Fatty acids, C16-18, reaction products with diethanolamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-07-25 to 2012-11-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18, reaction products with diethanolamine

EC Number:

293-014-3

EC Name:

Fatty acids, C16-18, reaction products with diethanolamine

Cas Number:

91032-08-5

Molecular formula:

C22H45NO3 to C52H101NO5

IUPAC Name:

Fatty acids, C16-18, reaction products with diethanolamine

Test material form:

solid: particulate/powder

Details on test material:

Name: Fatty acids, C16-18-, reaction products with diethanolamine
Product: Cepreton AT liq. conc.
Specification: Amide Intermediate of Cepreton AT (REACH)
CAS No.: 91032-08-5
Batch No.: ITA0058330
Physical State: solid
Colour: yellow
Molecular Weight: ca. 610-872
pH: 7-8
Active Components: > 99%
Storage Conditions: at room temperature, protected from light
Date of Analysis: 01.02.2012
Expiry Date: 11.01.2013
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (preliminary test: lot no. 0856, main study: lot no. 0856)
- Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control,
microbiological controls at re gular intervals)
- The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
(preliminary test: lot no. 300512, main study: lot no. 300512)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days) under laboratory conditions

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Based on the results observed in the preliminary test the following test item concentrations were selected for the main study:

12.5%, 25% and 50% (w/v), each diluted with AOO 4:1 (v/v).

No. of animals per dose:

5 animals/dose group, 5 animals/control group

Details on study design:

Preparation of the Test Item
Based on the results observed in the prescreen test the following test item concentrations were selected for the main study:
12.5%, 25% and 50% (w/v)
The preparations were made by subjecting them to ultrasonic bath at 37 oC immediately prior to each dosing.

Preparation of the Animals
The animals were randomly selected.
Identification was ensured by cage number and individual marking (tail).

Test Regime
Topical Application
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days.
Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein)
of 250 µL of 3H-methyl thymidine, diluted to a working concentration of 80 µCi/mL.
Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation.
The draining auricular lymph nodes were excised, weighed, individually pooled for each animal (2 lymph nodes per animal) and collected
in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation
through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant
was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of
macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this
solution was transferred into scintillation vials and stored at room temperature overnight.
Determination of Incorporated 3H -Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM).
Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually
for each animal.

Evaluation of Results
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations
per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of
test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined,
background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation,
EC3=c+[(3-d)/(b-d)]x(a c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three.
If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in
3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of
control group animals (Stimulation Index equal to or greater than 3.0).

Positive control substance(s):

other: Phenylenediamine

Statistics:

Outlier tests according to Dixon, Grubbs and Nalimov were performed for the values measured for the number of disintegrations per minute (DPM).
If outliers were identified, these values were not included in the calculation of the stimulation indices.
As at least four values per group are required for the evaluation of the results, the outlier test was not repeated to detect further outliers.

Results and discussion

Positive control results:

Reliability Check
The recent reliability check was performed in August 2012. The raw data of this study are kept in the BSL archives (BSL Project ID 121329S).
Positive-control substance: P-Phenylenediamine (CAS 106-50-3, Sigma, purity > 98%; Lot 060M0186V6) 1%
Vehicle: AOO (4:1 (v/v) acetone/olive oil)
Species/strain: healthy CBA/CaOlaHsd mice
Source: Harlan Winkelmann GmbH, 33178 Borchen, Germany
Concentrations: 1% on three consecutive days

The Stimulation Index was 10.7

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.7

Test group / Remarks:

50 % test group

Results 2

Key result

Parameter:

SI

Value:

2.5

Test group / Remarks:

50 % test group

Results 3

Key result

Parameter:

SI

Value:

2.1

Test group / Remarks:

12.5 % Test group

Any other information on results incl. tables

Ear Thickness – Preliminary Test (mm)

