Dihydro-3-(tripropenyl)furan-2,5-dione

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-05-23 to 2011-10-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

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:

Dihydro-3-(tripropenyl)furan-2,5-dione

EC Number:

295-556-6

EC Name:

Dihydro-3-(tripropenyl)furan-2,5-dione

Cas Number:

92077-08-2

Molecular formula:

R-C4H3O3 , whereas R=C8-C10-alkyl-(branched, unsaturated)

IUPAC Name:

3-[(1E)-2-methyloct-1-en-1-yl]oxolane-2,5-dione

Details on test material:

Name: Tripropenyl succinic anhydride
Product: Genopur ASA
CAS No.: 92077-08-2
Batch No.: ESD0010823
Certificate of Analysis: number or date will be reported in the final report
Chemical Name: dihydro-3-(tripropenyl)furan-2,5-dione
Molecular Weight: ca 220 (calculated value, the exact value will be
defined in the final report)
pH: 1-2
Physical State at RT: liquid
Density: 1.01 g/cm3 (20°C, DIN 51757)
Colour: yellow to brown
Expiry Date: 01.12.2012
Storage Conditions: at room temperature
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. 1125, 1130 and 1307 , main study: lot no. 1125)
- 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. 241110 and 141110, main study: lot no. 241110)
- 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:
6.25 %, 12.5 % and 25 % (v/v)
The preparations were made immediately prior to each dosing.
AOO was used as vehicle and served as negative control.

No. of animals per dose:

5 animals/dose group, 5 animals/control group
9 animals for the preliminary test

Details on study design:

Preliminary Test
In order to determine the highest tolerated and non-irritant test concentration a preliminary test was performed.
For this purpose, six animals were treated by topical application with the test item on three consecutive days at the
following concentrations to the entire dorsal surface of each ear:
Animal no. 1 and no. 2 were treated with a test item concentration of 100% (undiluted).
Animal no. 4 and no. 5 were treated with a test item concentration of 50%, diluted with AOO
(Acetone, Prolabo, lot no. K41154114, expiry date: 06/2015; olive oil highly refined, Sigma, lot no. BCBD1085, expiry date: 06/2011).
Animal no. 7 and no. 8 were treated with a test item concentration of 25%, diluted with AOO.
Three further animals were treated with 100% AOO and served as negative control.
Immediately before the first application, approximately 48 hours after the first application and shortly before
sacrificing the thickness of both ears of the surviving animals was measured.
Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions,
tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).
Animals no. 1 and no. 2 showed sticky fur at the application sites on day two of the preliminary test.
Neither signs of systemic toxicity nor signs of irritation at the application site could be detected in any animal.
On day 3 of the preliminary test, animal no. 1 was found dead and animal no. 2 was moribund and therefore
had to be euthanised due to animal welfare reasons.
Animals no. 4 and no. 5 showed hair loss, severe eschar and desquamation on day 6.
Animals no. 7 and no. 8 showed desquamation on day 6
One animal from the 25% group showed a weight loss of 3 g. All other animals showed the expected weight development,
which includes a weight loss of up to 2 g throughout the duration of the preliminary test.

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

Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity,
including dermal irritation at site of application.

Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

Dose Groups
3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.

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.
In certain situations where the dose response does not incorporate a data point lying below the SI value of three, provided the data are of
good quality (relatively close to an SI of three and evidence of a dose response), an EC3 value may be estimated by using the two doses
closest to the SI value of three. The EC3 value is estimated by log-linear interpolation between these two points on a plane where
the x-axis represents the dose level and the y-axis represents the SI. The point with the higher SI is denoted (a,b) and the point with
the lower SI is denoted (c,d). The formula for the EC3 estimate is as follows: EC3=2^{(log2(c)+(3-d)/(b-d)*[(log2(a)-log2(c)]},
by log-transforming the doses, EC3 estimates will never fall below zero.
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).
On the basis of the test results, the following risk phrases may be assigned in conformity with the criteria given in Commission
Regulation (EU) No 286/2011 as well as in OECD-GHS - Globally Harmonised System of Classification and Labelling of Chemicals,
third revised edition, July 2009:
Skin sensitiser
Category 1:
A substance is classified as a skin sensitiser
a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons, or
b) if there are positive results from an appropriate animal test.
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1A:
Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have
the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.
EC3 value ≤ 2%
WARNING, exclamation mark. May cause an allergic skin reaction.
 
Sub-category 1B:
Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals
can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.
EC3 value > 2%
WARNING, exclamation mark. May cause an allergic skin reaction.

