Dimethyl(octyl)amine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-20 to 2016-05-17

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:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl(octyl)amine

EC Number:

230-939-3

EC Name:

Dimethyl(octyl)amine

Cas Number:

7378-99-6

Molecular formula:

C10H23N

IUPAC Name:

dimethyl(octyl)amine

Test material form:

other: liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (prescreen test and main study: lot no. 0631)
- Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls
at regular intervals)
- The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
(prescreen test and main study: lot no. 02102150820)
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least five days) under laboratory conditions

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Based on the results observed in the prescreen test the following test item concentrations were selected for the main study:
12.5%, 25% and 50% (v/v)
The preparations were made immediately prior to each dosing.
AOO was used as vehicle and served as negative control.

No. of animals per dose:

5 animals/dose group, 5 animals/control group
10 mice / prescreen test

Details on study design:

Prescreen Test
Before the initiation of the prescreen test, a solubility test was performed to define the vehicle and the maximum concentration
which is technically applicable to the animals.
The maximum technically applicable concentration of the test item was found to be 100%.
In order to determine the highest tolerated and not excessively irritant test concentration a prescreen test was performed which was
conducted under the same conditions as the main study, except there was no assessment of lymph node proliferation. The mice were
observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test
and prior to termination. Both ears were observed for erythema and scored. Ear thickness measurements were performed
on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6. Excessive local irritation was indicated by an erythema
score ≥ 3 and/or ear swelling of ≥ 25%.
Two animals were treated by topical application with the test item on three consecutive days at a concentration of 100% (undiluted) to the
entire dorsal surface of each ear.
Four animals were treated by topical application with the test item on three consecutive days at a concentration of 50% (diluted in AOO) to the
entire dorsal surface of each ear.
Two animals were treated by topical application with the test item on three consecutive days at a concentration of 25% (diluted in AOO) to the
entire dorsal surface of each ear. Two further animals were treated with 100% AOO and served as negative control.
Immediately before the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of
both ears of the surviving animals was measured except for animal no. 7 where thickness of the left ear could not be measured
on day 3 and day 6 as the left ear was partially missing.
During this period also all clinical signs were recorded.
Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation,
diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).
One of the animals treated with the undiluted test item was found dead on the day of first application. The other animal treated with the
undiluted test item showed clinical signs of systemic toxicity 4 hours post-application and was euthanised for ethical reasons on the same day.
Neither signs of systemic toxicity nor signs of excessive irritation at any application site could be detected in any other animal.
The local effects observed such as wet fur are considered not to be signs of excessive irritation.
All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the duration of the prescreen test.

Preparation of the Animals
The animals were randomly selected using the validated departmental computerised system E WorkBook (version 9.4.0, ID Business Solutions Ltd.).
Identification was ensured by cage number and individual marking (tail).

Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation
at site of application.

Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

Dose Groups
3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.

Test Regime
Topical Application
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.
Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of
250 µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 µCi/mL.
Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph
nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS).
A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size).
After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended
with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of
macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this
solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H -Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM).
Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually
for each animal. 

Positive control substance(s):

other: Phenylenediamine

Statistics:

Outlier tests according to Dixon, Grubbs and Nalimov were performed for the values measured for the number of disintegrations per minute (DPM).
If outliers were identified, these values were not included in the calculation of the stimulation indices. As at least four values per group are required
for the evaluation of the results, the outlier test was not repeated to detect further outliers.

Results and discussion

Positive control results:

The recent reliability check was performed in December 2015. The raw data of this study are kept in the BSL archives (BSL Project ID 146632 X).
Positive-control substance: P-Phenylenediamine (CAS 106-50-3, Sigma, purity > 98%; Lot No.: WXBB8077V) 1%
Vehicle: AOO (4:1 (v/v) acetone/olive oil)
Species/strain: healthy CBA/CaOlaHsd mice
Source: Harlan Laboratories GmbH, 5800 AN Venray, The Netherlands
Concentrations: 1% on three consecutive days
The Stimulation Index was 9.1

The Stimulation Index was ...

In vivo (LLNA)

Results

Results 1

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 12.5 % = 1904.3 (MV) 25% = 7746.4 (MV) 50% = 7349.0 (MV) negative control = 1559.8 (MV)

Results 2

Parameter:

SI

Value:

5

Test group / Remarks:

25% concentration

Remarks on result:

other: exceeded the stimulation index of 3

Results 3

Parameter:

SI

Value:

1.2

Test group / Remarks:

12.5% concentration

Results 4

Parameter:

SI

Value:

4.8

Test group / Remarks:

50% concentration

Remarks on result:

other: exceeded the stimulation index of 3

Any other information on results incl. tables

All animals survived throughout the test period without showing any clinical signs.

All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

Migrated information

Conclusions:

The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 18% (the SI values are considered to
be dose dependent showing a saturation effect at 50% test item concentration).
Consequently, according to OECD 429 solutions or preparations containing more than 18% AD1 are expected to have a stimulation
index of >3 and are therefore considered to be dermal sensitisers.
According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test item AD1
has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.

Executive summary:

On the basis of the test results given below and in conformity with the criteria given inCommission Regulation (EU) No 286/2011 the substance should be:

classified into sub-category 1A
classified into sub-category 1B	X
unclassified

On the basis of the test results given below and in conformity with the criteria given inGHS (Globally Harmonized Classification System) ]the substance should be:

classified into sub-category 1A
classified into sub-category 1B	X
unclassified

Based on the results of the prescreen test the test item was assessed for sensitising properties at concentrations of 12.5%, 25% and 50X% (v/v), each diluted with AOO 4:1 (v/v), 4 parts acetone and 1 part olive oil.

At the daily clinical observation the animals did not show any visible clinical symptoms and no case of mortality was observed.

Species/strain: Mice, CBA/CaOlaHsd

Number of animals: 20/main test

Vehicle: AOO (4:1 (v/v) acetone/olive oil)

Summary Results

Two of the three tested concentrations exceeded the stimulation index of 3.

The stimulation index at a concentrationof        12.5% was     1.2

The stimulation index at a concentrationof        25%    was     5.0

The stimulation index at a concentrationof        50%    was     4.8

Conclusion

The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 18% (the SI values are considered to be dose dependent showing a saturation effect at 50% test item concentration).

Consequently, according to OECD 429 solutionsor preparations containing more than 18% AD1 are expected to have a stimulation index

of >3 and are therefore considered to be dermal sensitisers.

According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) ]the test item AD1

has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.