Ethanamine, N-ethyl-, reaction products with polyethylene-polypropylene glycol ether with trimethylolpropane (3:1) acrylate (>1 <6.5 mol EO and >1 < 6.5 mol PO)

Endpoint summary

Description of key information

LLNA: skin sensitiser, EC3 = 0.8% (BASF SE, 2018)

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:

28-04-2107 - 27-03-2018

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)

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

The analyses of the test item were carried out at the Competence Center Analytics, BASF SE, Ludwigshafen, Germany.


Name of test substance: Laromer LR 8889
Test substance No.: 16/0422-1
Batch identification: 160005P040
CAS No.: 173046-61-2
Purity: 98.2 area-% (HPLC, 201 nm)
98.4 area-% (HPLC, 231 nm)
(for details see Study code: 16L00510)
Content: > 99 g/100 g UVCB (unknown or variable composition, complex reaction products or biological materials; for details see Study code: 16L00510)
Identity: Confirmed (for details see Study code: 16L00510)
Homogeneity: The test substance was homogeneous by visual inspection.
Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility. The test facility is organizationally independent from the BASF SE sponsor division.
Physical state / color: Liquid / yellowish, clear
Storage conditions: Room temperature
Test-substance preparation: The test-substance preparation was produced on a weight per weight basis shortly before application. After stirring with a magnetic stirrer, the test substance was soluble in the vehicle.
Vehicle: MEK (methyl ethyl ketone)
Reason for the vehicle: MEK was used as vehicle because good solubility of the preparation was achieved.
Form of application: Solution


Stability of the test- substance preparation: Due to lack of a suitable method appropriate for low concentrations of the test subtstance the stability of the test substance mixture with the vehicle could not be confirmed. However, it was concluded that a good recovery rate could be achieved at the highest concentration (10%), while due to the limitations of the analytical methods, no conclusions concerning the recovery rates of the lower concentrations (0.5%, 2%) could be drawn (for details see analytical report Study code 17L00259).
Homogeneity of the test- substance preparation: The test-substance preparation was visually homogeneous (solution).
Concentration control analysis of the test- substance preparation: Due to lack of a suitable method appropriate for low concentrations of the test subtstance the correctness of the test substance concentrations of the test substance mixture with the vehicle could not be confirmed.
However, it was concluded that a good recovery rate could be achieved at the highest concentration (10%), while due to the limitations of the analytical methods, no conclusions concerning the recovery rates of the lower concentrations (0.5%, 2%) could be drawn (for details see analytical report Study code 17L00259).

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V.,Inc., Postbus 6174 5960 AD Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 8 weeks (pretest and main test)
- Weight at study initiation: 17.3 g – 19.3 g (pretest); 17.4 g – 20.6 g (main test)
- Housing: Polycarbonate cages type MII with mesh wire tops supplied by BECKER & Co., Castrop-Rauxel, Germany
- Diet (e.g. ad libitum): Kliba mouse/rat maintenance diet “GLP” supplied by Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum.
- Water (e.g. ad libitum): Drinking water ad libitum
- Acclimation period: at least 5 days

As group housing may increase oral exposure due to grooming of the animals and may interfere with clear observations of each individual animal, animals were single housed for the duration of the test.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20–24°C
- Humidity (%): 30 – 70%.
- Photoperiod (hrs dark / hrs light): 12 hours light from 06:00 h to 18:00 h; 12 hours darkness from 18:00 h to 06:00 h
- IN-LIFE DATES: From: 25-04-2017 To: 08-05-2017

Vehicle:

methyl ethyl ketone

Remarks:

MEK was used as vehicle because good solubility of the preparation was achieved.

Concentration:

Control group 1 Vehicle
Test group 2 0.5% in MEK
Test group 3 2% in MEK
Test group 4 10% in MEK

No. of animals per dose:

5

Details on study design:

A solubility experiment was performed according to the recommendations given by OECD 429. The highest test-substance concentration which was used in the pretest was a 50% test- substance preparation.

In order to determine the highest test-substance concentration that does not induce local signs of skin irritation and/or systemic toxicity, a pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed. Two mice were treated with test substance concentrations of 10% and 50% each on three consecutive days.
In the pretest, clinical signs were recorded after each application as well as on day 5. Signs of local irritation were recorded on day 1, 2 and 5. Prior to the first application of the test item (day 0), on day 2 and before sacrifice (day 5) the ear thickness was determined using a micrometer. Furthermore, the ears were punched after sacrifice at the apical area using a biopsy punch (Ø 0.8 cm) and were immediately pooled per animal and weighed using an analytical balance. Additionally, the weight of the pooled lymph nodes from both sides was determined for each animal.

No relevant signs of systemic toxicity were observed in the pretest. After application of the 50% concentration, the animals showed severe (>25%) increases in ear weights (compared to current vehicle values) and increases in ear thickness as indication of excessive ear skin irritation.
After application of the 10% test-substance concentration no relevant increases in ear weights (compared to current vehicle values) was noted. However, an increase (>25%) in ear thickness was noted in one animal on day 5.
Both concentrations caused swelling as well as scaling and erythema at the application sites during the study period. Additionally, test-substance residues (in the 50% concentration accompanied by erythema) in the head shoulder region were observed.

Therefore, the following dose levels were selected for the main study: 0.5%, 2% and 10%.

