N-benzylethylenediamine

Endpoint summary

Description of key information

It is concluded that the test substance exhibits a skin sensitizing potential in the LLNA (OECD 429; GLP)

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:

Nov 2018

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:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: R1640 Fr.16/199/17-18
- Expiration date of the lot/batch: 01 Mar 2020
- Purity: 98.8 corrected area %


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions: The stability of the test substance under storage conditions over the study period is guaranteed by the sponsor, and the sponsor holds this responsibility
- Solubility and stability of the test substance in the solvent/vehicle:
The test-substance preparations at different concentrations were solutions in propylene glycol (PG).
Physical state / color: Liquid / colorless, clear
Storage conditions: Room temperature


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 dissolved in the vehicle.

FORM AS APPLIED IN THE TEST
The test-substance preparations at different concentrations were solutions in propylene glycol (PG).

Species:

mouse

Strain:

other:

Remarks:

CBA/CAOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS GmbH, NL-5800 AN Venray
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 17.8 g – 20.9 g (pretest)
17.7 g – 21.4 g (main test)
- Housing: single housed
- Diet. ad libitum
- Water. ad libitum
- Acclimation period: at least 5 days before the first test substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24°C
- Humidity (%): 45 – 65%.
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

propylene glycol

Concentration:

1%, 2.5% and 5%

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The test-substance preparations at different concentrations were solutions in propylene glycol (PG).

MAIN STUDY
Form of application: Epicutaneous application is simulating dermal contact with the compound, which can occur under practical use conditions.
Application volume: 25 μL per ear
Site of application: Dorsal surface of both ears
Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
Body weight determination: Individual body weights on day 0 prior to the first application andon day 5 prior to the sacrifice of the animals.
Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
On study day five (about 66 to 72 hours after the last application of test substance to the ears), 20 μCi 3H-thymidine* in 250 μL sterile saline were injected into the tail vein of the mice.
The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation under Isoflurane anesthesia.
Determination of ear weight: Immediately after the death of each animal, a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal. These measurements serve for detecting a potential inflammatory ear swelling.
Removal and weight determination of the lymph nodes:
Immediately after removal of the ear punches, the left and right auricular lymph nodes were dissected. The weight of the pooled
lymph nodes from both sides was determined for each animal.
Preparation of cellsuspension and determination of cell count:
After weight determination, the pooled lymph nodes of each animal were stored in phosphate-buffered saline (PBS) in an ice
water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully
passing the lymph nodes through an iron mesh (mesh size 200 μm) into 8 mL phosphate-buffered physiological saline. For
determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500 or 1:1000 (Test
group 4). The cell count was determined using a Casy® Counter.
Measurement of3H-thymidine incorporation of the lymph node cells:
The remaining cell suspensions were washed twice with PBS and precipitated with 5% trichloro-acetic acid (TCA). Each
precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a β-scintillation
counter.
Evaluation of results:
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.

Positive control substance(s):

other: A concurrent positive control (reliability check) with a known sensitizer was not included in this study.

Statistics:

For the statistical analysis of the parameters: 3H-thymidine incorporation, cell count, lymph node weight and ear weight, Wilcoxon-Test was used

Parameter:

SI

Value:

2.57

Test group / Remarks:

1% preparation of test substance

Parameter:

SI

Value:

16.99

Test group / Remarks:

2.5% preparation of test substance

Parameter:

SI

Value:

46.34

Test group / Remarks:

5 % preparation of test substance

Parameter:

EC3

Value:

1.05

Remarks on result:

other:

Remarks:

given in percent

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
When applied as 5% and 2.5% test-substance preparation in propylene glycol, the test substance induced a biologically relevant (increase to 3-fold or above of control value =
stimulation index (SI) ≥ 3), statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. The increase of the
1% test-substance preparation was statistically significant but failed to reach the cut-off.

DETAILS ON STIMULATION INDEX CALCULATION
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.

