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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Analytical purity: 97%
- Source: Aldrich Chemical Company, Milwaukee, WI, USA
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:DSM Engineering Plastics
- Expiration date of the lot/batch:SNO620150908
- Purity test date:100%



STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2—8°C)
- Stability under test conditions: temp < 150 C

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Source: Janvier, Le Genest-Saint—lsle, France
- Females (if applicable) nulliparous and non-pregnant: [yes/]

- Age at study initiation:approx. 10 weeks old
- Weight at study initiation: Body weight
variation was within +/- 20% of the sex mean.
- Housing: Animals were group housed in labeled Makrolon cages (Mlll type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Sbhne GmbH + CO. KG,Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon Mll type cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet : Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiaten GmbH, Soest, Germany).
- Water : Free access to tap water.
- Acclimation period:
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%,
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle.

Study design: in vivo (non-LLNA)

No. of animals per dose:
Three groups of five animals were treated with one test item concentration per group.
Positive control substance(s):
yes

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
No irritation was observed in any of the pre-screen animals and variations in ear thickness during the
observation period were less than 25% from Day 1 pre-dose values.
Based on these results, the highest test item concentration selected for the main study was a 10%
5% and 10% was considered not
to have a toxicologically significant effect on the activity of the nodes.
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility:
- Irritation:
- Systemic toxicity:5% and 10% was considered not to have a toxicologically significant effect on the activity of the nodes.
- Ear thickness measurements:
- Erythema scores:

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:

Results and discussion

In vivo (LLNA)

Results
Key result
Parameter:
SI
Value:
0.8
Variability:
+/_ 0. 2

Any other information on results incl. tables

8.1. Pre-screen Test

Both animals treated at 25% were found dead on Day 2.

No irritation was observed in any of the pre-screen animals and variations in ear thickness during the

observation period were less than 25% from Day 1 pre-dose values.

Based on these results, the highest test item concentration selected for the main study was a 10%

concentration.

8.2. Main Study

8.2.1. Skin Reactions I Irritation

The slight irritation of the ears as shown by some animals treated at 5% and 10% was considered not

to have a toxicologically significant effect on the activity of the nodes.

8.2.2. 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.

8.2.3. Macroscopic Examination of the Auricular 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.

8.2.4. Radioactivity Measurements and SI Values

Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and

10% were 730, 952 and 974 DPM, respectively. The mean DPM/animal value for the vehicle control

group was 866 DPM. The SI values calculated for the test item concentrations 2, 5 and 10% were 0.8,

1.1 and 1.1, respectively.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Since there was no indication that Succinonitrile could elicit a SI 2 3 when tested up to 10%, it was
established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any)
exceeds 10%.
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node
Assay as performed at WIL Research Europe is an appropriate model for testing for contact
hypersensitivity (see APPENDIX 2).
Based on these results, Succinonitrile 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 (2011) (including all
amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of
items and mixtures (including all amendments).
Executive summary:

Assessment of skin sensitization to Succinonitrile in the Mouse (Local Lymph Node Assay).

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

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

EC, No 440/2008; B42: "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.

In the main study, three experimental groups of five female CBA/J mice were treated with test item

concentrations of 2, 5 or 10% w/w on three consecutive days, by open application on the ears. Five

vehicle control animals were similarly treated, but with the vehicle alone (N,N-Dimethyl formamide).

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.

The slight irritation of the ears as shown by some animals treated at 5% and 10% was considered not

to have a toxicologically significant effect on the activity of the nodes.

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 2, 5 and

10% were 730, 952 and 974 DPM, respectively. The mean DPM/animal value for the vehicle control

group was 866 DPM. The SI values calculated for the test item concentrations 2, 5 and 10% were 0.8,

1.1 and 1.1, respectively.

Since there was no indication that Succinonitrile could elicit a SI 2 3 when tested up to 10%, it was

established that the EC3 value (the estimated test item concentration that will give a Si =3) (if any)

exceeds 10%.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node

Assay as performed at WlL Research Europe is an appropriate model for testing for contact

hypersensitivity.

Based on these results, Succinonitrile 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 (2011) (including all

amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of

items and mixtures (including all amendments).