Succinic anhydride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-01-26 to 2010-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

24 April 2002

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Succinic anhydride

EC Number:

203-570-0

EC Name:

Succinic anhydride

Cas Number:

108-30-5

Molecular formula:

C4H4O3

IUPAC Name:

oxolane-2,5-dione

Specific details on test material used for the study:

- White flakes with pungent odour.
- Solubility in water of 50 g/L (20° C, hydrolised)
- Density of 1.2340.
- Melting point of 119.6 C.
- Stable at room temperature.
- Name of test material (as cited in study report):succinic anhydride
- Substance type: dihydro-2,5-furandion cyclic acid anhydride
- Physical state: solid
- Analytical purity: 99.5%
- Purity test date: 22 October 2009
- Lot/batch No.: LEBA5A3070
- Expiration date of the lot/batch: 30 June 2010
- Stability under test conditions: stable
- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy, 1-33049 San Pietro all \latisone (UD).
- Females nulliparous and non-pregnant: yes
- Age at study initiation: About 8 weeks at the first administration.
- Weight at study initiation: 17.8 -22.2 g.
- Housing: Mice were individually housed in Makrolon cages type II, (22 cm x 16.S cm ground area, 15 cm high). Bedding material consisted of Aspen wood chips (Fa. ABEDD Dominik Mayr KEG, A-8580 Köflach). Germ reduction by autoclaving; Changed 1/week. Random samples of the bedding material are analysed for contaminants by the supplier. One sample is analysed also for contaminants in addition by an independent external laboratory. The limits of tolerance are the same as with the feed.
- Diet: Maintenance diet for rats and mice R/M-H (item V1534-300), autoclavable, ad libitum.
Random samples of the feed are analysed for contaminants by the manufacturer: Ssniff Spezialdiaten GmbH, D-59494 Soest. One sample is analysed also for contaminants in addition by an independent external laboratory. The limits of tolerance are derived from the "Deutsche Futtermittelverordnung" (German feed regulation).
- Water (e.g. ad libitum): Tap water from Makrolon-bottles with stainless steel canules, ad libitum.
Random samples of the water are analysed for contaminants and germ content by the "AGES", A-1226 Vienna. The limits of tolerance are identical with that used for drinking water for humans in Austria.
- Acclimation period: Mice were acclimated for 13 days
- Indication of any skin lesions: not specifed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Average of 21.2°C (continuous monitoring and recording).
- Humidity (%): Average of 58.9 % (continuous monitoring and recording).
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): Only artificial light from 6.00 a.m. to 6.00 p.m.
- IN-LIFE DATES: From: 2010-01-13 To: 2010-02-01

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

10, 25, and 31.3% (w/w) succinic anhydride

No. of animals per dose:

5 animals/ group (including spare animals). The spare animal of the low dose group died and therefore, the spare animals of other groups were not used. The number of animals/group used for lymph node examination was 4.

Details on study design:

Solubility testing of the test substance showed the highest concentration suitable for application of the test substance can be achieved with DMF (31.3%, w/w).

A range finding study was performed with 2 animals/concentration at 31.3 and 25% succinic anhydride. None of the animals showed overt systemic toxicity, excessive local skin irritation at the application sites or an important increase in ear thickness in the range finding study. Therefore, 31.3% was chosen as the highest test substance concentration and ear thickness measurement was not performed in the main study.

Main study: Test solutions were prepared fresh on each day of administration and administered in 3 concentrations to the dorsal surfaces of the ears of mice. One negative control group and one positive control group were treated with dimethylformamide (DMF) and hexyl cinnamic aldehyde (HCA), respectively. Each animal was treated for 3 consecutive days. 3 days after the last topical administration, each animal received 20 uCi 3HTdR by slow intravenous administration via the tail vein. Approximately 5 hours after 3HTdR injection, all animals were sacrificed by carbon dioxide asphyxiation and the draining auricular lymph nodes were rapidly excised. The proliferation of the lymphocytes of the draining auricular lymph node was measured by the determination of the amounts of incorporated 3HTdR.

Positive control substance(s):

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

Statistics:

Stimulation Indices were calculated according to the standard method (see more details in section "Any other information on materials and methods incl. tables")

Results and discussion

Positive control results:

Application of 25% HCA in acetone:olive oil (4:1 v/v) resulted in an SI of 7.3.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Test group / Remarks:

negative control

Remarks on result:

other: dpm: 4733

Results 2

Key result

Parameter:

SI

Value:

9.2

Test group / Remarks:

low dose (10% test substance)

Remarks on result:

other: dpm: 43520

Results 3

Key result

Parameter:

SI

Value:

11.6

Test group / Remarks:

mid dose (25% test substance)

Remarks on result:

other: dpm: 54949

Results 4

Key result

Parameter:

SI

Value:

11

Test group / Remarks:

high dose (31.3% test substance)

Remarks on result:

other: dpm: 52036

Results 5

Parameter:

SI

Value:

7.3

Test group / Remarks:

positive control (25% HCA)

Remarks on result:

other: 34509

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION :
- The SIs of the test substance groups were between 9.2 and 11.6.