Positive control substance(s):

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

Statistics:

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 measuring 3H-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 of 3H-thymidine at least 3-fold or greater than that recorded 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.

Positive control results:

The sensitivity of mice (CBA/CaOlaHsd, Envigo RMS B.V.,Inc., Postbus 6174, 5960 AD Horst, The Netherlands) and the reliability of experimental techniques were assessed by regularly using a known sensitizer as recommended by the test guidelines.


Periodic positive control data (Vehicle: MEK)





Project No. Date of performance Concentrations Stimulation Index Stimulation Index EC 3.0 EC 1.5
3H-thymidine Cell counts
incorporation
58V0288/98A008 Jun 2016 2.60, 7.08, 14.77 1.50, 2.21, 3.01 1.4 1
58V0288/98A009 Jan 2017 1.56, 3.20, 9.16 1.22, 2.0, 3.42 4.5 2.4
58V0165/16A074 Jul 2016
58V0174/16A060 Jul 2016 7.99 2.24
58V0010/16A057 Sep 2016 10.97 2.28

Key result

Parameter:

EC3

Value:

0.8

Key result

Parameter:

SI

Value:

1.68

Test group / Remarks:

0.5%

Key result

Parameter:

SI

Value:

8.86

Test group / Remarks:

2%

Key result

Parameter:

SI

Value:

15.55

Test group / Remarks:

10%

Cellular proliferation data / Observations:

3H-thymidine incorporation, cell count and lymph node weight

When applied as 10% or 2% preparation in MEK, the test substance induced a biologically relevant (increase above the cutoff Stimulation Index of 3), statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes.
Concomitantly, the 10% and 2% test-substance preparation induced a biologically relevant, statistically significant and concentration-dependent response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts.
The SI of the 0.5% test-substance preparation lies at the border of biological relevance and was statistically significant.
In addition, statistically significant and concentration-dependent increases in lymph node weights were noted at all concentrations.

Table 1: Stimulation indices

Test Group Treatment ³H-thymidine incorporation Cell Count Stimulation Lymph Node Weight Ear Weight Stimulation
Stimulation Index Index Stimulation Index Index
1 vehicle MEK 1.00 1.00 1.00 1.00
2 0.5% in MEK 1.68 1.49## 1.38## 0.98
3 2% in MEK 8.86## 2.71## 2.29## 1.03
4 10% in MEK 15.55## 3.63## 3.38## 1.33##

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

Ear weights
The 2% and 0.5% test-substance concentration did not cause increases (SI ≥ 1.25) in ear weights demonstrating the absence of relevant ear skin irritation.
A statistically significant increase (SI ≥ 1.25) in ear weights was noted at the 10% concentration.

Body weights
The expected body weight gain was generally observed during the study.

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

Local findings
Slight swelling on study day 2 and 5 and very slight erythema on study day 2 were noted in all animals of the 10% concentration group.

Interpretation of results:

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

Conclusions:

It is concluded that Laromer LR 8889 exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen

The threshold concentration for sensitization induction was >0.5% <2%. 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 0.8% and 0.5%, respectively.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitizing potential of Laromer LR8889 was assessed using the radioactive Murine Local Lymph Node Assay according to OECD guideline 429. Groups of 5 female CBA/CaOlaHsd mice each were treated on the dorsal skin of the ears with 0.5%, 2% and 10% (w/w) preparations of the test substance in methylethylketone (MEK),or with the vehicle alone.The high concentration was selected based on the presence of ear irritation in a pretest at 50%. Lymphnode 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 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, cell count, lymph node weight and ear weight are summarized for each test group in the table below.

Table 1: Stimulation indices

 

Test Group	Treatment	³H-thymidine incorporation Stimulation Index		Cell Count Stimulation Index		Lymph Node Weight Stimulation Index		Ear Weight Stimulation Index
1	vehicle MEK	1.00		1.00		1.00		1.00
2	0.5% in MEK	1.68		1.49	##	1.38	##	0.98
3	2% in MEK	8.86	##	2.71	##	2.29	##	1.03
4	10% in MEK	15.55	##	3.63	##	3.38	##	1.33	##

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

 

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

When applied as 10% or 2% preparation in MEK, the test substance induced a biologically relevant (increase above the cutoff Stimulation Index of 3), statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. Concomitantly, the 10% and 2% test-substance preparation induced a biologically relevant, statistically significant and concentration-dependent response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. The SI of the 0.5% test-substance preparation lies at the border of biological relevance and was statistically significant. In addition, statistically significant and concentration-dependent increases in lymph node weights were noted at all tested concentrations. The 2% and 0.5% test-substance concentration did not cause increases (SI ≥ 1.25) in ear weights demonstrating the absence of relevant ear skin irritation.However, slight swelling on study day 2 and 5 and very slight erythema on study day 2 were noted in all animals of the 10% concentration group. Moreover, a statistically significant increase (SI ≥ 1.25) in ear weights was noted at this concentration.

Thus, the threshold for sensitization induction was >0.5% <2%. The EC 3 for3H-thymidine incorporation and the EC 1.5 for cell count was calculated by linear regression from the results of these concentrations to be 0.8% and 0.5%, respectively.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the skin sensitization testing, the test item is classified as skin sensitization cat. 1A (H317) according to Regulation (EC) No 1272/2008 (CLP).