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Description of key information

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
Study period:
02 - 28 Apr 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP-Guideline study (RL1 is considered appropriate as a) exposure of the animals was performed according to OECD GL 429; b) the used methodology was validated in the test facility according to the performance standards defined in OECD GL 429; c) a publication is included as reference for the validity of the methodology as required according to OECD 429 and d) the threshold used in the study is defined more stringent compared to the threshold provided in the publication)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
lymphocyte proliferation was measured based on cell counting instead of thymidine incorporation and hence, a different cut off value (EC1.4 instead of EC3) was used for evaluation of results
Qualifier:
equivalent or similar to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
lymphocyte proliferation was measured based on cell counting instead of thymidine incorporation and hence, a different cut off value (EC1.4 instead of EC3) was used for evaluation of results
GLP compliance:
yes (incl. certificate)
Remarks:
(GROUPE INTERMINISTERIEL DES PRODUITS CHIMIQUES)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J (CBA/J@Rj)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage Janvier Labs, Le Genest Saint Isle, France
- Age at study initiation: 8 weeks
- Weight at study initiation: 19.1 - 21.8 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: A04, SAFE (Augy, France), ad libitum
- Water: tap water from public distribution system, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50 and 100%
No. of animals per dose:
4 (+1 additional animal/group as reserve)
Details on study design:
RANGE FINDING TESTS:
In a preliminary screening test, the dorsal surface of each ear from 1 mouse was treated daily with 25 µL undiluted test substance for 3 consecutive days. The mouse was observed for signs of toxicity or excessive local irritation. Ear thickness was recorded on Day 1, Day 3 and Day 6. Bodyweight was recorded on Day 1 (prior to dosing) and Day 6. Application of undiluted test substance did not induce clinical signs of systemic toxicity or cutaneous reactions including ear swelling or induction of erythema. Moreover, no irritation was observed due to test substance application. Therefore, no signs indicative for skin irritation were determined and hence, the undiluted test substance was included in the main study as highest concentration.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: lymphocyte cell counting
- Criteria used to consider a positive response: The proliferative response of lymph node cells was expressed as the number of lymphocytes per lymph node and as ratio of lymphocytes of test nodes relative to that recorded for control nodes :

SI = cell count of treated group/cell count of control group

The test item will be regarded as a sensitiser if at least one concentration of the test item results is
greater than 1.4 compared to control values (SI ≥ 1.4) . Other relevant criteria such as dose-response and irritation level were also taken into account for the interpretation of the results. Any test item failing to produce a SI < 1.4 will be classified as a "non-sensitiser".


TREATMENT PREPARATION AND ADMINISTRATION:
25 µL of the test item was applied on the entire dorsal surface of each ear. The application was repeated once daily over 3 consecutive days. Body weights were recored on Day 1 (prior to dosing) and Day 6 (prior to termination). Local irritation reactions were assessed and ear thickness measurements were performed with a micrometer on Day 1 and Day 3 before application and on Day 6 after sacrifice. Furthermore, punch biopsies of both ears were prepared for weighing on Day 6 after sacrifice. The draining auricular lymph nodes of each ear were excised, weighed and pooled for each experimental group. A single cell suspension was prepared by gentle mechanical tissue disaggregation through a 200-meshcell strainers. 10 µL of this cell suspension was diluted in 10 mL of physiological saline solution before lymphocyte cells were counted using a cell counter (Beckman Coulter Z2). A size range of 5 - 15 µm was selected for counting which covers the average size of a lymphocyte of 8 µm.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
No significant (SI ≥ 1.4) lymphoproliferative response was noted for the tested concentrations. The calculated stimulation index values were 0.98, 1.02 and 1.32 at test item concentrations of 25%, 50% and 100%, respectively. Thus, as all SI indexes were below 1.4, an EC1.4 value could not be determined.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The positive control substance induced a significant lymphoproliferative response as shown by a stimulation index value of 7.3.

Mortality and clinical signs of toxicity

Application of the test substance did not induce mortaility or clinical signs of systemic toxicity. Body weights of control and test animals were comparable over the study period.

Local skin irritation

No cutaneous reactions were noted during the main test. Dose-dependent increases in ear and lymph node weights were determined which did not result in statistically significant differences. Values obtained for ear thickness were comparable among the groups. Therefore, the test item was not considered as excessively irritant at these concentrations.