CLINICAL OBSERVATIONS:
- No abnormal behaviour or clinical signs were detected during the experiment in the animals.
- No local irritations were observed at the application sites of all animals of all test substance groups and the negative control group throughout the whole study. On Days 3 and 4 all animals of the positive control group had slight erythema on the application sites indicating slight irritative skin reactions.
- One animal (No. 10) of the low dosed group died on Day 6 near the application of 3H-methyl thymidine. Since this animal showed no adverse effects and no body mass loss during the study and the gross necropsy was without observation this death is assumed as random event which is not associated with the test substance application.

BODY WEIGHTS:
- Body masses and body mass gains of all animals were in the range to be expected from animals of the same strain, sex and age.

Any other information on results incl. tables

Table 1. Body mass index of animals on days 1 and 6

Negative control				Low dose				Mid dose				High dose				Positive control
No.	b.m. day 1	b.m. day 6	b.m. gain	No.	b.m.day 1	b.m.day 6	b.m.gain	No.	b.m.day 1	b.m.day 6	b.mgain	No.	b.m.day 1	b.m.day 6	b.m.gain	No.	b.m.day 1	b.m.day 6	b.m.gain
1	19.8	21.2	1.4	6	19.1	20.1	1.0	11	21.5	22.8	1.3	16	22.2	23.2	1.0	21	19.8	20.9	1.1
2	19.0	19.5	0.5	7	17.8	19.4	1.6	12	19.2	20.3	1.1	17	20.0	20.7	0.7	22	20.2	21.2	1.0
3	20.7	21.6	0.9	8	20.6	21.6	1.0	13	18.9	20.3	1.4	18	18.3	20.0	1.7	23	19.0	20.7	1.7
4	19.1	20.3	1.2	9	20.8	21.6	0.8	14	19.6	20.9	1.3	19	20.2	20.9	0.7	24	19.3	20.9	1.6
Mean	19.7	20.7	1.0	Mean	19.6	20.7	1.1	Mean	19.8	21.1	1.3	Mean	20.2	21.2	1.0	Mean	19.6	20.9	1.4
SD	0.8	0.9	0.4	SD	1.4	1.1	0.3	SD	1.2	1.2	0.1	SD	1.6	1.4	0.5	SD	0.5	0.2	0.4
n	4	4	4	n	4	4	4	n	4	4	4	n	4	4	4	n	4	4	4
No. = Animal number; b.m.= body mass in g

Table 2. General observations and observations of the application sites on days 1 -6

Finding	Group	No. of the affected animals	Observation time (day)
General behavior normal	Negative control	1, 2, 3, 4	1 -6
	Low dose	6, 7, 8, 9	1 -6
	Mid dose	11, 12, 13, 14	1 -6
	High dose	16, 17, 18, 19	1 -6
	Positive control	21, 22, 23, 24	1 -6
Application sites normal	Negative control	1, 2, 3, 4	1 -6
	Low dose	6, 7, 8, 9	1 -6
	Mid dose	11, 12, 13, 14	1 -6
	High dose	16, 17, 18, 19	1 -6
Slight erythma	Positive control	21, 22, 23, 24	3 -4

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The results from this local lymph node assay (OECD 429) indicate that succinic anhydride is a skin sensitizer based on stimulation indices of 9.2, 11.6, and 11.0, calculated for the low(10%), mid (25%) and high dose (31.3%) groups, respectively.
No clear dose response relationship was observed. Potency categorisation for skin sensitisation according to ECHA Guidance R.8, Appendix R. 8 -10, Table R. 8 -23 was not possible, since no EC3 value could be calculated for succinic anhydride from the data obtained in this study.

Executive summary:

In a dermal sensitization study conducted according to OECD TG 429 with succinic anhydride (99.5%) in dimethlformamide, three groups of 4 female young adult CBA/CaOlaHsd mice/dose group were tested in the local lymph node assay.

Succinic anhydride was administered epicutaneously to the dorsal surface of both ears, once a day on three consecutive days, at concentrations of 10%, 25%, or 31.3% (w/w). The volume administered was 25 uL per ear. Positive (25% hexyl cinnamic aldehyde in acetone:olive oil) and negative control (dimethylformamide) groups were tested concurrently under identical conditions. Five days after topical administration, 3H-methyl thymidine was administered intravenously to all mice via a tail vein. Approximately 5 hours later, the draining auricular lymph nodes were excised, incorporation of radiolabelled 3HTdR was determined and compared with negative controls, and the SI index was calculated.

No adverse effects were noted in any animal. The SIs of the test substance concentrations were 9.2 (low dose), 11.6 (mid dose), and 11.0 (high dose). Although no clear dose response was observed, the SIs of all test substance concentrations were greater than 3 and succinic anhydride is regarded as a sensitizer in the LLNA.

Based on these results, classification of succinic anhydride as skin sensitizer Category 1 according to the CLP criteria set out in Regulation (EC) 1272/2008 is warranted.