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Description of key information

In vitro and in chemico alternatives to an in vivo test could not be performed because of the low solubility of v134434 in the test solutions. Only a QSAR analysis was available which alone was not sufficient to conclude on the skin sensitisation potential of v134434. Therefore, a LLNA test was performed.

The LLNA for V134434 resulted in a SI slightly above 3 (3.2), indicating potential for skin sensitisation. In combination with the DEREK QSAR result (plausible skin sensitizer), v134434 should be classified as Skin Sensitisation Cat. 1B according to GHS rules.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Qualifier:
according to guideline
Guideline:
other: REACH Guidance on QSARs R.6
Specific details on test material used for the study:
Smiles: C1(C=CC(C(=C1C(C)=O)C2=CC=C(C=C2)OCCCCCCCCCCCCCCCC)=O)=O
Remarks on result:
other: QSAR Prediction: plausible skin sensitizer

DEREK NEXUS version 6.0.1 yielded an alert for V134434 for skin sensitization based on the presence of a quinone. V134434 is predicted to be sensitizing to the skin (plausible). DEREK NEXUS could not predict an EC3 for V134434 due to insufficient data regarding structurally related substances.

Interpretation of results:
other: alert for plausible skin sensitizer
Conclusions:
DEREK NEXUS version 6.0.1 yielded an alert for V134434 for skin sensitization based on the presence of a quinone. V134434 is predicted to be sensitizing to the skin (plausible). DEREK NEXUS could not predict an EC3 for V134434 due to insufficient data regarding structurally related substances.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
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)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2012
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: unknown
- Age at study initiation: approximately 10 weeks old
- Weight at study initiation: 19.0 to 23.5 g
- Housing:
On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled.
- Diet (e.g. ad libitum):
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum):
Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period:
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.
- Indication of any skin lesions:
Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 42 to 61%.
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle
- IN-LIFE DATES: From: 16 Oct 2018 To: 19 Nov 2018
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
test item concentrations 1, 2 and 5%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility:
The vehicle was chosen from the vehicles specified in the test guideline (in order of preference): Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol, dimethylsulfoxide and 1% Pluronic© L92 in Elix water (in case an aqueous vehicle is suitable). The vehicle was selected on the basis of maximizing the solubility based on trial preparations performed at Charles River Den Bosch and on information provided by the Sponsor. Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure.
- Irritation: /
- Systemic toxicity:
At a 10%, 25% and 50% test item concentration, clinical signs of systemic toxicity were observed. At a 5% test item concentration, no signs of systemic toxicity were observed
- Ear thickness measurements:
At a 25% and 50% test item concentration, ear thickness increased more than 25% during the observation period from Day 1 pre-dose values. No ear thickness increase above 25% was observed at lower doses.
- Erythema scores: /

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Determination of cellular proliferation (incorporated radioactivity), individual animal approach
- Criteria used to consider a positive response: erythema scores, Disintegrations Per Minute

TREATMENT PREPARATION AND ADMINISTRATION:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item.
The dosing formulations were kept at room temperature until dosing.
No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test item, since the test method requires a logical concentration range rather than specific dose levels.
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day (day 1-3). The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The EC3 value (the estimated test item concentration that will give a SI =3) was determined, using linear interpolation.
Positive control results:
The SI values calculated for the item concentrations 5, 10 and 25% were 1.1, 2.0 and 5.5 respectively. An EC3 value of 14.3% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.4, 14.1, 17.3, 9.8, 17.8 and 19.2%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
Parameter:
SI
Value:
2.2
Variability:
SE = 0.2
Test group / Remarks:
1% test item
Parameter:
SI
Value:
2.7
Variability:
SE = 0.4
Test group / Remarks:
2% test item
Parameter:
SI
Value:
3.2
Variability:
SE = 0.7
Test group / Remarks:
5% test item
Parameter:
EC3
Value:
3.8
Remarks on result:
other: determined through linear interpolation
Cellular proliferation data / Observations:
All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size.

No erythema was observed in any of the animals.

Red test item remnants were present on the dorsal surface of the ears of several animals throughout the test item treated groups between Days 1 and 3, which did not hamper scoring of the skin reactions.

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Body weights and body weight gains of experimental animals remained in the same range as controls over the study period. 

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
These results indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 3.8% was calculated using linear interpolation. Based on these results:
• according to the recommendations made in the test guidelines (including all amendments), V134434 would be regarded as skin sensitizer.
• according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), V134434 should be classified as skin sensitizer (Category 1B).
• according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), V134434 should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.
Executive summary:

The objective of this study was to evaluate whether V134434 induces skin sensitization in mice after three epidermal exposures of the animals under the conditions described in this report.

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

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

·      EC No 640/2012, Part B: "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. At a 25% and 50% test item concentration, ear thickness increased more than 25% during the observation period from Day 1 pre-dose values and clinical signs of systemic toxicity wereobserved[KV1] . At a 10% test item concentration, clinical signs of systemic toxicity were also observed. Overall, these concentrations did not meet the selection criteria. At a 5% test item concentration, no signs of systemic toxicity were observed. The observed increase in ear thickness exceeding 25% was considered an incidental finding with no relevance since it was seen for one ear of one animal on Day 6 only and no relevant increase was seen at a higher concentration of 10%. Therefore the 5% concentration was selected as highest concentration for the main study.

 

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 1, 2 or 5% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with3H-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.

 

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 were 1040, 1285 and 1495 DPM for the experimental groups treated with test item concentrations 1, 2 and 5%, respectively. The mean DPM/animal value for the vehicle control group was 470 DPM. The SI values were 2.2, 2.7 and 3.2 for the test item concentrations 1, 2 and 5%, respectively.


 

These results indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 3.8% was calculated.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Based on these results:

·      according to the recommendations made in the test guidelines (including all amendments), V134434 would be regarded as skin sensitizer.

·      according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), V134434 should be classified as skin sensitizer (Category 1B).

·       according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), V134434 should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification