Reaction product of propylidynetrimethanol, propoxylated, reaction products with ammonia and 2,2-Dimethyl-3-(4-morpholinyl)propanal

Endpoint summary

Description of key information

Under the conditions of the present Local Lymph Node Assay, the substance tested at concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v) as formulations in a suitable vehicle (DMF) was shown to have skin sensitization potential.

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:

2013-03-06 to 2013-03-14

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:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 16.6 - 20.9 g
- Housing: Grouped caging (5 animals/cage)
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 – 70 %
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 2013-03-06 To: 2013-03-14

Vehicle:

dimethylformamide

Concentration:

25 % (w/v)
10 % (w/v)
5 % (w/v)
2.5 % (w/v)

No. of animals per dose:

5 animals per dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:
The test item was adequately miscible with all evaluated vehicles (Acetone: Olive oil 4:1 mixture (v/v) (AOO), Dimethylformamide (DMF), Methyl ethyl ketone (MEK), Dimethyl sulfoxide (DMSO)). Since N,N-Dimethylformamide (DMF) is one of the most preferred one by the relevant guidelines this vehicle was selected to be used for the test item formulations in the main test.
- Mortality:
Mortality was observed in all test groups on Day 3 before the treatment (2/2, 2/2 and 1/2 animals in the 100 %, 75 % and 50 % (w/v) dose groups, respectively).
No mortality was observed during the second preliminary test (test item concentrations: 25 %, 10%, 5 and 2.5 % (w/v). No significant, treatment related effect on the body weights was observed in any treatment groups. No signs of systemic toxicity were observed in any treatment group.
- Irritation: No significant sign of local irritation (indicated by an erythema score ≥ 3) was observed in any treatment group.
- Lymph node proliferation response: No significant sign of local irritation (indicated by an increase of ear thickness ≥ 25 %) were observed at the treatment site (ears) at the tested concentrations of the second preliminary test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Individual Approach of Local Lymph Node Assay
- Criteria used to consider a positive response:
Exposure to at least one concentration (non-irritating, non-toxic) of the test item which resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and PC responses may also be used when determining whether a borderline result is declared positive.

TREATMENT PREPARATION AND ADMINISTRATION:
In vivo Treatment
Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, of the positive control substance (positive control group) or of the vehicles (AOO or DMF as negative control groups). The formulations were applied, with a pipette, on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Positive control substance(s):

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

Statistics:

Statistical analysis was performed by SPSS/PC+ (4.0.1) software package.
The measured DPM values corrected with the mean background value were used for statistical analysis of the proliferation data. The heterogeneity of variance between the groups treated with the test item and the relevant vehicle control (DMF) was checked by Bartlett's test. Since significant heterogeneity was detected, the normal distribution of data was examined by Kolmogorow-Smirnow test followed by the non-parametric method of Kruskal-Wallis One-Way analysis of variance. As a results of this analysis the inter-group comparisons was performed using Mann-Whitney U-test to asses the significance of inter-group differences. Significance of the positive control response was evaluated by T-test versus the relevant vehicle control (AOO).

Positive control results:

The positive control group animals were treated with 25 % (w/v) HCA solution (formulated in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.
The positive control substance induced the appropriate, statistically significant stimulation compared to the control (SI = 17.06; p < 0.01, T-test versus AOO control). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed sensitivity and validity of the assay.

Parameter:

SI

Value:

0.95

Test group / Remarks:

concentration of 2.5 %

Parameter:

SI

Value:

4.88

Test group / Remarks:

concentration of 10 %

Parameter:

SI

Value:

18.78

Test group / Remarks:

concentration of 25 %

Key result

Parameter:

EC3

Value:

6.8

Test group / Remarks:

25% (w/v): 18.78, 10% (w/v): 4.88, 5% (w/v): 1.95 2.5% (w/v): 0.95 %. The calculated EC3 value was 6.8 %.

Body Weight Measurement

No significant, treatment related effect on the body weights was observed in any treatment group.

 

Clinical Observations and Signs of Irritation

No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of significant irritation (indicated by an erythema score ≥ 3) or any other local effect was observed in any treatment groups.

 

Proliferation Assay

The lymph nodes in the positive control group were larger than the relevant vehicle control (AOO). Larger lymph nodes than the relevant vehicle control (DMF) was observed in the 25 % and the 10 % (w/v) test item treated groups. Visual appearance of the lymph nodes was normal in the negative (vehicle) control groups (both AOO and DMF) and in the 5 % and 2.5 % (w/v) test item treated groups.

Significantly increased lymphoproliferation (SI ≥ 3) was observed for the test item at concentrations of 25 % and 10 % (w/v). No significantly increased lymphoproliferation was observed at concentrations of 5 % and 2.5 % (w/v). The corresponding stimulation index values were 18.78, 4.88, 1.95 and 0.95 at treatment concentrations of 25 %, 10 %, 5% or 2.5 % (w/v), respectively. Significance of the dose-response was evaluated by linear regression using the SI values. Statistically significant dose-related response was observed (p = 0.006, r = 0.99).

