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

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Apr 2018 to 24 Apr 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline 429. Skin Sensitization: Local Lymph Node Assay, July 2010.

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

EC No 640/2012 Part B. Skin Sensitization: Local Lymph Node Assay, July 2012.

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

EPA Health Effects Test Guideline OPPTS 870.2600. Skin Sensitization, March 2003.

Deviations:

not specified

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

268-594-6

EC Name:

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

Cas Number:

68130-51-8

Molecular formula:

C25H44O8 to C45H84O8 (in CH2 unit increments)

IUPAC Name:

3-(heptanoyloxy)-2-[(octanoyloxy)methyl]-2-[(pentanoyloxy)methyl]propyl decanoate; 3-(heptanoyloxy)-2-{[(2-methylbutanoyl)oxy]methyl}-2-[(octanoyloxy)methyl]propyl decanoate

Test material form:

liquid

Details on test material:

Name Hatcol 1570
Batch no. B29855
Appearance slight yellow, clear liquid
CAS No. 68130-51-8
EINECS-No. 268-594-6
Molecular formula C10-H20-O2.C8-H16-O2.C7-H14-O2.C5-H12-O4.C5-H10-O2
Molecular weight not stated
Purity 99.7 %
Homogeneity homogeneous
Vapour pressure not stated
Stability H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated
Solubility H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated
Production date 14. Jun. 2017
Expiry date 14. Jun. 2019
Storage Room Temperature (20 ± 5°C)

Specific details on test material used for the study:

No furthe details specified in the study report.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Species: Mouse
Strain: CBA/J
Condition: Inbred, SPF-Quality
Source: Janvier, Le Genest-Saint-Isle, France
Number of Animals: 20 Females (nulliparous and non-pregnant). Five females per group.
Age at the Initiation of Dosing: Young adult animals (approximately 9 weeks old) were selected.
Weight at the Initiation of Dosing: 17.4 to 21.2 g.

Animal Identification
At study assignment, each animal was identified using a tail mark with indelible ink.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to the study at the discretion of the coordinating biotechnician according to body weights, with all animals within ± 20% of the sex mean. Animals in poor health or at extremes of body weight range were not assigned to the study.
Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.
The disposition of all animals was documented in the study records.

Housing
On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 22 to 23°C with an actual daily mean relative humidity of 41 to 48%. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA), except when interrupted by study procedures/activities.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Pre-screen Test
Two test item concentrations were tested; a 50% and 100% concentration.

Main Study
Three groups of five animals were treated with one test item concentration per group, 10%, 25% and 50%.

No. of animals per dose:

Pre-screen Test
Two young adult animals per concentration were selected.

Main Study
Four groups of five animals (control, 10%, 25% and 50%)

Details on study design:

Pre-screen Test
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Two test item concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines.
The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 10 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.

Main Study
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

Excision of the Nodes - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.

Tissue Processing for Radioactivity - Day 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 μm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

Radioactivity Measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

In-life Procedures, Observations, and Measurements
Mortality/Moribundity Checks
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations
Postdose Observations
Postdose observations were performed once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
All the animals were examined for reaction to dosing. The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing.

Body Weights
Animals were weighed individually on Day 1 (predose) and 6 (prior to necropsy).

Irritation
Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing). According to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.

Terminal procedures
No necropsy was performed, since all animals survived until the end of the observation period.

Positive control substance(s):

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

Statistics:

Not specified

Results and discussion

Positive control results:

An EC3 value of 19.2% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.2, 14.1, 17.3, 9.8, 17.8, 18.0, 14.7 and 13.2%
Based on the results, it was concluded that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

2

Test group / Remarks:

50%

Cellular proliferation data / Observations:

Pre-screen Test
At a 100% test item concentration, variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values and therefore this concentration did not meet the selection criteria.
At a 50% test item concentration, no signs of systemic toxicity were noted and no irritation was observed and therefore this concentration was selected as highest concentration for the main study.

Main Study
Skin Reactions / Irritation
The very slight erythema as noted in two animals treated at 25% and three animals treated at 50% on Days 1 and/or 3 was considered not to have a toxicologically significant effect on the activity of the nodes.

Systemic Toxicity
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Macroscopic Examination of the Lymph Nodes and Surrounding Area
All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity Measurements and SI Values
Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 1147, 1253 and 1542 DPM, respectively. The mean DPM/animal value for the vehicle control group was 765 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.5, 1.6 and 2.0, respectively.

Any other information on results incl. tables

Pre-Screen Test: Body Weight and Skin Reactions

TS^1(%)	Animal	Day 1			Day 2		Day 3		Day 4		Day 5		Day 6
		bw (g)^2	Erythema^3		Erythema		Erythema		Erythema		Erythema		Erythema		bw (g)
			Left	Right	Left	Right	Left	Right	Left	Right	Left	Right	Left	Right
50	1 2	21.6 22.1	0 0	0 0	1 1	1 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	22.7 23.2
100	3 4	20.3 19.7	0 0	0 0	1 1	1 1	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	22.2 20.4

¹TS = test item (% w/w)

²Body weight (grams)

³Grading erythema and eschar formation (left = dorsal surface of left ears; right = dorsal surface of right ear): 0 = No erythema; 1 = Very slight erythema (barely perceptible).