EC3 CALCULATION
The threshold concentration for sensitization induction was around 1%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation was calculated by linear regression from the results of the 1% and 2.5% concentrations to be 1.05%. The EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by semilogarithmical regression from the results of the 1% and 2.5% concentrations to be 0.95%.

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

BODY WEIGHTS
Slightly reduced mean body weight was noted in test group 3 (2.5% concentration) during the
study. No influence on the mean body weights was observed in all other grou

Pretest / Irritation Screening

No signs of systemic toxicity were observed in the pretest.

After application of the 10% concentration, the animals showed considerable increases (>25%) in ear weights (compared to historical vehicle values) and increases (≥ 25%) in ear thickness as indication of excessive ear skin irritation.

At the concentration of 5%, the animals showed some increases in ear weights (compared to historical vehicle values) and ear thickness as indication of slight ear skin irritation.

At the 1% concentration, no signs of local irritation were observed.

Interpretation of results:

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

Conclusions:

It is concluded that N-benzylethylenediamine exhibits a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The skin sensitizing potential of N-benzylethylenediamine 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 CBA/CaOlaHsd mice each were treated with 1%, 2.5% and 5% (w/w)

preparations of the test substance in propylene glycol (PG) 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

by measuring 3H-thymidine incorporation (indicator of cell proliferation). 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.

The mean stimulation indices (expressed as multiples of the vehicle control) for 3H-thymidine incorporation, cell count, lymph node weight and ear weight are summarized for each test group in the table below.

Test group	Treatment	3H-thymidine incorporation SI²	Cell count SI²	Lymph Node Weight SI²	Ear Weight SI²
1	vehicle propylene glycol	1.00	1.00	1.00	1.00
2	1% in  propylene glycol	2.57**	1.58**	1.51*	1.04
3	2.5% in  propylene glycol	16.99**	3.06**	2.09**	1.03
4	5% in  propylene glycol	46.34**	5.58**	3.90**	1.21**

SI = Stimulation index; ² = test group x / test group 1 (vehicle control); * for p≤ 0.05,** for p≤ 0.01

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

When applied as 5% and 2.5% test-substance preparation in propylene glycol, the test substance induced a biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3), statistically significant and concentration-dependent increase of

3H-thymidine incorporation into the cells from the auricular lymph nodes. The increase of the 1% test-substance preparation was statistically significant but failed to reach the cut-off.

Concomitantly, the 5% and 2.5% test-substance preparation induced a biologically relevant and statistically significant response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. The increase induced by the 1% testsubstance preparation lies just above the border of biological relevance and was statistically significant.

In addition, statistically significant increases in lymph node weights were noted at all tested concentrations.

The test-substance concentrations did not indicate increases in ear weights above 25% compared to vehicle control (SI ≥ 1.25), demonstrating the absence of excessive ear skin irritation. However, the highest concentration (5%) caused statistically significant and

considerably increased ear weights (mean SI 1.21) at the border of this cutoff value.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitizing potantial of N-benzylethylenediamine was assessed in a stepwise approach:

1st LLNA

The skin sensitizing potential of N-benzylethylenediamine was assessed using the radioactive Local Lymph Node Assay (BASF SE, 2019). 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 1%, 2.5% and 5% (w/w) preparations of the test substance in propylene glycol (PG) or with the vehicle alone. No signs of systemic toxicity were noticed in all animals during general observation.

When applied as 5% and 2.5% test-substance preparation in propylene glycol, the test substance induced a biologically relevant (increase to 3-fold or above of control value = stimulation index (SI) ≥ 3), statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. The increase of the 1% test-substance preparation was statistically significant but failed to reach the cut-off. Concomitantly, the 5% and 2.5% test-substance preparation induced a biologically relevant and statistically significant response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. The increase induced by the 1% testsubstance preparation lies just above the border of biological relevance and was statistically significant. In addition, statistically significant increases in lymph node weights were noted at all tested concentrations. The test-substance concentrations did not indicate increases in ear weights above 25% compared to vehicle control (SI ≥ 1.25), demonstrating the absence of excessive ear skin irritation. However, the highest concentration (5%) caused statistically significant and considerably increased ear weights (mean SI 1.21) at the border of this cutoff value.

