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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Feb - 29 Feb 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethyl(propyl)amine
EC Number:
213-139-9
EC Name:
Dimethyl(propyl)amine
Cas Number:
926-63-6
Molecular formula:
C5H13N
IUPAC Name:
dimethyl(propyl)amine
Specific details on test material used for the study:
- Lot/batch no.: B921

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain as given in study report: CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Horst, The Netherlands
- Age at study initiation: pretest: 10-11 weeks; main test: 8-9 weeks
- Weight at study initiation: 19 - 23.5 g
- Housing: in groups, in Makrolon type II cages during pretest and in Makrolon type III cages during main test
- Diet: pelleted standard diet, ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 33 - 65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25%, 50%, and 100% (undiluted test item)
No. of animals per dose:
Pretest: 2 animals/group
Main test: 5 animals/group
Details on study design:
PREPARATION OF TEST MATERIAL
The test item was placed into an appropriate container on a tared balance and vehicle was added to achieve the required test item concentration. The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion. Vortexing was used to formulate the test item.

TREATMENT
1. Pretest:
The aim of the pretest was to determine the highest non-irritant test concentration that does not induce systemic toxicity at the same time. For this purpose, 2 animals were treated by topical application to the dorsal surface of each ear with test item concentrations of 50% (w/w) and 100% (undiluted test item) once daily each on 3 consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch with a diameter of 8 mm corresponding to an area of 0.5 cm2, and were immediately pooled per animal and weighed. Eventual ear irritation was considered important if an erythema of the ear skin of a score value >= 3 was observed at any observation time and/or if an increase in ear thickness of >= 25% was recorded on day 3 or day 6. The measured ear weight was also considered in this evaluation. The test concentrations for the main test were selected on the basis of the results of this pretest
2. Main test:
Three groups each of five animals were treated with the different concentrations of the test item by topical application (25 µL) at the dorsum of each ear once daily each on 3 consecutive days. Test item concentrations was 25%, 50% (each in acetone:olive oil (4+1 v/v)), and 100% (undiluted test item). The control group was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (³H-methyl thymidine, ³HTdR; 20 µCi in 250 µL phosphate buffer saline PBS). Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised, pooled per animal and immediately weighed. Furthermore, both ears of mice were punched at the apical area using a biopsy punch and the punches were immediately weighed pooled per animal.

CLINICAL OBSERVATIONS
The animals were observed and examined for mortality, clinical signs of toxicity and local signs of skin reaction; body weights were recorded.

PARAMETERS
For identification of possible induction of sensitization, the response in the draining lymph node after epicutaneous application of several concentrations of the test substance to the skin of the dorsal part of the ear is determined. The parameters used to characterize the response are lymph node cell count, 3H-thymidine incorporation into the lymph node cells and to a certain extent lymph node weight. Because not only sensitization induction but also irritation of the ear skin by the test substance may induce lymph node responses, the weight of ear punches taken from the area of test-substance application is determined as a parameter for inflammatory ear swelling serving as an indicator for the irritant action of the test substance.
Thus, the stimulation indices (SI) of cell count, ³H-thymidine incorporation, lymph node weight and ear weight were calculated as the ratio of the test group values for these parameters divided by those of the vehicle control group.

1. PREPARATION OF CELL SUSPENSION AND DETERMINATION OF CELL COUNT
After washing two times with PBS, the lymph node cells were resuspended in 5 % trichloroacetic acid and incubated at approximately +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid and transferred to plastic scintillation vials with ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, Rodgau, Germany) and thoroughly mixed. The level of ³HTdR incorporation was then measured on a ß-scintillation counter. Similarly, background ³HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses ³HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
2. LYMPH NODE WEIGHING AND CELL COUNT
After excision, the lymph nodes were pooled per animal and weighed immediately. Furthermore, the lymph node cell count was determined for each animal.. Therefore, the volume of the cell suspensions mentioned above was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined by means of a cell counter.
3. EAR WEIGHING
After the lymph nodes were excised, both ears of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal).

EVALUATION CRITERIA
- A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
- Furthermore, an index was calculated for the lymph node weight and -cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle control group. In more detail, the proliferative response of the lymph node cells is expressed as DPM/animal and as the ratio of ³HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before determination of the DPM/animal values, mean scintillation-background DPM needs to be subtracted from test and control raw data. For BALB/c mice, a cutoff-value for the lymph node cell count index of 1.55 was reported for a positive response (Ehling et al., Toxicology, 212: 69–79, 2005) and the cutoff-value for the ear weight index regarding a positive response (ear irritation) was reported to be 1.1 (Ulrich et al., Archives of Toxicol, 74: 733-744, 2001). However, these cutoff-values mentioned in the respective papers have been determined using a different strain of mice and can thus not be implicitly adopted.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated for body weight, ear weight and lymph node weight data, as well as for lymph node cell count, and for the DPM values. 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 test groups and negative control group. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. 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; one was identified.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.01
Test group / Remarks:
25% test item
Key result
Parameter:
SI
Value:
0.62
Test group / Remarks:
50% test item
Key result
Parameter:
SI
Value:
1.41
Test group / Remarks:
100% test item
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Please refer to "any other information on results".

EC3 CALCULATION: An EC3 value could not be calculated because none of the S.I.s exceeded the threshold values of 3.

CLINICAL OBSERVATIONS: No deaths occurred during the study period. The animals did not show any signs of systemic toxicity or local skin irritation during the
course of the study.

