N-[3-(dimethylamino)propyl]stearamide monoacetate

Endpoint summary

Description of key information

One valid study is available for the test item NDPSa (Cas n.: 13282-70-7,other identifier: H-31339). 

GHS Cat. 1; LLNA in mice; OECD TG 429; E.L. Wilkinson (2015)

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:

From: 2014-11-11 To: 2015-01-20

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)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Remarks:

CBA/JHsd mice

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:Harlan Sprague Dawley, Frederick, Maryland, U.S.A.
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Microbiological status of animals, when known:Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants. Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers.
- Age at study initiation: 9 weeks old.
- Weight at study initiation: between 19.8 and 21.5 grams.
- Housing: solid-bottom cages with appropriate bedding and nestlets toys as enrichment.
- Diet (e.g. ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum.
- Water (e.g. ad libitum): tap water ad libitum.
- Acclimation period:quarantined for 8 days.
- Indication of any skin lesions: none.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26
- Humidity (%): 30-70
- Air changes (per hr): n/a
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle.
- IN-LIFE DATES: From: 12 November 2014 To: 17 November 2014

Vehicle:

propylene glycol

Concentration:

The 70% concentration, prepared in propylene glycol, was chosen as the high dose based on solubility testing. (insoluble) Hence, concentrations greater than 70% were not evaluated.

No. of animals per dose:

All females.
Group 1 : 6
Group 2: 5
Group 3: 5
Group 4: 5
Group 5: 5
Group 6: 6

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility:Due to insolubility of the test substance, concentrations greater than 70% were not evaluated.

MAIN STUDY
Five groups of 5 or
6 female CBA/JHsd mice were dosed for 3 consecutive days with 0% (vehicle), 5%, 25%, 50%, or 70% H-31339 on both ears. Propylene glycol (PG) was used as the diluting vehicle. One group of 6 female mice was dosed for 3 consecutive days with 25% hexylcinnamaldehyde (HCA) in PG as a positive control. On test day 6 of the assay, mice received 3H-thymidine by tail vein injection and were sacrificed approximately 5 hours later. The cell proliferation in the draining auricular lymph nodes of the ears from the test substance groups was then evaluated and compared to the vehicle group.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:Statistically significant increases in cell proliferation measurements compared to the vehicle group were observed at all test concentrations of H-31339. Stimulation indices (SIs) of greater than 3.0 were observed at all test concentrations of H-31339. The EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI = 3) for the test substance under the conditions of this study was calculated to be 0.6%. A 25% concentration of the positive control, HCA, produced a dermal sensitization response in mice. Therefore, the LLNA test system was valid for this study with H-31339.

TREATMENT PREPARATION AND ADMINISTRATION:Twenty-five μL of vehicle, test substance preparation, or positive control were administered topically to the dorsum of each mouse ear for 3 consecutive days (test days 1-3) at concentrations listed in the Study Design. Test days 4-5 were days of rest followed by intravenous injection of 20 μCi of 3H-thymidine in PBS per mouse on test day 6. Two mice (359 in the 25% test substance group and 558 in the 70% test substance group) were not injected with the appropriate amount of radioactive material and the lymph nodes for these mice were analyzed but not reported.

Positive control substance(s):

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

Statistics:

Significance was judged at p < 0.01. Lymph node dpm data were transformed to Log to obtain normality or homogenous variances.

Positive control results:

The positive control, hexylcinnamaldehyde (HCA), was purchased commercially. Any available information on the positive control was included in the study records. Impurities in the positive control were not expected to interfere with the study results. The positive control appeared to be stable under the conditions of the study. No evidence of instability, such as a change in color or physical state, was observed.
A 25% HCA solution in the vehicle was blended using a vortex mixer and stored in a vial protected from light until dosing was completed.

Key result

Parameter:

SI

Value:

0

Variability:

threshold positive response

Test group / Remarks:

1

Key result

Parameter:

SI

Value:

19.98

Variability:

threshold positive response

Test group / Remarks:

2

Key result

Parameter:

SI

Value:

32.71

Variability:

threshold positive response

Test group / Remarks:

3

Key result

Parameter:

SI

Value:

28.57

Variability:

threshold positive response

Test group / Remarks:

4

Key result

Parameter:

SI

Value:

21.37

Variability:

threshold positive response

Test group / Remarks:

5

Key result

Parameter:

SI

Value:

9.74

Variability:

threshold positive response

Test group / Remarks:

6

Cellular proliferation data / Observations:

Statistically significant increases in cell proliferation measurements compared to the vehicle group were observed at all test concentrations of H-31339. Stimulation indices (SIs) of greater than 3.0 were observed at all test concentrations of H-31339. The EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI = 3) for the test substance under the conditions of this study was calculated to be 0.6%. A 25% concentration of the positive control, HCA, produced a dermal sensitization response in mice. Therefore, the LLNA test system was valid for this study with H-31339. Under the conditions of this study, H-31339 produced a dermal sensitization response in mice.

Category EC3 Value (%)
Extreme <0.1
Strong ≥ 0.1 - <1
Moderate ≥1 - <10
Weak ≥10 - ≤ 100

CLINICAL OBSERVATIONS: Careful clinical observations were performed on test day 1, prior to each dose (at approximately the same time ± 2 hours) on test day 2 and 3, and on the day of sacrifice.

