Registration Dossier

Administrative data

Description of key information

The SI values calculated for the substance concentrations 5, 10 and 25 % were 1.5, 1.1 and 1.5 respectively.

Since there was no indication that the test substance could elicit an SI >= 3 when tested up to 25 %, it was established that the EC3 value (if any) exceeds 25 %.

The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

Based on these results:

-       according to the recommendations made in the test guidelines, R507 -2 would not be regarded as skin sensitizer.

-       according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2004), R507 -2 does not have to be classified for sensitization by skin contact

-       according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), R507-2 does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact.

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
Study period:
03 December 2007 - 07 January 2008
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:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CSA strain
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River France, L'Arbresle Cedex, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:Young adult animals (approx. 12 weeks old)
- Weight at study initiation:Body weight variation was within +/- 20 % of the sex mean.
- Housing:Individual housing in labelled Macrolon cages (Ml type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment. The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.
- Diet (e.g. ad libitum):Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiaten GmbH, Soest, Germany).
- Water (e.g. ad libitum):Free access to tap water.
- Acclimation period:The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
- Indication of any skin lesions:A health inspection was performed prior to treatment, to ensure that the animals are in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.

ENVIRONMENTAL CONDITIONS
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0 °C (actual range: 19.0-23.1 °C), a relative humidity of 30-70 % (actual range: 43 - 71 %) and 12 hours artificial fluorescent light and 12 hours darkness per day.
Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70 % for relative humidity. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

- IN-LIFE DATES: From: 03 December 2007 To: 07 January 2008
Vehicle:
dimethylformamide
Concentration:
0 %, 5 %, 10 %, 25 %
No. of animals per dose:
5 animals per dose
Details on study design:
PRE-SCREEN TESTS:
A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (grade 2) at the highest.

A series of two test substance concentrations was tested, selected from the series: 100 % (undiluted), 50 %, 25 %, 10 %, 5 %, 2.5 %, 1 % and if needed further lower concentrations using the same steps. The highest concentration, selected from this series, was the maximum concentration that could technically be applied.
The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (8-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the ear was cleaned of residual test substance with tap water and the irritation was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.


MAIN STUDY:
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the preliminary irritation study.
One group of five animals was treated with vehicle.
Red staining by the test substance prevented scoring for erythema. As no signs of necrosis or oedema were present, a 25 % concentration was selected as the highest concentration that could be used in the main study.



ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:Assessment for Contact Hypersensitivity to R507-2 in the Mouse (Local Lymph Node Assay)
- Criteria used to consider a positive response:If the results indicate a SI >=3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM.

TREATMENT PREPARATION AND ADMINISTRATION: The dorsal surface of both ears was epidermally treated (25 µL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing.

The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
5% test substance
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
10% test substance
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
25 % test substance
Cellular proliferation data / Observations:


CLINICAL OBSERVATIONS: Red staining by the test substance prevented scoring for erythema. No oedema was observed in any of the animals examined. One or both nodes of two animals at 5%, four animals at 10% and four animals at 25% were considered enlarged. The other nodes of the experimental and control groups were considered normal in size.
No macroscopic abnormalities of the surrounding area were noted.
No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
Interpretation of results:
GHS criteria not met
Conclusions:
The SI values calculated for the substance concentrations 5, 10 and 25 % were 1.5, 1.1 and 1.5 respectively.

Since there was no indication that the test substance could elicit an SI >= 3 when tested up to 25 %, it was established that the EC3 value (if any) exceeds 25 %.

The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

Based on these results:
- according to the recommendations made in the test guidelines, R507-2 would not be regarded as skin sensitizer.

- according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2004), R507-2 does not have to be classified for sensitization by skin contact

- according to the EC criteria for classification and labeling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), R507-2 does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact.
Executive summary:

Test substance concentrations selected for the main study were based on the results of a preliminary study.

 

In the main study, three groups of five experimental animals were treated with test substance concentrations of 5 %, 10 % or 25 % on three consecutive days, by open application on the ears.

Five vehicle control animals were similarly treated, but with vehicle alone (Dimethyl formamide). Three days after the last exposure, all animals were injected with3H-methyl thymidine and after

five hours the draining (auricular) lymph nodes were excised.

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.

 

Red staining by the test substance prevented scoring for erythema. No oedema was observed in any of the animals examined.

 

One or both nodes of two animals at 5 %, four animals at 10 % and four animals at 25 % were considered enlarged. The other nodes of the experimental and control groups were considered normal in size.

No macroscopic abnormalities of the surrounding area were noted.

 

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5 %, 10 % and 25 % were 609, 433 and 630 respectively.

The mean DPM/animal value for the vehicle control group was 410.

 

The SI values calculated for the substance concentrations 5 %, 10 % and 25 % were 1.5, 1.1 and 1.5 respectively.

 

Since there was no indication that the test substance could elicit an SI 3 when tested up to 25 %, it was established that the EC3 value (if any) exceeds 25 %.

 

Based on these results:

- according to the recommendations made in the test guidelines, R507-2 would not be regarded as skin sensitizer.

- according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2004), R507-2 does not have to be classified for sensitization by skin contact.

- according to the EC criteria for classification and labeling requirements for dangerous substances and preparations (Council Directive 67/548/EEC), R507-2 does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Justification for classification or non-classification

Since there was no indication that the test substance could elicit an SI >= 3 when tested up to 25%, it was established that the EC3 value (if any) exceeds 25%. Since the EC3 values are over the threshold values listed in the CLP criteria it can be confirmed that the test substance R507 -2 is not classified for sensitisation.