BODY WEIGHTS: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Any other information on results incl. tables

PRE-SCREEN TEST:

- Compound solubility: Test item solution at different concentrations could be prepared using acetone:olive oil (4+1 v/v) as vehicle.

- Irritation: From day 2 to day 4, the animal treated with 100% of the undiluted test item showed an erythema of the ear skin (Score 1). At 50% test item concentration, no signs of local skin irritation were observed.

- Systemic toxicity: At the tested concentrations (50% and 100%) the animals did not show any signs of systemic toxicity.

- Ear thickness measurements: At none of the tested concentrations, the measured increase in ear thickness exceeded the threshold value of 25% for excessive local skin irritation mentioned in OECD guideline 429.

- Erythema scores: Score 1 at 100% test item

MAIN STUDY

STIMULATION INDEX

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of ³HTdR compared with the negative control, as indicated by the Stimulation Index (S.I.); the estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value. In present case, stimulation Indices (S.I.) of 1.01, 0.62, and 1.41 were determined for the test concentrations 25, 50 and 100%, respectively. Thus, compared to the negative control (S.I. set at 1.00), an EC3 value could not be calculated because none of the obtained S.I. exceeded the threshold value of 3 for a positive response. For further details please see table below.

EAR WEIGHT

An increase in ear weights was only observed in the mid dose group in comparison to the vehicle control group. However, this was regarded as not biologically relevant since the observed increase of 6.6% compared to control was below the threshold value of 25% (increase in ear weights for excessive local skin irritation as mentioned in the OECD TG 429).

LYMPH NODE WEIGHT AND COUNT

A statistically significant or biologically relevant increase in lymph node weight and –cell count was not obtained in any test group in comparison to the negative control group. A clear dose response was also not obtained.

MORTALITY, CLINICAL SYMPTOMS, SKIN REACTION, BODY WEIGHT

Neither mortality nor clinical signs of toxicity were observed. No skin reaction was noticed. Body weight data were as expected and thus, inconspicuous.

No statistically significant or biologically relevant increase in DPM value was observed in the test group in comparison to the vehicle control group. A clear dose response was also not obtained.

Individual DPM values and Stimulation Indices for the control and treated groups:

Test item concentration (%)

Animal

DPM values (measured)

DPM values corrected for background

Stimulation Index (S.I.), relative to the mean of control group

Background in duplicate

18 and 34 (mean 26)

-

-

Negative Control group (0%)

1

498

472

-

2

1201

1175

-

3

700

674

-

4

605

579

-

5

973

947

-

25 %

1

666

640

0.8

2

461

435

0.6

3

656

630

0.8

4

751

725

0.9

5

1499

1473

1.9

50%

1

1033

1007

1.3

2

134

108

0.1

3

488

462

0.6

4

651

625

0.8

5

225

199

0.3

100%

1

2074

2048

2.7

2

923

897

1.2

3

640

614

0.8

4

1154

1128

1.5

5

768

742

1.0

Stimulation Indices (S.I.) per dose group:

Test item concentration

Group calculation

Mean DPM/animal (N = 5)

Standard Deviation

S.I.

Negative control

769.4

287.2

1.00

25%

780.6

401.4

1.01

50%

480.2

359.3

0.62

100%

1085.8

571.0

1.41

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Neither mortality nor clinical signs of toxicity were observed. No skin reaction was noticed. Body weight data were as expected and thus, inconspicuous.
Based on the results described above, it is concluded that, DMPA is not a skin sensitizer in the LLNA.
Executive summary:

The skin sensitizing potential of Dimethyl(propyl)amine (DMPA) was assessed in the Local Lymph Node Assay (LLNA) according to the OECD TG 429. For this purpose groups of 5 female CBA mice each were treated with 0, 25, 50 and 100% of the test item; acetone:olive oil (4+1 v/v) was used as vehicle. The negative control group received vehicle only. Treatment consisted of topical application of the test item to the dorsum of each ear of each animal for three consecutive days. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). After 5 hours the animals were sacrificed. Ear weight was determined and the left and right auricular lymph nodes were collected and the weight of the pooled lymph nodes from both sides was determined for each animal. Single cell suspensions of lymph node cells were prepared from lymph nodes pooled per animal and used for lymph node cell count. Subsequently the suspensions were washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a ß-scintillation counter.The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of ³HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.).

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results in a 3-fold or greater increase in incorporation of ³HTdR compared with the negative control, as indicated by the Stimulation Index (S.I.); the estimated test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

No statistically significant or biologically relevant increase in DPM value was observed in the test groups in comparison to the vehicle control group; a clear dose response was also not obtained. The respective S.I. values of 1.01, 0.62 and 1.41 obtained for the 25, 50 and 100% test item concentrations and compared to the negative control (S.I. 1.00) did not exceed the threshold value of 3 for a positive response. An increase in ear weights was only observed in the mid dose group in comparison to the vehicle control group. However, this was regarded as not biologically relevant since the observed increase of 6.6% compared to control was below the threshold value of 25% (increase in ear weights for excessive local skin irritation as mentioned in the OECD TG 429). A statistically significant or biologically relevant increase in lymph node weight and –cell count was not obtained in any test group in comparison to the negative control group.

Neither mortality nor clinical signs of toxicity were observed. No skin reaction was noticed. Body weight data were as expected and thus, inconspicuous.

Based on the results described above, it is concluded that, DMPA is not a skin sensitizer in the LLNA.