Fatty acids, vegetable-oil, esters with dipropylene glycol

Skin sensitisation

Administrative data

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

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

Test guideline 2

Qualifier:

equivalent or similar to guideline

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. QA statement)

Remarks:

(GROUPE INTERMINISTERIEL DES PRODUITS CHIMIQUES)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, vegetable-oil, esters with dipropylene glycol

EC Number:

305-769-9

EC Name:

Fatty acids, vegetable-oil, esters with dipropylene glycol

Cas Number:

95009-41-9

Molecular formula:

not available (UVCB)

IUPAC Name:

Fatty acids, vegetable-oil, esters with dipropylene glycol

Details on test material:

- Name of test material (as cited in study report): Fatty acids, vegetable-oil, esters with dipropyleneglycol
- Physical state: liquid
- Analytical purity: 100% (UVCB)
- Lot/batch No.: ES 110680
- Expiration date of the lot/batch: 25 October 2016
- Storage condition of test material: at room temperature

In vivo test system

Test animals

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

Study design: in vivo (LLNA)

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.

Results and discussion

In vivo (LLNA)

Results

Results 1

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.

Results 2

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.

Any other information on results incl. tables

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 10^6cells /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

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: non classified
DSD: non classified