Decanoic acid, mixed esters with heptanoic acid, octanoic acid and pentaerythritol

Endpoint summary

Description of key information

Skin Sensitisation - LLNA

The test item Hatcol 3178 is not considered a skin sensitizer under the test conditions of the study.

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:

25 January 2017 to 14 February 2017 (test commissioned October 2016)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for the testing of chemicals 429. Skin Sensitisation: Local Lymph Node Assay (Adopted: 22 July 2010).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

No further details specified in the study report.

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Species: Mice CBA/Ca
Source: AnLab Prague, Czech Republic
Number and Sex of Animals: 29 females, non-pregnant and nulliparous, were used
Age at First Dose: 8-11 weeks,
Animal Health: The health condition of animals was examined by a veterinarian before initiation of the study
Acclimation: The animals were acclimated in identical conditions as during the experiment for 6 days prior to the start of treatment. Acclimation was conducted according to standard operation procedures.
Housing Condition: The animals were housed in polypropylene cages (5 animals per cage) suspended on stainless steel racks, in a room equipped with central air-conditioning. The room temperature was within the range of 22 ± 3°C, relative humidity was within the range of 50 - 60 %. The light regimen was set to a 12-hour light / 12-hour dark cycle. Sanitation procedures were conducted in accordance with standard operating procedures.
Diet: A laboratory food ssniff (ssniff Spezialdiäten GmbH, Germany) was served ad libitum, each day approximately at the same time.
Water: The animals received tap water for human consumption. Supply of drinking water was unlimited.
Bedding: Lignocel S3/4, Lufa - ITL GmbH, Germany
Animals Identification: Each animal was marked with permanent pen on the tail. Each cage was affixed with a cage card containing pertinent animal and study
information.
Justification for the Choice of Species: The CBA/Ca mice are the standard experimental rodent of choice and recommended by OECD Guideline No. 429.

Vehicle:

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

Concentration:

Based on the observations recorded in the Pre-screen test, the concentrations of 50, 25 and 10% were selected for the main test.

No. of animals per dose:

5 females – control (vehicle)
5 females – positive control
15 females – test item (5 per concentration)
4 females - pre-screen test (2 per concentration)

Details on study design:

Preparation of solutions
25% α-Hexylcinnamaldehyde
0.5 mL of α-Hexylcinnamaldehyde at a concentration of 85% was diluted in 1.2 mL vehicle (Acetone/Olive Oil, 4:1, v/v). Stock and diluted solution of α-Hexylcinnamaldehyde were kept at room temperature (20-25 °C).

5% Trichloroacetic acid
2.5 g TCA was dissolved in 50 mL of deionized water. The solution was kept under refrigeration (2-8 °C).

Phosphate buffered saline
One tablet (1906.0 mg) dissolved in 200 mL of deionized water yields 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4. The solution was kept under refrigeration (2-8 °C).

125I-iododeoxyuridine
125I-iododeoxyuridine (1 mCi/mL) was diluted for final radioactivity of  2 μCi (7.4 x 104 Bq) in 250 μL of PBS buffer. Diluted solution of 125I-iododeoxyuridine was kept under refrigeration (2-8 °C).

Dose Preparation
The required amount of the test item (according to the concentration) was mixed in vehicle shortly before the administration (i.e. 50% concentration was obtained by mixing of 1mL of test item with 1mL of vehicle).
The preparations were prepared prior to each dosing.

Dose Administration
The test item was administered at a volume of 25 μL to the dorsum of each ear. The α-Hexyl cinnamaldehyde (25%) as positive control and vehicle as a negative control were administered at the same volume.

Pre-screen test
The pre-screen test was conducted under the same conditions as the main LLNA study, except there was no assessment of lymph node proliferation. The test item was administered undiluted at a concentration of 100%.

Test procedure of Pre-screen test
All mice (2 mice/group) were observed daily for any clinical signs of toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to the termination (day 6). Both ears of each mouse were assessed for any signs of irritation.
Ear thickness was measured by a calliper on Day 1 (pre-dose), Day 3 and Day 6. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥25%.

Main study
Day 1: Each animal was identified and the body weight was recorded. To the dorsum of each ear 25μL of the appropriate dilution of the test item, or the vehicle alone was applied.
Days 2 and 3: The application procedure carried out on day 1 was repeated.
Days 4 and 5: No treatment.
Day 6: The body weight of each animal was recorded. 250μL of phosphate-buffered saline (PBS) containing 2 μCi (7.4 x 104 Bq) of 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine was injected into all test and control mice via the tail vein.
Animals were sacrificed 5 hours post administration of 125I-iododeoxyuridine and fluorodeoxyuridine. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group, (pooled treatment group approach) and weighted.