 

Mean DPM and SD
25 % (w/v): 18841.0 ± 4113.0
10 % (w/v): 4896.8 ± 947.3
5 % (w/v): 1959.0 ± 867.9
2.5 % (w/v): 956.6 ± 53.6

Vehicle control (DMF): 1003.4 ± 1270.0
Vehicle control (AOO): 491.4 ± 465.3
Positive control (25 % w/v HCA): 8385.0 ± 33

 

Statistical Analysis

Individual DPM values (corrected with the mean background value) were statistically evaluated. Statistically significant increase of the proliferation value was observed in the positive control group (p < 0.01, T-test versus AOO control). Statistically significant increase of the proliferation values compared to the relevant vehicle control (DMF) was observed in the 25 % and the 10 % (w/v) test item treated groups (p < 0.01; Mann-Whitney U-test versus DMF control). No statistically significant increase of the proliferation values compared to the relevant vehicle control (DMF) was observed in the 5 % and the 2.5 % (w/v) test item treated groups (Mann-Whitney U-test versus DMF control).

 

Interpretation of Observations

Selection of test item concentrations based on the results of a formulation evaluation and also results of two preliminary irritation/toxicity tests according to the relevant guidelines. The test item was a liquid hence miscible with an appropriate vehicle was evaluated. The test item was adequately miscible with all evaluated vehicles. Since N,N-Dimethylformamide (DMF) is one of the most preferred one by the relevant guidelines this vehicle was selected for the test item formulations in the main test.

Due to mortality and significant systemic toxicity observed in the first preliminary irritation/toxicity test sensitization potential of the test item SIKA Hardener MTJ was examined as 25 % (as the maximum applicable concentration) and as 10 %, 5 % and 2.5 % (w/v) formulations in the selected vehicle (DMF) in the LLNA.

Since the test was valid and no sign of systemic toxicity or excessive irritation was observed, the proliferation values obtained are considered to reflect the real potential of the test item to cause/not cause lymphoproliferation in the Local Lymph Node Assay.

Visually larger lymph nodes than the relevant control (AOO or DMF) was observed in the positive control, the 25 % and the 10 % (w/v) test item treated groups.

Significantly increased lymphoproliferation (≥ 3) was observed for the test item at concentrations of 25 % and 10 % (w/v). The observed SI values were 18.78, 4.88, 1.95 and 0.95 at concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. The observed proliferation values at concentrations of 25 % and 10 % (w/v) statistically significantly differed from the relevant vehicle (DMF) control (p < 0.01; Mann-Whitney U-test). Significant dose-related response was observed (linear regression, p = 0.006, r = 0.99).

According to evaluation criteria of the relevant guidelines, the significantly increased proliferation values observed at concentrations of 25 % and 10 % (w/v) and the strong dose-response relationship are considered evidence that SIKA Hardener MTJ is a skin sensitizer.

The EC3 (dose calculated to induce a stimulation index of 3) was calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve based on published method. The calculated EC3 value of SIKA Hardener MTJ was 6.8 % in this LLNA. Using the calculated EC3 values SIKA Hardener MTJ can be ranked among moderate skin sensitizers in this LLNA according to the published data for classification of contact allergens.

Interpretation of results:

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

Conclusions:

Under the conditions of the present Local Lymph Node Assay, SIKA Hardener MTJ tested at concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v) as formulations in a suitable vehicle (DMF) was shown to have skin sensitization potential. Based on the EC3 value SIKA Hardener MTJ was considered a moderate skin sensitizer.

Executive summary:

The aim of this study was to determine the skin sensitization potential of SIKA Hardener MTJ in the Local Lymph Node Assay (LLNA). Individual approach was used in this test.

The maximum dose selection was performed according to the relevant guidelines and based on results of the formulation evaluation and two preliminary irritation / toxicity tests. Due to mortality and significant systemic toxicity observed in the first preliminary test at concentrations of 100 % (the undiluted test item), 75 % and 50 % (w/v) the test item was examined in the main test at concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v) as formulations in N,N-Dimethylformamide (DMF).

In the main test, 35 female CBA/Ca mice were allocated to seven groups of five animals each:

- four groups received SIKA Hardener MTJ at 25 %, 10 %, 5 % or 2.5 % (w/v) in DMF,

- one control group (used as negative control for the test item treated groups) received the vehicle of the test item (DMF) only,

- one control group (used as negative control for the positive control substance treated group) received the vehicle of the positive control substance (AOO) only,

- the positive control group received 25 % (w/v) α-Hexylcinnamaldehyde (HCA) in AOO.

Each substance was applied on the external surface of each ear (25 μL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6 animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR), than sacrificed approximately 5 hours after the injection. Auricular lymph nodes were removed and processed. The cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the obtained values were used to calculate stimulation indices (SI).

The positive control substance (25 % (w/v) HCA in AOO) induced the appropriate, statistically significant stimulation compared to the relevant control (SI = 17.06; p < 0.01, T-test versus the relevant vehicle (AOO) control), thus confirming the validity of the assay.

