N,N-bis(3-aminopropyl)methylamine

Endpoint summary

Description of key information

LLNA: not sensitising

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

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: Harlan Laboratories B.V. Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: 1st pre-test: 9 - 10 weeks; 2nd pre-test: 10 - 11 weeks; Main study: 11 - 12 weeks
- Housing: group (Makrolon Type II (pre-test) / III (main study), with wire mesh top)
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethylformamide

Concentration:

1, 2 and 5 %

No. of animals per dose:

5

Details on study design:

Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 1, 2, and 5% in DMF. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 19.8 μCi of 3H-methyl thymidine (equivalent to 79.3 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.

Positive control substance(s):

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

Statistics:

A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations validated statistical program R Script DecisionTree_2.Rnw was used. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers (performed with validated program R Script Outlier.Rnw). No statistical outlier was identified. However, both biological and statistical significance were considered together.

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in April 2015.

Parameter:

SI

Remarks on result:

other: In this study Stimulation Indices (S.I.) of 1.32, 1.05 and 2.04 were determined with the test item at concentrations of 1, 2, and 5% (w/w) in DMF, respectively. A clear dose response was not observed.

Lymph Node Weights and Cell Counts

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant increase in lymph node weights was observed for the high group in comparison to the vehicle control group. A statistically significant or biologically relevant increase in lymph node cell counts was not observed in any of the test item treated groups in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count did not exceed this threshold.

Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A statistically significant increase in ear weights was not observed in any of the test item treated groups in comparison to the vehicle control group.

Test item concentration	Group Calculation
	Mean DPM       per animal          (2 lymph nodes)^a)	SD	S.I.
Vehicle Control Group (DMF)	1140.5	771.3	1.00
1% N,N-Bis(3-aminopropyl)methylamine	1894.3	442.5	1.32
2% N,N-Bis(3-aminopropyl)methylamine	1512.3	447.5	1.05
5% N,N-Bis(3-aminopropyl)methylamine	2933.1	841.0	2.04^$

a) Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

$ Mean DPM value for the group was according to the ANOVA (Dunnett-test) significantly higher than the corresponding control value. The p value for the analysis was

Conclusions:

The test item N,N-Bis(3-aminopropyl)methylamine was thus not a skin sensitiser under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

N,N-Bis(3-aminopropyl)methylamine was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item solutions at different concentrations were prepared in the vehicle dimethylformamide (DMF). For this purpose a local lymph node assay was performed using test item concentrations of 1, 2, and 5% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by two pre-experiments). The animals showed neither signs of systemic toxicity nor mortality during the course of the study. From day 2 to day 5 one animal treated with a test item concentration of 5% showed scabby ear skin. Also from day 3 to day 5, two animals treated with a test item concentration of 2% showed scabby ear skin. Animals treated with 1% test item concentration did not show any signs of local skin irritation. Erythema of the ear skin was not observed in any animal. A statistically significant increase in ear weights was not observed in any treated group in comparison to the vehicle control group (p<0.05). For BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. The indices determined for the low and mid dose group slightly exceeded this threshold (indices of 1.14 and 1.13). However, the increased ear weights were considered to be not biologically relevant, as the threshold mentioned in the OECD guideline of 1.25 was not exceeded. In this study Stimulation Indices (S.I.) of 1.32, 1.05 and 2.04 were determined with the test item at concentrations of 1, 2, and 5% (w/w) in DMF, respectively. A clear dose response was not observed. A statistically significant increase in DPM value and also in lymph node weights was observed in the highest dose group in comparison to the vehicle control group. This was not considered to be biologically relevant as the S.I. determined for this concentration did not exceed the threshold value of 3. A statistically significant or biologically relevant increase in lymph node cell counts was not observed in any treated group in comparison to the vehicle control group. N,N-Bis(3-aminopropyl)methylamine was thus not a skin sensitizer under the test conditions of this study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The classification criteria according to Regulation (EC) No 1272/2008 (CLP) are not met.