Dihexyl fumarate

Endpoint summary

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

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)

Deviations:

yes

Remarks:

During the acclimation phase the relative humidity in the animal room was between approximately 25 - 65 % instead of 45 - 65% for several hours. This deviation to the study plan, however, does not affect the validity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Identity: Dihexyl Fumarate
Batch No.: VE00180024
Purity: 98.1 % dose calculation not adjusted to purity
Stability in solvent: Not indicated by the sponsor
Storage: At room temperature, light protected
Expiration Date: January 07, 2014

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

Test system: Mice, CBA/CaOlaHsd

Rationale: Recognised as the recommended test system

Source: Harlan Laboratories B.V. Postbus 6174
5960 AD Horst / The Netherlands

Number of animals for
the pre-tests: 2 females

Number of animals for the main study:
25 females

Number of animals per group 5 females (nulliparous and non-pregnant) Number of test groups: 3
Number of control (vehicle) groups: 1
Number of positive control groups: 1

Age: 1st pre-test: 10 - 11 weeks (beginning of treatment) 2nd pre-test: 11 - 12 weeks (beginning of treatment) Main study: 9 – 10 weeks (beginning of treatment)

Body weight: See Annex 1

Identification: The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.

Acclimatisation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Husbandry
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: group

Cage Type: Makrolon Type II / III, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)

Bedding: granulated soft wood bedding
(Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)

Feed: pelleted standard diet, ad libitum
(Harlan Laboratories B.V. 5960 AD Horst / Netherlands)

Water: tap water, ad libitum
(Gemeindewerke, 64380 Rossdorf, Germany)

Environment:
temperature 22 + 2°C relative humidity 25-65%
artificial light 6.00 a.m. - 6.00 p.m.

Vehicle:

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

Concentration:

1, 10, 25%

No. of animals per dose:

5

Details on study design:

Allocation
The animals were distributed as follows:

Control Group: 0%(vehicle only), 5 animals
Low Dose Group: 1%, 5 animals
Mid Dose Group: 10 %, 5 animals
High Dose Group: 25%, 5 animals
Positive control Group: 25%, 5 animals

Positive control substance(s):

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

Key result

Parameter:

SI

Value:

0.99

Test group / Remarks:

1% Dihexyl Fumarate

Key result

Parameter:

SI

Value:

1.25

Test group / Remarks:

10% Dihexyl Fumarate

Key result

Parameter:

SI

Value:

2.28

Test group / Remarks:

25% Dihexyl Fumarate

RESULTS

Table 1: Calculation and Results of Individual Data

Vehicle for the test item and positive control: acetone:olive oil

 

Test item concentration	Group No.	Animal No.	DPM values measured	DPM-BG per animal (2 lymph nodes)a)	S.I.b)
% (w/w)
---		BG I	16	---	---
---		BG II	16	---	---
0	1	1	2065	2049	---
0		2	1483	1467	---
0		3	1301	1285	---
0		4	1324	1308	---
0		5	946	930	---
1	2	6	733	717	0.5
1		7	1464	1448	1.0
1		8	2354	2338	1.7
1		9	929	913	0.6
1		10	1573	1557	1.1
10	3	11	814	798	0.6
10		12	1980	1964	1.4
10		13	2491	2475	1.8
10		14	1138	1122	0.8
10		15	2421	2405	1.7
25	4	16	3046	3030	2.2
25		17	1713	1697	1.2
25		18	3265	3249	2.3
25		19	4871	4855	3.4
25		20	3237	3221	2.3
25 (HCA)	5	21	3922	3906	2.8
25 (HCA)		22	12200	12184	8.7
25 (HCA)		23	12672	12656	9.0
25 (HCA)		24	10954	10938	7.8
25 (HCA)		25	10545	10529	7.5

BG =  Background (1 ml 5% trichloroacetic acid) in duplicate

1   =  Control Group for the test item and for the positive control item

2-4 =  Test Groups (w/w)

5   =  Positive Control Group (w/v)