	Animal No.	Measurement of Ear Thickness (mm)
Concentration		Day 1		Day 3		Day 6
		left	right	left	right	left	right
Fatty acids, C16-18-,  reaction products  with diethanolamine 50% in AOO	1	0.22	0.22	0.22	0.20	0.21	0.20
Fatty acids, C16-18-,  reaction products  with diethanolamine 50% in AOO	2	0.18	0.20	0.22	0.19	0.20	0.19
Negative Control 100% AOO	3	0.22	0.20	0.21	0.23	0.20	0.21

Absolute Body Weights – Preliminary Test (g)

Concentration	Animal No.	Start of Study	End of Study	Weight Gain
Fatty acids, C16-18-,  reaction products  with diethanolamine 50% in AOO	1	22	22	0
Fatty acids, C16-18-,  reaction products  with diethanolamine 50% in AOO	2	24	23	-1
Negative Control 100% AOO	3	23	24	1

Radioactive Determination of the Positive-Control Group of the Recent Study

POS	CPM			Test Item	Conc. [%]	Animal number	DPM	DPM- mean back- ground	DPM/ Node	Stimu-lation Index
21	437.0			Negative		21	881.0	847.6	423.8
22	825.0			Control		22	1656.0	1622.6	811.3
23	825.0					23	1684.0	1650.6	825.3
24	1163.0					24	2382.0	2348.6	1174.3
25	956.0					25	1939.0	1905.6	952.8
MV	841.2					MV	1708.4	1675.0	837.5	1.0
SD	236.9					SD	488.8	488.8	244.4
26	5415.0			Phenylene-	1	26	11113.0	11079.6	5539.8	6.6
27	3832.0			diamine		27	7978.0	7944.6	3972.3	4.7
28	8496.0					28	17266.0	17232.6	8616.3	10.3
29	8479.0					29	17464.0	17430.6	8715.3	10.4
30	8535.0					30	17683.0	17649.6	8824.8	10.5
MV	6951.4					MV	14300.8	14267.4	7133.7	8.5
SD	1965.6					SD	4009.4	4009.4	2004.7	2.4
116	19.0			Background			38.0
117	15.0			Szinti and			30.0
118	19.0			TCA			39.0
119	16.0						33.0
120	13.0						27.0
MV	16.4					MV	33.4	0.0	0.0	0.0
SD	2.3					SD	4.6

Radioactive Determination of the Test Substance Groups

POS	CPM	Test Item	Conc. [%]	Animal number	DPM	DPM- mean back- ground	DPM/ Node	Stimu-lation Index
16	524.0	Negative		16	1081.0	1066.0	533.0
17	612.0	Control		17	1278.0	1263.0	631.5
18	562.0			18	1169.0	1154.0	577.0
19	563.0			19	1147.0	1132.0	566.0
20	446.0			20	921.0	906.0	453.0
MV	541.4			MV	1119.2	1104.2	552.1	1.0
SD	55.3			SD	117.7	117.7	58.8
11	882.0	Fatty acids, C16-18-,	12.5	1	1838.0	1823.0	911.5	1.7
12	843.0	reaction products		2	1726.0	1711.0	855.5	1.5
13	433.0	with diethanolamine		3	885.0	870.0	435.0	0.8
14	1898.0	in AOO		4	3938.0	3923.0	1961.5	3.6
15	1639.0			5	3418.0	3403.0	1701.5	3.1
MV	1139.0			MV	2361.0	2346.0	1173.0	2.1
SD	543.7			SD	1136.6	1136.6	568.3	1.0
6	1675.0	Fatty acids, C16-18-,	25	6	3507.0	3492.0	1746.0	3.2
7	1595.0	reaction products		7	3261.0	3246.0	1623.0	2.9
8	1166.0	with diethanolamine		8	2427.0	2412.0	1206.0	2.2
9	1160.0	in AOO		9	2388.0	2373.0	1186.5	2.1
10	1011.0			10	2092.0	2077.0	1038.5	1.9
MV	1321.4			MV	2735.0	2720.0	1360.0	2.5
SD	263.2			SD	548.0	548.0	274.0	0.5
1	959.0	Fatty acids, C16-18-,	50	11	1970.0	1955.0	977.5	1.8
2	737.0	reaction products		12	1503.0	1488.0	744.0	1.3
3	1019.0	with diethanolamine		13	2114.0	2099.0	1049.5	1.9
4	1334.0*	in AOO		14	2780.0*	n.d.	n.d.	n.d.
5	867.0			15	1773.0	1758.0	879.0	1.6
MV	895.5			MV	1840.0	1825.0	912.5	1.7
SD	106.3			SD	229.1	229.1	114.6	0.2
96	8.0	Background			17.0
97	8.0	Szinti and			15.0
98	6.0	TCA			12.0
99	9.0				17.0
100	7.0				14.0
MV	7.6			MV	15.0	0.0	0.0	0.0
SD	1.0			SD	1.9
* = outlier, failed Nalimov;  n.d. = not determined