Preparation of cell suspensions
Cell suspension of lymph node cells from pooled treatment groups were prepared by gentle mechanical disaggregation by glass homogenizer. Lymph node cells were centrifuged (SOPA-00156- BIO) at 600 x g at 4 °C for 10 min. Cell suspensions were precipitated with 5% trichloroacetic acid (TCA) at 4 °C for 18-20h. Pellets were centrifuged by 2000g at 4 °C for 5 min., re-suspended in 1 ml TCA and transferred to gamma counting tubes for 125I-counting.

Determination of cellular proliferation (incorporated radioactivity)
Incorporation of 125I-iododeoxyuridine was measured by 125I-counting on Automatic Gamma Counter Wizard2 (Perkin Elmer) as counts per minute (CPM). The incorporation is expressed as disintegrations per minute (DPM)/treatment group. DPM were calculated according to formula:
DPM= CPM / absolute detector efficient
(Absolute detector efficiency for Gamma Counter Wizard2 2470 = 66.73%)

Clinical Observation
Animals were carefully observed once daily for any clinical symptoms, either of local irritation at the application site or of the systemic toxicity. All observations were systematically recorded with individual records being maintained for each animal.

Body Weight
The animal body weights were measured at the start of the test and at the scheduled day of euthanasia of the animals.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Not specified

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

1.85

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

50%

Cellular proliferation data / Observations:

In comparison with the control group, an increase of the pooled lymph node weight was recorded at all used concentrations. The increase was not dose dependent. The pooled lymph node weights of treated groups were 0.0359g for 10% concentration, 0.0413g for 25% concentration and 0.0364g for 50% concentration of tested item. The lymph node weight of control group and positive control group were 0.0333g and 0.0570g, respectively. The DPM values for the three treated groups were 2499 (10%), 2635 (25%) and 2275 (50%), respectively. The EC3 value could not be calculated, as all measured points were below the stimulation index of three.

Clinical Observations (Hatcol 3178 100%)

Signs/Day

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Mouse 1

Mouse 2

Mouse 1

Mouse 2

Mouse 1

Mouse 2

Mouse 1

Mouse 2

Mouse 1

Mouse 2

Mouse 1

Mouse 2

Apathy

-

-

-

-

++

-

++

-

-

-

-

-

Ataxia

-

-

-

-

-

-

-

-

-

-

-

-

Intake of food

-

-

-

-

No

-

No

-

No

-

-

-

Intake of water

-

-

-

-

No

-

No

-

No

-

-

-

Tremor

-

-

-

-

-

-

-

-

-

-

-

-

Change in grooming activity

-

-

-

-

-

-

-

-

-

-

-

-

Pilo-erection

-

-

-

-

-

-

-

-

-

-

-

-

 

Initial and terminal body weights (g)

Mouse no.	Initial body weight	Terminal body weight
1	17.86	17.16
2	19.17	18.90
Mean	18.52	18.03
S.D.	0.926	1.230

 

Erythema Score

Mouse no.	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
1	0	0	0	0	0	0
2	0	0	0	0	0	0

 

Ear Thickness (mm)

Mouse no.	Day 1 (pre dose)		Day 3		Day 6
1	0.18	0.18	0.18	0.17	0.17	0.17
2	0.18	0.19	0.18	0.19	0.18	0.19
Mean	0.182		0.180		0.178
S.D.	0.005		0.008		0.009

 

Clinical Observations (Hatcol 3178 50%)

Signs/Day

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Mouse 1

Mouse 2

Mouse 1

Mouse 2

Mouse 1

Mouse 2

Mouse 1

Mouse 2

Mouse 1

Mouse 2

Mouse 1

Mouse 2

Apathy

-

-

-

-

-

-

-

-

-

-

-

-

Ataxia

-

-

-

-

-

-

-

-

-

-

-

-

Intake of food

-

-

-

-

-

-

-

-

-

-

-

-

Intake of water

-

-

-

-

-

-

-

-

-

-

-

-

Tremor

-

-

-

-

-

-

-

-

-

-

-

-

Change in grooming activity

-

-

-

-

-

-

-

-

-

-

-

-

Pilo-erection

-

-

-

-

-

-

-

-

-

-

-

-

 

Initial and terminal body weights (g)

Mouse no.	Initial body weight	Terminal body weight
1	19.86	20.17
2	20.93	20.66
Mean	20.40	20.42
S.D.	0.757	0.346

 

Erythema Score

Mouse no.	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
1	0	0	0	0	0	0
2	0	0	0	0	0	0

 

Ear Thickness (mm)

Mouse no.	Day 1 (pre dose)		Day 3		Day 6
1	0.19	0.18	0.19	0.18	0.19	0.19
2	0.19	0.20	0.19	0.19	0.19	0.19
Mean	0.190		0.188		0.190
S.D.	0.008		0.005		0.000

 

Individual body weights (g)

Control (vehicle)