 

Pre-Screen Test: Ear Thickness Measurements

TS^1(%)	Animal	Day 1		Day 3				Day 6
		left (mm)	Right (mm)	Left		Right		Left		Right
				(mm)	%^2	(mm)	%^2	(mm)	%^2	(mm)	%^2
50	1 2	0.225 0.230	0.215 0.235	0.265 0.270	18 17	0.260 0.255	21 9	0.220 0.235	-2 -2	0.230 0.230	7 -2
100	3 4	0.220 0.220	0.220 0.230	0.280 0.285	27 30	0.280 0.270	27 17	0.230 0.225	5 2	0.240 0.225	9 -2

Left (mm) = thickness of left ear in millimeters; right (mm) = thickness of right ear in millimeters.

¹TS = test item (% w/w)

²Percent increase compared to Day 1 pre-dose value. A 25% value is used as the threshold for selection for use in the main study.

 

Main Study: Body Weights and Skin Reactions

Group	TS^1(%)	Animal	Day 1			Day 2		Day 3		Day 4		Day 5		Day 6
			bw (g)^2	Erythema^3		Erythema		Erythema		Erythema		Erythema		Erythema		bw (g)
				Left	Right	Left	Right	Left	Right	Left	Right	Left	Right	Left	Right
1	0	1 2 3 4 5	19.8 17.9 19.4 19.8 19.6	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	21.4 19.2 20.4 22.0 21.0
2	10	6 7 8 9 10	21.2 20.8 18.3 18.8 20.2	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	21.8 21.4 20.0 19.7 21.1
3	25	11 12 13 14 15	19.6 17.4 18.1 21.2 20.6	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 1 0 0 0	1 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	20.8 19.3 18.9 22.0 22.3
4	50	16 17 18 19 20	19.0 19.2 18.9 18.8 18.9	1 0 0 1 0	0 0 0 1 0	0 0 0 0 0	0 0 0 0 0	1 1 0 0 0	1 0 0 1 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	0 0 0 0 0	19.8 20.3 19.2 19.5 19.3

¹TS = test item (% w/w)

²Body weight (grams)

³Grading erythema and eschar formation (Left = dorsal surface of left ear; Right = dorsal surface of right ear): 0 = No erythema; 1 = Very slight erythema (barely perceptible)

 

Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and Stimulation Index (SI)

Group	TS^1(%)	Animal	Size nodes^2		DPM^3/ animal	Mean DPM ± SEM^4	Mean SI ± SEM
			Left	Right
1	0	1 2 3 4 5	n n n n n	n n n n n	755 719 638 1579^5 947	765 ± 65	1.0 ± 0.1
2	10	6 7 8 9 10	n n n n n	n n n n n	1119 1700 1062 864 992	1147 ± 145	1.5 ± 0.2
3	25	11 12 13 14 15	n n n n n	n n n n n	935 2092 1307 1145 786	1253 ± 228	1.6 ± 0.3
4	50	16 17 18 19 20	n n n n n	n n n n n	1196 1202 1349 6202^5 2420	1542 ± 295	2.0 ± 0.4

¹TS = test item (% w/w)

²Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +, ++ or +++: degree of enlargement, n: considered to be normal).

³DPM = Disintegrations per minute

⁴SEM = Standard Error of the Mean.

⁵Value rejected and not used for interpretation (outlier based on the Dixon’s Q-test).

 

SUMMARY RELIABILITY CHECK

Group^1	%HCA	Mean DPM ± SEM	SI ± SEM
1 2 3 4	0% (AcOO) 5% 10% 25%	352 ± 64 485 ± 41 785 ± 142 1243 ± 210	1.0 ± 0.2 1.4 ± 0.1 2.2 ± 0.4 3.5 ± 0.6

¹Five females per group

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 50%, Hatcol 1570 was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 50%.
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
Based on these results, Hatcol 1570 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

The objective of the study was to evaluate whether Hatcol 1570 induces skin sensitization in mice after three epidermal exposures of the animals under the conditions described in this study plan.

The study was carried out based on the guidelines described in:

-OECD, Section 4, Health Effects, No.429 (2010),

-EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay"

-EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

 

Test item concentrations selected for the main study were based on the results of a pre-screen test. The 100% test item concentration did not meet the selection criteria. The 50% test item concentration was selected as highest concentration for the main study.

 

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

 

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 1147, 1253 and 1542 DPM, respectively. The mean DPM/animal value for the vehicle control group was 765 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.5, 1.6 and 2.0, respectively.

 

Since there was no indication that the test item elicits a SI≥3 when tested up to 50%, Hatcol 1570 was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 50%.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Based on these results, Hatcol 1570 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).