BODY WEIGHTS: No biologically relevant changes in body weights were observed at any test concentration.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness).
No clinical signs of toxicity were observed in the study.

Stimulation Index Data

Group	Material Tested	n	Mean (dpm)	S.D. (dpm)	SI
1	0 % Vehicle	6	717.92	163.58	n/a
2	5 %	5	14341.45 #	2684.63	19.98
3	25 %	4^b	23481.00 #	7188.52	32.71
4	50 %	5^c	20513.45 #	10008.19	28.57
5	70 %	4^b	15345.00 #	2911.64	21.37
6	25 % Positive Control	6	6992.58	2073.54	9.74

a Data were not included in the statistical analysis of the test substance groups.

b One mouse was not injected with the appropriate amount of radioactive material and the lymph nodes for this mouse were not analysed.

c The dpm value for one mouse was deemed an outlier by statistical analysis; however, the dpm data for this mouse were included in the statistical analysis. The group mean calculated stimulation index remained >3.0 whether the outlier was included or excluded in the calculation; therefore, the data were reported.

 

# Statistically significant increase in dpm data from vehicle at p < 0.01 by Jonckheere-Terpstra trend test.

* Statistically significant increase in dpm data from vehicle at p < 0.01 by Dunnett/Tamhane-Dunnett test.

@ Statistically significant increase in dpm data from vehicle at p < 0.01 by Dunn's test.

~ Due to lack of control group values or variability among group means, statistical analyses were unable to be performed.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on these data, and according to the guidance provided by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC), H-31339 is considered a strong dermal sensitizer in mice.

Executive summary:

The objective of this study was to evaluate the potential of H-31339 to produce a dermal sensitization response in mice using the local lymph node assay (LLNA). Five groups of 5 or 6 female CBA/JHsd mice were dosed for 3 consecutive days with 0% (vehicle), 5%, 25%, 50%, or 70% H-31339 on both ears. Propylene glycol (PG) was used as the diluting vehicle. One group of 6 female mice was dosed for 3 consecutive days with 25% hexylcinnamaldehyde (HCA) in PG as a positive control. On test day 6 of the assay, mice received 3H-thymidine by tail vein injection and were sacrificed approximately 5 hours later. The cell proliferation in the draining auricular lymph nodes of the ears from the test substance groups was then evaluated and compared to the vehicle group.

No biologically relevant changes in body weights were observed at any test concentration. No clinical signs of toxicity were observed in the study.

Statistically significant increases in cell proliferation measurements compared to the vehicle group were observed at all test concentrations of H-31339. Stimulation indices (SIs) of greater than 3.0 were observed at all test concentrations of H-31339. The EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI = 3) for the test substance under the conditions of this study was calculated to be 0.6%. A 25% concentration of the positive control, HCA, produced a dermal sensitization response in mice. Therefore, the LLNA test system was valid for this study with H-31339. Under the conditions of this study, H-31339 produced a dermal sensitization response in mice.

Based on these data, and according to the guidance provided by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC), H-31339 is considered a strong dermal sensitizer in mice.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

LLNA skin sensitisation:

The objective of this study was to evaluate the potential of H-31339 to produce a dermal sensitization response in mice using the local lymph node assay (LLNA). Five groups of 5 or 6 female CBA/JHsd mice were dosed for 3 consecutive days with 0% (vehicle), 5%, 25%, 50%, or 70% H-31339 on both ears. Propylene glycol (PG) was used as the diluting vehicle. One group of 6 female mice was dosed for 3 consecutive days with 25% hexylcinnamaldehyde (HCA) in PG as a positive control. On test day 6 of the assay, mice received 3H-thymidine by tail vein injection and were sacrificed approximately 5 hours later. The cell proliferation in the draining auricular lymph nodes of the ears from the test substance groups was then evaluated and compared to the vehicle group.

No biologically relevant changes in body weights were observed at any test concentration. No clinical signs of toxicity were observed in the study.

Statistically significant increases in cell proliferation measurements compared to the vehicle group were observed at all test concentrations of H-31339. Stimulation indices (SIs) of greater than 3.0 were observed at all test concentrations of H-31339. The EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI = 3) for the test substance under the conditions of this study was calculated to be 0.6%. A 25% concentration of the positive control, HCA, produced a dermal sensitization response in mice. Therefore, the LLNA test system was valid for this study with H-31339. Under the conditions of this study, H-31339 produced a dermal sensitization response in mice.

Based on these data, and according to the guidance provided by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC), H-31339 is considered a strong dermal sensitizer in mice.

 

 

Justification for selection of Skin sensitisation toxicity endpoint -
one key 1 study used

 

Harmonised classification:

The substance has harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP). 

 

Self-classification:

Based on the available information on test item, self-classification is proposed according to the CLP or GHS.

GHS 1.

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Due to the results of the LLNA toxicity study, with a conclusive outcome of being a strong dermal sensitizer, a classification is  justified for category GHS 1.

This classification has been selected on the adverse effects outcome of the key 1 study report.

 

GHS Category 1