Thus, it is concluded that N-benzylethylenediamine exhibits a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen. The threshold concentration for sensitization induction was around 1%.

DPRA

The test substance was incubated with synthetic peptides for 24 hours at 25 ± 2.5 °C and the remaining non-depleted peptide concentration was determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm (BASF SE, 2019). The peptides are custom-made material containing phenylalanine to facilitate UV-detection and either cysteine or lysine as the reactive center. Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model, it was concluded that N-benzylethylenediamine shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

2nd LLNA

In a second LLNA the skin sensitizing potential of the registered substance was reevaluated using basically the same set up as in the first LLNA.

The skin sensitizing potential of N-benzylethylenediamine was assessed using the radioactive Local Lymph Node Assay (BASF SE; 2019). 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 1%, 2.5% and 5% (w/w) concentrations of the test substance in propylene glycol (PG) or with the vehicle alone. The highest concentration was selected based on the presence of ear skin irritation in a pretest using a 10% test-substance concentration.

No signs of systemic toxicity were noticed in all animals during general observation. However, one animal of test group 3 (2.5% concentration) died spontaneously before 2nd induction (1 day after beginning of application). When applied as 2.5% and 5% concentration in propylene glycol, 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. Concomitantly, the 2.5% and 5% test-substance concentrations induced a biologically relevant and statistically significant response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. In addition, statistically significant increases in lymph node weights were noted at the 2.5% and 5% concentrations. No relevant lymph node response was caused by the 1% concentration. The 5% concentration caused statistically significant and considerably increased ear weights (increase to stimulation index SI ≥ 1.25), indicating excessive ear skin irritation. The 1% and 2.5% test-substance concentrations did not cause any increase in ear weights demonstrating the absence of relevant ear skin irritation.

Thus, it is concluded that N-benzylethylenediamine exhibits a skin sensitizing potential in the Local Lymph Node Assay under the test conditions chosen. The threshold concentration for sensitization induction was >1% <2.5%. The EC 3 for 3H-thymidine incorporation and the EC 1.5 for cell count was calculated by linear regression from the results of these concentrations to be 1.2% and 1.4%, respectively.

discussion

The skin sensitizing potantial of N-benzylethylenediamine was assessed in two LLNA, since the test substance in the first LLNA was shown to be instable under formation of an impurity (see also genetic toxicity). The reanalysis of the substance batch used in this test after study conduction showed a decrease of the purity from > 99% to 93.754% under unexpected formation of an impurity. It has to be considered that N-benzylethylenediamine could undergo degradation to benzaldehyde and ethylene diamine. Benzaldehyde can then easily react with N-benzylethylenediamine under formation of a dibenzylated mono-imine. Further analytical investigation regarding the structure of the impurity confirmed that postulation. It is well kown, that imines are capable to react with proteins under induction of allergic skin reactions. Therefore, it could not be excluded that the test substance instability or the impurity is the cause of the positive response. For this reason, the substance was redistilled and puified to a content of > 99% again. To prove a lack of protein reactivity, a DPRA was conducted before the second LLNA. Only in case the redistilled test substance does not show direct protein reactivity anymore, a repetition of the first LLNA would have been possible. Based on the observed results of the DPRA, it was concluded that N-benzylethylenediamine shows minimal or no chemical reactivity under the test conditions chosen. For that reason, a second LLNA was ordered and performed in the same way than the first LLNA. However, the second LLNA reproduced the results of the first assay. In conclusion, N-benzylethylenediamine is considered to cause skin sensitizing effects.

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. As a result the substance is considered to be classified for skin sensitization GHS, category 1A, H317 under Regulation (EC) No. 1272/2008.