% test-substance preparation was statistically significant.

Concomitantly, the 25% test-substance preparation induced a biologically relevant (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) and statistically significant response in the auricular lymph node cell counts. The SI of the 10% test-substance preparation lies below the border of biological relevance and was statistically significant.

In addition, statistically significant increases in lymph node weights were noted at the 25% and 10% concentration.

The test-substance concentrations did not cause increases (SI ≥ 1.25) in ear weights demonstrating the absence of excessive ear skin irritation. However, slight swelling of the ear skin on study days 1 and 2 and slight scaling on study day 5 were observed in all animals of the 25% concentration. In addition, application of the 25% concentration caused ear skin lesions in 2 animals on study day 5. Moreover, a statistically significant increase in ear weight was noted at this concentration.

Nevertheless, the concentration of 25% has to be considered to indicate a skin sensitization potential beause of lymph node responses exceeding the respective cutoff values in the absence of sufficiently strong ear skin irritation.

 

 

Thus, it is concluded that the test substance exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay underthe test conditions chosen. The threshold concentration for sensitization induction was >10% <25%. The EC 3 (estimated concentration that leads to the SI of 3.0) for³H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 18.4% and 12.6%, respectively (BASF SE, 2019).

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) 1272/2008. As a result, the substance is considered to be classified for skin sensitization category 1B according to Regulation (EC) No. 1272/2008.