HCA=  25% -α-Hexyl cinnamaldehyde, tech. 85%

S.I. =  Stimulation Index

a)     =  values corrected for mean background value (BGI andBGII).

b)     =  Stimulation Indices relative to the mean of the control group (Group1)

 

Table 2: Calculation of Stimulation Indices per Dose Group

 

Test item concentration	Group Calculation	SD	S.I.
	Mean DPM per animal (2 lymph nodes)a)
Vehicle Control Group for the Test Item (acetone:olive oil)	1407.8	408.6	1.00
1% Dihexyl Fumarate	1394.6	634.5	0.99
10% Dihexyl Fumarate	1752.8	758.5	1.25
25% Dihexyl Fumarate	3210.4	1121.3	2.28
Positive Control Group (25%α-HCA)	10042.6	3539.5	7.13

a)     Mean DPM/animal was determined by dividing the sum of the measured valuesfromlymphnodesofallanimalswithinagroupbythenumberofanimalsinthatgroup(5animals)

The EC3 value could not be calculated, since all S.I.´s, for the test item, are below the threshold value of 3.

 

Viability /Mortality

No deaths occurred during the study period.

 

ClinicalSigns

From day 3 to 6 the animals treated with a test item concentration of 25% showed an erythema of the ear skin (Score 1). On day 4 also the animals treated with 10% test item concentration showed an erythema of the ear skin (Score 1). Animals treated with 1% test item concentration did not show any signs of local skin irritation.

 

BodyWeights

The body weight of the animals, recorded prior to the first application and prior to treatment with3HTdR, was within the range commonly recorded for animals of this strain and age.

 

EarWeight

The measured ear weight of all animals treated was recorded on the day of preparation. An increase in ear weight was observed in the groups treated with 10 and 25% test item concentration in comparison to the vehicle control group. A dose-dependance was not observed. A statistically significant increase in ear weights was observed in all dose groups in comparison to the vehicle control group(p<0.05).

 

EarThickness

The measured ear thickness of all animals treated was recorded prior to the 1st application, on study day 3 and prior to necropsy (day 6). A statistically significant increase in ear thickness was observed in the high dose group in comparison to the vehicle control group (p<0.05).

 

DISCUSSION

In order to study a possible skin sensitising potential of Dihexyl Fumarate, three groups each of five female mice were treated once daily with the test item at a concentrations of 1, 10 and 25% (w/w) in acetone:olive oil (4:1 v/v) by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by the two pre-experiments. One further group of five mice was treated with the vehicle for the test item and positive control (acetone:olive oil (4+1 v/v)) and another group of five mice was treated with the positive control at 25%. Five days after the first topical application the mice were injected intravenously into a tail vein with radio- labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of3H-methyl thymidine measured in ab-scintillationcounter.

From day 3 to day 6, the animals treated with a test item concentration of 25% showed an erythema of the ear skin (Score 1). Animals treated with 10% test item concentration showed an erythema of the ear skin (Score 1) on day 4. The animals treated with 1% did not show any signs of local skin irritation. Animal 24 belonging to the positive control group showed ruffled fur on day 2 and 3. A statistically significant increase in ear weights was observed in all dose groups in comparison to the vehicle control group (p<0.05). A statistically significant increase in ear thickness was observed in the high dose group in comparison to the vehicle control group (p<0.05).

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

 

In this study Stimulation Indices of 0.99, 1.25 and 2.28 were determined with the test item at concentrations of 1, 10 and 25% (w/w) in acetone:olive oil (4:1 v/v). A clear dose response was observed.

 

The S.I. determined for the positive control was 7.13, demonstrating the validity of the study. An outlier was identified in the positive control group (DPM value determined for animal number 21). However, the exclusion of the outlier did not change the overall test result and the value in question was not excluded from calculation.

 

CONCLUSION

The test item Dihexyl Fumarate was not a skin sensitiser under the test conditions of this study.

 

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Dihexyl Fumarate was not a skin sensitiser under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available