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:

The recent reliability check was performed in July 2011.
The raw data of this study are kept in the BSL archives (BSL Project ID 110336 T).
Positive-control substance: P-Phenylenediamine (CAS 106-50-3, Sigma, purity > 98%; Lot 069K0076) 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 14.3

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Each of the three tested concentrations exceeded the stimulation index of 3. The stimulation index at a concentration of 6.25% was 32.6 The stimulation index at a concentration of 12.5% was 43.5 The stimulation index at a concentration of 25% was 40.1 All animals survived throughout the test period without showing any clinical signs.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Test Item Conc. [%] Animal number DPM Negative 16 1068.0 Control 17 875.0 18 3101.0* 19 1230.0 20 1139.0 MV 1078.0 SD 130.5 Tripropenyl 6.25 1 29128.0 2 34742.0 3 24534.0 4 48423.0 5 36803.0 MV 34726.0 SD 8088.7 Tripropenyl 12.5 6 47698.0 7 46643.0 9 43765.0 10 54537.0 MV 46328.4 SD 5092.1 Tripropenyl 25 11 41976.0 12 36813.0 13 50026.0 14 39867.0 15 44499.0 MV 42636.2 SD 4474.2 * = outlier, failed Grubbs, Nalimov and Dixon; n.d. = not determined

Any other information on results incl. tables

Table Ear Thickness – Preliminary Test (mm)

		Measurement of Ear Thickness (mm)
Group	Animal	Day 1		Day 3		Day 6
	No.	left	right	left	right	left	right
Tripropenyl succinic anhydride 100%	1	0.18	0.18	†	*	*	*
Tripropenyl succinic anhydride 100%	2	0.18	0.19	††	*	*	*
Negative Control 100% AOO	3	0.18	0.19	*	*	*	*
Tripropenyl succinic anhydride 50% in AOO	4	0.20	0.21	0.21	0.21	0.22	0.22
Tripropenyl succinic anhydride 50% in AOO	5	0.22	0.21	0.22	0.21	0.23	0.22
Negative Control 100% AOO	6	0.21	0.21	0.21	0.21	0.21	0.21
Tripropenyl succinic anhydride 25% in AOO	7	0.18	0.17	0.19	0.18	0.20	0.20
Tripropenyl succinic anhydride 25% in AOO	8	0.17	0.17	0.19	0.20	0.20	0.18
Negative Control 100% AOO	9	0.20	0.19	0.19	0.19	0.19	0.19
†= animal was found dead;††= animal was euthanised due to animal welfare reasons; * = no data available

Table Absolute Body Weights – Preliminary Test (g)

Concentration	Animal No.	Start of Study	End of Study	Weight Gain
Tripropenyl succinic anhydride 100%	1	20	†	*
Tripropenyl succinic anhydride 100%	2	19	††	*
Negative Control 100% AOO	3	21	*	*
Tripropenyl succinic anhydride 50% in AOO	4	18	18	0
Tripropenyl succinic anhydride 50% in AOO	5	19	19	0
Negative Control 100% AOO	6	19	20	1
Tripropenyl succinic anhydride 25% in AOO	7	24	21	-3
Tripropenyl succinic anhydride 25% in AOO	8	23	22	-1
Negative Control 100% AOO	9	20	20	0
†= animal was found dead;††= animal was euthanised due to animal welfare reasons;*= no data available

Table 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
6	722.0			Negative		6	1492.0	1478.0	739.0
7	660.0			Control		7	1368.0	1354.0	677.0
8	906.0					8	1881.0	1867.0	933.5
9	487.0					9	1007.0	993.0	496.5
10	919.0					10	1904.0	1890.0	945.0
MV	738.8					MV	1530.4	1516.4	758.2	1.0
SD	161.4					SD	335.9	335.9	168.0
13	13285.0			Phenylene-	1	11	27709.0	27695.0	13847.5	18.3
14	8590.0			diamine		12	17850.0	17836.0	8918.0	11.8
15	12542.0					13	26145.0	26131.0	13065.5	17.2
16	10239.0					14	21597.0	21583.0	10791.5	14.2
17	7404.0					15	15359.0	15345.0	7672.5	10.1
MV	10412.0					MV	21732.0	21718.0	10859.0	14.3
SD	2244.5					SD	4709.6	4709.6	2354.8	3.1
66	7.0			Background			15.0
67	8.0			Szinti and			16.0
68	7.0			TCA			13.0
69	7.0						14.0
70	6.0						12.0
MV	7.0					MV	14.0	0.0	0.0	0.0
SD	0.6					SD	1.4