Mouse no.	Initial body weight	Terminal body weight
1	17.68	18.63
2	16.79	17.89
3	18.40	19.17
4	18.58	18.94
5	16.24	16.86
Mean	17.54	18.30
S.D.	0.473	0.938

Positive control

Mouse no.	Initial body weight	Terminal body weight
1	17.22	19.37
2	16.93	20.48
3	16.99	20.12
4	17.48	20.80
5	16.76	20.75
Mean	17.08	20.30
S.D.	0.280	0.588

Hatcol 3178 (50%)

Mouse no.	Initial body weight	Terminal body weight
1	20.75	22.17
2	21.29	22.94
3	19.17	22.17
4	21.45	22.26
5	20.88	21.48
Mean	20.71	22.20
S.D.	0.906	0.518

Hatcol 3178 (25%)

Mouse no.	Initial body weight	Terminal body weight
1	18.66	22.32
2	19.76	21.85
3	19.13	21.49
4	19.45	21.29
5	18.71	22.11
Mean	19.14	21.82
S.D.	0.473	0.426

Hatcol 3178 (10%)

Mouse no.	Initial body weight	Terminal body weight
1	18.37	20.43
2	18.47	21.03
3	17.80	20.28
4	18.11	20.40
5	18.95	21.19
Mean	18.34	20.67
S.D.	0.428	0.413

 

Lymph node weight, DPM, SI, EC3 values

	Lymph node weight (g)	Number of lymph nodes	DPM	SI	EC3
Control	0.0333	10	1425	-	-
Positive control	0.0570	10	9076	6.37
Hatcol 3178 10%	0.0359	10	2499	1.75
Hatcol 3178 25%	0.0413	10	2635	1.85
Hatcol 3178 50%	0.0364	10	2275	1.60

 

Interpretation of results:

GHS criteria not met

Conclusions:

The skin sensitizing potential of Hatcol 3178 was assessed using the murine local lymph node assay (LLNA), according to OECD Guideline No. 429.
Based on the results of this study, Hatcol 3178 is not considered a skin sensitizer under the conditions of this study.

Executive summary:

The sensitization potential of Hatcol 3178 was evaluated using the Local Lymph Node Assay (LLNA). The LLNA has been developed to determine the allergic contact sensitization potential of chemicals.

Based on the recommendations of the OECD Guideline 429, the test item was dissolved in acetone:olive oil 4:1 (v/v). The positive control (α-Hexylcinnamic aldehyde) (25%) was dissolved in the same vehicle.

The Pre-screen test was performed using doses of 100 % (undiluted form) and 50 % (v/v). Based on the observations recorded in the preliminary test, the concentration of 50 % was selected as top dose for the main test.

Five female mice (CBA/Ca) per group were topically exposed (dorsum of both ears) to the test item at concentrations of 10%, 25% and 50%, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/pooled treatment group and compared with DPM value from the vehicle control group and expressed as the Stimulation Index (SI).

After application of the test item at three concentrations (10%, 25% and 50% v/v) the animals did not show visible clinical symptoms of either local irritation or systemic toxicity.

The Stimulation Indices (SI) of 1.75, 1.85 and 1.60 were determined with the test item at concentration of 10, 25, and 50% in acetone:olive oil 4:1, respectively. The EC3 value could not be calculated, as all measured points were below the Stimulation Index of three.

The test item Hatcol 3178 is not considered a skin sensitizer under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin Sensitiation - LLNA

The sensitization potential of Hatcol 3178 was evaluated using the Local Lymph Node Assay (LLNA).

Based on the recommendations of the OECD Guideline 429, the test item was dissolved in acetone:olive oil 4:1 (v/v). The positive control (α-Hexylcinnamic aldehyde) (25%) was dissolved in the same vehicle.

The Pre-screen test was performed using doses of 100 % (undiluted form) and 50 % (v/v). Based on the observations recorded in the preliminary test, the concentration of 50 % was selected as top dose for the main test.

Five female mice (CBA/Ca) per group were topically exposed (dorsum of both ears) to the test item at concentrations of 10%, 25% and 50%, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive 125I-iododeoxyuridine and 10-5M fluorodeoxyuridine in the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/pooled treatment group and compared with DPM value from the vehicle control group and expressed as the Stimulation Index (SI).

After application of the test item at three concentrations (10%, 25% and 50% v/v) the animals did not show visible clinical symptoms of either local irritation or systemic toxicity.

The Stimulation Indices (SI) of 1.75, 1.85 and 1.60 were determined with the test item at concentration of 10, 25, and 50% in acetone:olive oil 4:1, respectively. The EC3 value could not be calculated, as all measured points were below the Stimulation Index of three.

The test item Hatcol 3178 is not considered a skin sensitizer under the test conditions of this study.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test item Hatcol 3178 is not considered a skin sensitizer under the test conditions of the study and therefore does not fulfill the criteria for classification in accordance with CLP.