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Toxicological information

Skin sensitisation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N,N-Bis(3-aminopropyl)methylamine
- Physical state: liquid, colorless, clear
- Analytical purity: 99.6 area %
- Lot/batch No.: O 2903

In vivo test system

Test animals

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

Study design: in vivo (LLNA)

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.

Results and discussion

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.

In vivo (LLNA)

Results
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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