No mortality, significant, treatment related effect on the body weights or any other sign of systemic toxicity was observed in any treatment group. No sign of significant irritation (indicated by an erythema score ≥ 3) or any other local effect was observed in any treatment group.

Visually larger lymph nodes than the relevant vehicle control (AOO or DMF) was observed in the positive control, the 25 % and the 10 % (w/v) test item treated groups.

Significantly increased lymphoproliferation (≥ 3) was observed for the test item at concentrations of 25 % and 10 % (w/v). No significantly increased lymphoproliferation was observed at concentrations of 5 % and 2.5 % (w/v). The observed SI values were 18.78, 4.88, 1.95 and 0.95 at concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. The observed proliferation values at concentrations of 25 % and 10 % (w/v) statistically significantly differed from the relevant vehicle (DMF) control (p < 0.01; Mann-Whitney U-test). Significant dose-related response was observed (linear regression, p = 0.006, r = 0.99).

Since the test was valid and no sign of systemic toxicity or significant irritation was observed, the proliferation values obtained are considered to reflect the real potential of the test item to cause/not cause lymphoproliferation in the Local Lymph Node Assay.

According to evaluation criteria of the relevant guidelines, the significantly increased proliferation values observed at concentrations of 25% and 10 % (w/v) and the strong dose-response relationship are considered evidence that SIKA Hardener MTJ is a skin sensitizer.

The EC3 (dose calculated to induce a stimulation index of 3) was calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve based on published method. The calculated EC3 value of SIKA Hardener MTJ was 6.8 % in this LLNA. Using the calculated EC3 values SIKA Hardener MTJ can be ranked among moderate skin sensitizers in this LLNA according to the published data for classification of contact allergens.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The aim of the study was to determine the skin sensitization potential of SIKA Hardener MTJ in the Local Lymph Node Assay (LLNA). Individual approach was used in this test.

The maximum dose selection was performed according to the relevant guidelines and based on results of the formulation evaluation and two preliminary irritation / toxicity tests. Due to mortality and significant systemic toxicity observed in the first preliminary test at concentrations of 100 % (the undiluted test item), 75 % and 50 % (w/v) the test item was examined in the main test at concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v) as formulations in N,N-Dimethylformamide (DMF).

In the main test, 35 female CBA/Ca mice were allocated to seven groups of five animals each:

- four groups received SIKA Hardener MTJ at 25 %, 10 %, 5 % or 2.5 % (w/v) in DMF,

- one control group (used as negative control for the test item treated groups) received the vehicle of the test item (DMF) only,

- one control group (used as negative control for the positive control substance treated group) received the vehicle of the positive control substance (AOO) only,

- the positive control group received 25 % (w/v) α-Hexylcinnamaldehyde (HCA) in AOO.

Each substance was applied on the external surface of each ear (25 μL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6 animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR), than sacrificed approximately 5 hours after the injection. Auricular lymph nodes were removed and processed. The cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the obtained values were used to calculate stimulation indices (SI).

The positive control substance (25 % (w/v) HCA in AOO) induced the appropriate, statistically significant stimulation compared to the relevant control (SI = 17.06; p < 0.01, T-test versus the relevant vehicle (AOO) control), thus confirming the validity of the assay.

No mortality, significant, treatment related effect on the body weights or any other sign of systemic toxicity was observed in any treatment group. No sign of significant irritation (indicated by an erythema score ≥ 3) or any other local effect was observed in any treatment group.

Visually larger lymph nodes than the relevant vehicle control (AOO or DMF) was observed in the positive control, the 25 % and the 10 % (w/v) test item treated groups.

Significantly increased lymphoproliferation (≥ 3) was observed for the test item at concentrations of 25 % and 10 % (w/v). No significantly increased lymphoproliferation was observed at concentrations of 5 % and 2.5 % (w/v). The observed SI values were 18.78, 4.88, 1.95 and 0.95 at concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. The observed proliferation values at concentrations of 25 % and 10 % (w/v) statistically significantly differed from the relevant vehicle (DMF) control (p < 0.01; Mann-Whitney U-test). Significant dose-related response was observed (linear regression, p = 0.006, r = 0.99).

Since the test was valid and no sign of systemic toxicity or significant irritation was observed, the proliferation values obtained are considered to reflect the real potential of the test item to cause/not cause lymphoproliferation in the Local Lymph Node Assay.

According to evaluation criteria of the relevant guidelines, the significantly increased proliferation values observed at concentrations of 25% and 10 % (w/v) and the strong dose-response relationship are considered evidence that SIKA Hardener MTJ is a skin sensitizer.

The EC3 (dose calculated to induce a stimulation index of 3) was calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve based on published method. The calculated EC3 value of SIKA Hardener MTJ was 6.8 % in this LLNA. Using the calculated EC3 values SIKA Hardener MTJ can be ranked among moderate skin sensitizers in this LLNA according to the published data for classification of contact allergens.

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 (EC) No 1272/2008. As a result the substance is considered to be classified as skin sens. Cat. 1B, H317 for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) No 2020/1182.