Table Radioactive Determination of the Test Substance Groups

POS	CPM			Test Item	Conc. [%]	Animal number	DPM	DPM- mean back- ground	DPM/ Node	Stimu-lation Index
6	511.0			Negative		16	1068.0	1053.6	526.8
7	421.0			Control		17	875.0	860.6	430.3
8	1477.0*					18	3101.0*	n.d.	n.d.
9	588.0					19	1230.0	1215.6	607.8
10	547.0					20	1139.0	1124.6	562.3
MV	516.8					MV	1078.0	1063.6	531.8	1.0
SD	61.6					SD	130.5	130.5	65.3
49	13757.0			Tripropenyl	6.25	1	29128.0	29113.6	14556.8	27.4
50	16315.0			succinic		2	34742.0	34727.6	17363.8	32.7
51	11626.0			anhydride		3	24534.0	24519.6	12259.8	23.1
52	22916.0			in AOO		4	48423.0	48408.6	24204.3	45.5
53	17112.0					5	36803.0	36788.6	18394.3	34.6
MV	16345.2					MV	34726.0	34711.6	17355.8	32.6
SD	3814.0					SD	8088.7	8088.7	4044.4	7.6
54	22276.0			Tripropenyl	12.5	6	47698.0	47683.6	23841.8	44.8
55	22057.0			succinic		7	46643.0	46628.6	23314.3	43.8
56	18459.0			anhydride		8	38999.0	38984.6	19492.3	36.7
57	20419.0			in AOO		9	43765.0	43750.6	21875.3	41.1
58	25646.0					10	54537.0	54522.6	27261.3	51.3
MV	21771.4					MV	46328.4	46314.0	23157.0	43.5
SD	2372.7					SD	5092.1	5092.1	2546.0	4.8
61	19812.0			Tripropenyl	25	11	41976.0	41961.6	20980.8	39.5
62	17891.0			succinic		12	36813.0	36798.6	18399.3	34.6
63	23553.0			anhydride		13	50026.0	50011.6	25005.8	47.0
64	18665.0			in AOO		14	39867.0	39852.6	19926.3	37.5
65	20915.0					15	44499.0	44484.6	22242.3	41.8
MV	20167.2					MV	42636.2	42621.8	21310.9	40.1
SD	1979.2					SD	4474.2	4474.2	2237.1	4.2
66	6.0			Background			13.0
67	8.0			Szinti and			17.0
68	8.0			TCA			16.0
69	6.0						12.0
70	7.0						14.0
MV	7.0					MV	14.4	0.0	0.0	0.0
SD	0.9					SD	1.9
* = outlier, failed Grubbs, Nalimov and Dixon;  n.d. = not determined

Table Absolute Body Weights in g

Concentration	Animal No.	Start of Study	End of Study	Weight Gain
Tripropenyl succinic	1	21	22	1
anhydride	2	19	21	2
	3	18	20	2
	4	19	20	1
6.25% in AOO	5	20	21	1
Tripropenyl succinic	6	20	21	1
anhydride	7	17	18	1
	8	20	23	3
	9	17	19	2
12.5% in AOO	10	17	19	2
Tripropenyl succinic	11	20	21	1
anhydride	12	19	21	2
	13	19	20	1
	14	19	21	2
25% in AOO	15	20	22	2
	16	18	20	2
Negative	17	21	21	0
Control	18	22	23	1
100% AOO	19	20	23	3
	20	18	20	2

Individual Weight of Each Lymph Node and Means of Animals and Groups

Group	Animal	Left lymph	Right lymph	Mean of	Mean of
	No.	node weight (mg)	node weight (mg)	lymph node weights per animal (mg)	test group (mg)
	1	10.8	11.7	11.3
Tripropenyl succinic	2	10.5	9.0	9.8
anhydride	3	10.7	9.2	10.0	11.5
6.25% in AOO	4	13.3	12.4	12.9
	5	12.7	14.2	13.5
	6	14.2	17.5	15.9
Tripropenyl succinic	7	12.5	13.0	12.8
anhydride	8	11.2	14.5	12.9	13.8
12.5% in AOO	9	13.8	13.1	13.5
	10	14.5	13.6	14.1
	11	13.3	14.0	13.7
Tripropenyl succinic	12	12.4	15.7	14.1
anhydride	13	14.5	14.6	14.6	14.2
25% in AOO	14	12.5	17.4	15.0
	15	13.0	14.1	13.6
	16	2.4	3.1	2.8
Negative Control	17	2.1	3.1	2.6
	18	4.3	2.9	3.6	2.9
100% AOO	19	2.4	3.0	2.7
	20	2.6	3.1	2.9

Table Clinical Observation

Time of Observation	Systemic Effects	Local Effects
Group 1, animals no. 1 – 5 / test item at a concentration of 6.25% 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 2, animals no. 6 – 10 / 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 3, animals no. 11 – 15 / 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 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:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The registered substance was considered to be a skin sensitizer. The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 0.03%.
The results of the radioactivity determination are supported by the second endpoint, the means of the lymph node weights per group,
which showed dose-related increased values compared to the negative control values.

Executive summary:

The skin sensitisation potential of the registered substance was investigated in a local lymph node assay.

Based on the results of the preliminary test the test item was assessed for sensitising properties at concentrations of 6.25 %, 12.5 % and 25 % (v/v) in AOO (4:1 (v/v) acetone/olive oil). At the daily clinical observation the animals did not show any visible clinical symptoms and no case of mortality was observed.

Summary Results:

The mean weight of the lymph nodes

for the 6.25% test group was 11.5 mg

for the 12.5% test group was 13.8 mg

for the 25% test group was 14.2 mg

for the negative-control group was 2.9 mg

Each of the three tested concentrations exceeded the stimulation index of 3.

The stimulation index at a concentration of 6.25% was 32.6

The stimulation index at a concentration of 12.5% was 43.5

The stimulation index at a concentration of 25% was 40.1

Conclusion

On the basis of the test results obtained in the study, the test item is considered to be a skin sensitiser. The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 0.03%.

The results of the radioactivity determination are supported by the second endpoint, the means of the lymph node weights per group,

which showed dose-related increased values compared to the negative control values.