Absolute Body Weights in g

Concentration	Animal No.	Start of Study	End of Study	Weight Gain
Fatty acids, C16-18-,	1	19	19	0
reaction products	2	20	20	0
with diethanolamine	3	19	19	0
	4	21	21	0
12.5% in AOO	5	23	24	1
Fatty acids, C16-18-,	6	20	21	1
reaction products	7	23	24	1
with diethanolamine	8	20	21	1
	9	21	22	1
25% in AOO	10	21	22	1
Fatty acids, C16-18-,	11	23	24	1
reaction products	12	21	22	1
with diethanolamine	13	17	19	2
	14	22	24	2
50% in AOO	15	20	21	1
	16	21	22	1
Negative Control	17	21	23	2
	18	22	23	1
100% AOO	19	22	23	1
	20	21	22	1

Clinical Observation

Time of Observation	Systemic Effects	Local Effects
Group 1, animals no. 1 – 5 / test item at a concentration of 12.5% in AOO
Day 1	nsf	nsf
Day 2	nsf	nsf
Day 3	nsf	nsf
Day 4	nsf	nsf
Day 5	nsf	nsf
Day 6	nsf	nsf
Group 2, animals no. 6 – 10 / test item at a concentration of 25% in AOO
Day 1	nsf	nsf
Day 2	nsf	nsf
Day 3	nsf	nsf
Day 4	nsf	nsf
Day 5	nsf	nsf
Day 6	nsf	nsf
Group 3, animals no. 11 – 15 / test item at a concentration of 50% in AOO
Day 1	nsf	nsf
Day 2	nsf	nsf
Day 3	nsf	nsf
Day 4	nsf	nsf
Day 5	nsf	nsf
Day 6	nsf	nsf
Group 4, animals no. 16 – 20 / negative control AOO
Day 1	nsf	nsf
Day 2	nsf	nsf
Day 3	nsf	nsf
Day 4	nsf	nsf
Day 5	nsf	nsf
Day 6	nsf	nsf

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3.
Consequently, according to OECD 429 the test item Fatty acids, C16-18-, reaction products with diethanolamine is expected to have no sensitising properties and therefore should not be regarded as a dermal sensitiser.
The results of radioactivity determination are supported by means of the lymph node weights per group, which showed no relevant
difference compared to the negative control.

Executive summary:

Summary Results

The mean weight of the lymph nodes

for the 12.5% test group was 3.1 mg

for the 25% test group was 3.3 mg

for the 50% test group was 2.8 mg

for the negative-control group was 2.8 mg

None of the three tested concentrations of the test item reached the stimulation index of 3.

The stimulation index at a concentration of 12.5% was 2.1

The stimulation index at a concentration of 25% was 2.5

The stimulation index at a concentration of 50% was 1.7

Conclusion

The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations

were below 3. Consequently, according to OECD 429[3]the test item Fatty acids, C16-18-, reaction products with diethanolamine as described in this report is expected to have no sensitising properties and therefore should not be regarded as a dermal sensitiser.

The results of radioactivity determination are supported by the means of the lymph node weights per group, which showed no relevant difference compared to the negative control.