Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 February 2018 - 29 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordacen with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
Clear, light yellow liquid
Storage: 2 to 8 °C, in the dark
Expiry date: 01-Jan-18
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
N/A
- Specific activity:
N/A
- Locations of the label:
N/A
- Expiration date of radiochemical substance:
N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 ºC, protected from light
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Soluble in acetone: olive oil, 4:1 v/v
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dissolved in acetone: olive oil, 4:1 v/v
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: Test item dose solutions prepared at 50, 25, 10 % in acetone: olive oil, 4:1 v/v
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as a liquid

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A

OTHER SPECIFICS: N/A

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: Yes
- Microbiological status of animals, when known: Not reported
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: The animals were housed in suspended solid floor polypropylene cages furnished with softwood wood flakes
- Diet (e.g. ad libitum): Free access to food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains tap water) was allowed throughout the study.
- Acclimation period: ≥ 5 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): ≥15
- Photoperiod (hrs dark / hrs light): 12:12
- IN-LIFE DATES: From: 22 February 2018 To: 29 March 2018

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Prelim test: Undiluted, 50, 25 % v/v
Main test: 50, 25, 10 % v/v
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Soluble in vehicle at 50 %
- Irritation: No signs of irratation observed in any of the test groups.
- Systemic toxicity: Animal in the undiluted test item group showed severe signs of systemic toxicity and was humanely killed 1-hour post dose on Day 1. No other animals showed signs of sstemic toxicity.
- Ear thickness measurements: No abnormal ear thickness measurements were recorded in any of the test groups.
- Erythema scores: No sign of erythema obsered in any of thes test groups (all values = 0)

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Mouse Local Lymph Node Assay
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitizer".


TREATMENT PREPARATION AND ADMINISTRATION:

For the purpose of the study, the test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.

The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non parametric Kruskal Wallis Rank Sum and Mann Whitney U test procedures were used.

Results and discussion

Positive control results:
The positive control article produced a Stimulation Index of 4.59 (positive indication)

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
2.6
Variability:
SD: ±1620
Test group / Remarks:
10 % v/v
Key result
Parameter:
SI
Value:
11.61
Variability:
SD: ±4145
Test group / Remarks:
25 % v/v
Key result
Parameter:
SI
Value:
17.2
Variability:
SD: ±4728
Test group / Remarks:
50 % v/v
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA

Not reported

DETAILS ON STIMULATION INDEX CALCULATION

The scintillation counter provided data including the DPM value (disintegrations per minute during a ten minute period) for each individual animal. The mean DPM value for each test group was divided by the mean DPM for the control group to provide the Stimulation Index (SI) value for each test group.

EC3 CALCULATION

The EC3 value is the concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation. The equation used for the calculation of EC3 is:

EC3 = c + [[(3-d) / (b-d)] x (a-c)]

where;

a = lowest concentration giving stimulation index >3
b = actual stimulation index caused by 'a'
c = highest concentration failing to produce stimulation index of 3
d = actual stimulation index caused by 'c'

in this test;

a = 25
b = 11.61
c = 10
d = 2.60

therefore;

EC3 = 10.7 %

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 10.7 %.

CLINICAL OBSERVATIONS:

There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or with 10, 25 or 50 % v/v formulations of the test article. No irritation was noted in any of the test groups.

BODY WEIGHTS

There was no indication of a treatment related effect on body weight.

Any other information on results incl. tables

Table 1       Stimulation index results

 

Test Group

Animal #

Individual animal DPM

Mean group DPM

(std. dev.)

Stimulation index

(SI)a

Solvent control

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

1-1

781.37

1250.67

(±305.22)

n/a

1-2

1255.20

1-3

1631.04

1-4

1247.48

1-5

1338.26

10 % test item

2-1

3853.82

3257.11

(±1620.02)

2.60

2-2

5239.57

2-3

2204.47

2-4

1090.55

2-5

3897.13

25 % test item

3-1

18597.07

14522.23**

(±4727.53)

11.61

3-2

14353.77

3-3

14063.90

3-4

17590.18

3-5

8006.25

50 % test item

4-1

24202.56

21510.59**

(±4727.53)

17.20

4-2

2199.93

4-3

26536.65

4-4

14016.35

4-5

20806.45

Positive control

5-1

7916.88

5745.41**

(±1879.73)

4.59

5-2

6709.41

5-3

6290.52

5-4

3062.95

5-5

4747.28

a Stimulation Index (SI) of ≥ 3.0 indicates a positive result

** p<0.01

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Based on the condition of this study, the test item was classified as a Category 1B sensitiser in accordance with Regulation (EC) No 1272/2008.
Executive summary:

OECD 429 (2018) - In a dermal sensitization study with Reaction mass of N,N,N’,N’-tetrabutylmethylenediamine and dibutylamine in acetone: olive oil (4:1 v/v) young female adult mice (CBA/CaCrl) were tested using the Local Lymph Node Assay (LLNA).

 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50 %, this concentration was selected as the highest dose investigated in the main test. Three groups, each of five animals, were treated with 25 μL of the test item in solution in the vehicle at concentrations of 50, 25 and 10 % v/v. A further group of five animals was treated with the vehicle alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser, α-Hexylcinnamaldehyde at a concentration of 25 % v/v in the vehicle.

 

There were no signs of systemic toxicity or skin irritation through the test period. Ear thickness were also within the normal range. There were no clinical abnormalities or macroscopic abnormalities of the surrounding area were noted for any of the animals and no mortality reported during the study. The Stimulation Index, expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, was determined for each treatment group.

 

The test item did elicit a Stimulation Index ≥ 3 when tested at ≥ 25 % v/v with SI values of 2.60, 11.61 and 17.20 in the 10, 25 and 50 % w/v test groups, respectively. The test item was therefore considered to be a sensitiser under the conditions of the test. The EC3value, the concentration of test item expected to cause a three-fold increase in3HTdR incorporation (EC3value) was calculated to be 10.7 %.

 

In this study, N,N,N’,N’-tetrabutylmethylenediamine and dibutyylamine was a dermal sensitiser.

Based on the condition of this study, the test item was classified as a Category 1B sensitiser in accordance with Regulation (EC) No 1272/2008.