Table 2: Cell count. Stimulation index and calculation of EC1.4

 

Group

Cell count/group (x 106cells /mL)

SI

Control (AOO)

36.02

 

25%

35.34

0.98

50%

36.66

1.02

100%

47.38

1.32

 

Table 3: Individual ear thickness

 

Group

Ear thickness (mm)

Increase in ear thickness (%)

Increase in ear thickness (%)

Day 1

Day 3

Day 6

D3/D1

D6/D1

Control (AOO)

0.21

0.22

0.18

4.8

-14.3

0.21

0.22

0.20

4.8

-4.8

0.23

0.23

0.20

0.0

-13.0

0.22

0.23

0.20

3.5

-10.3

Mean

0.22 ± 0.01

0.23 ± 0.01

0.20 ± 0.01

3.5 ± 2.3

-10.3 ± 4.3

25 %

0.20

0.21

0.21

5.0

5.0

0.23

0.23

0.19

0.0

-17.4

0.23

0.23

0.19

0.0

-17.4

0.23

0.23

0.20

0.0

-13.0

Mean

0.22 ± 0.02

0.23 ± 0.01

0.20 ± 0.01

1.3 ± 2.5

-10.7 ± 10.7

50%

0.21

0.21

0.19

0.0

-9.5

0.24

0.22

0.20

-8.3

-16.7

0.22

0.22

0.21

0.0

-4.5

0.24

0.24

0.21

0.0

-12.5

Mean

0.23 ± 0.02

0.22 ± 0.01

0.20 ± 0.01

-2.1 ± 4.2

-10.8 ± 5.1

100%

0.24

0.22

0.21

-8.3

-12.5

0.22

0.22

0.21

0.0

-4.5

0.23

0.23

0.20

0.0

-13.0

0.21

0.21

0.19

0.0

-9.5

Mean

0.23 ± 0.01

0.22 ± 0.01

0.20 ± 0.01

-2.1 ± 4.2

-9.9 ± 3.9

 

Table 4: Individual ear biopsy and lymph node weight

 

Group

Ear weight (Day 6 (mg)

% of control

Lmph nodes (mg)

% of control

Control (AOO)

27.8

 

5.2

 

27.9

4.8

25.8

4.9

25.9

4.5

Mean

26.9 ± 1.2

 

4.9 ± 0.3

 

25 %

26.0

 

5.4

 

27.3

6.5

29.4

4.8

26.8

4.9

Mean

27.4 ± 1.5

2.0

5.7 ± 0.7

16.3

50%

26.4

 

6.4

 

30.0

6.3

27.9

5.9

31.3

5.9

Mean

28.9 ± 2.2

7.6

6.1 ± 0.3

24.5

100%

29.2

 

6.8

 

32.9

6.2

30.0

6.6

28.1

5.7

Mean

28.1 ± 2.1

11.9

6.3 ± 0.5

28.6

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: non classified
DSD: non classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin sensitization

Sensitizing properties of Fatty acids, vegetable-oil, esters with dipropylene glycol were determined in a GLP-conducted local lymph node assay performed similar to OECD 429 in female CBA/J (CBA/J@Rj) (4 animals/group) (Richeux, F., 2014). In accordance with OECD 429, which states that “other endpoints for assessment of the number of proliferating cells may be employed provided by the performance standards”, lymphocyte proliferation was determined by cell counting using a cell counter (Beckman Coulter Z2) with a selected size range of 5 – 15 µm in regard to an average size of a lymphocyte of 8 µm. Depending on the implemented methodology, a lab-internal cut-off value of SI ≥ 1.4 was validated for a positive sensitization reaction in accordance to the performance standards defined in OECD 429. As shown by Ehling et al., the cut-off values indicating a sensitization reaction, were lower as the traditional cut-off value of SI ≥ 3. Based on the results of a preliminary test, animals were exposed to 25, 50 and 100% (w/v) test substance diluted in a acetone - olive oil (AOO). Application of the test substance did not induce mortaility or clinical signs of systemic toxicity. Body weights of control and test animals were comparable over the study period. Furthermore, no cutaneous reactions were noted during the main test which may indicate irritation of the skin. Dose-dependent increases in ear and lymph node weights were determined which did not result in statistically significant differences for any dose and are therefore not considered as indicative for a sensitization reaction. Further, lymphoproliferative responses leading to stimulation indices 0.98, 1.02 and 1.32 were determined at test item concentrations of 25%, 50% and 100%, respectively, thereby not exceeding the internal validated cut-off value of SI ≥ 1.4. Moreover, taking into consideration that application of the undiluted test substance (100%) does not induce a SI value exceeding the defined cut off value, the test result does not provide sufficient evidence for classification and Fatty acids, vegetable-oil, esters with dipropylene glycol are therefore not considered as sensitizing.


Justification for selection of skin sensitisation endpoint:
The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.5, of Regulation (EC) No 1907/2006.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation of Fatty acids, vegetable-oil, esters with dipropylene glycol do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.