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Description of key information

Based on the study results, the test substance was considered to be a strong skin sensitiser.

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:
From 4 April, 2012 to 7 May, 2012
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: CBA/J
Sex:
female
Details on test animals and environmental conditions:
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx 11 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (20 - 23 g)
- Housing: Animals were group housed in labeld makrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 d

ENVIRONMENTAL CONDITIONS

Deviations from the minimum level of daily mean relative humidity occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.


IN-LIFE DATES: From 04 April 2012 to 07 May 2012
Vehicle:
dimethylformamide
Concentration:
0, 0.5, 1 , 2.5%
No. of animals per dose:
5
Details on study design:
The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

RANGE FINDING TESTS:
In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed prior to start of this study. All available information was evaluated (e.g. existing human and animal data, literature, substance data supplied by the sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer. The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (20011) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on Classification, Labelling and Packaging of substances and mixtures.

ANIMAL ASSIGNMENT
3 groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance formulations (w/w) were prepared within 4 h prior to each treatment. Homogeneity was obtained to visually acceptable levels.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 h after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: all animals were killed by intraperitoneal injection (0.2 mL/animal) with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The six-month reliability check with alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity. See attached document 'Reliability check'.
Key result
Parameter:
EC3
Value:
> 0 - <= 0.5
Test group / Remarks:
EC3 value was determined to be between the test concentrations of 0 to 0.5%, which will result in SI of 3.
Key result
Parameter:
SI
Value:
ca. 6.2
Test group / Remarks:
0.5% test substance
Key result
Parameter:
SI
Value:
ca. 7.6
Test group / Remarks:
1% test substance
Key result
Parameter:
SI
Value:
ca. 11.1
Test group / Remarks:
2.5% test substance

Results pre-screen test:

Erythema scores and variations in ear thickness are given in detail in Table 1 and 2.

Based on these results, the highest test substance concentration selected for the main study was a 2.5% concentration.

Other results - main study:

-Skin reactions / Irritation: No irritation of the ears was observed in any of the animals examined.

-Systemic toxicity/body weights: 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. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.

-Macroscopy of the auricular lymph nodes and surrounding area: The auricular lymph nodes of all the animals at 1 and 2.5% and of some animals (3/5) at 0.5% appeared larger in size as compared to the other treated groups. The largest auricular lymph nodes were found in the higher dose groups. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Interpretation of results:
other: Category 1A (indication of significant skin sensitising potential) based on EU CLP criteria
Conclusions:
Under the study conditions, the test substance is considered to be a strong skin sensitizer.
Executive summary:

A study was conducted to assess the contact hypersensitivity of DPHA in the mouse local lymph node assay according to OECD Guideline 429, EU Method B.42 and EPA Guideline OPPTS 870.2600 in compliance with GLP. Test substance 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 concentrations of 0.5, 1 or 2.5% w/w on three consecutive days by application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (N,N-Dimethylformamide). 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. No irritation was observed in any of the animals examined. The auricular lymph nodes of all animals at 1 and 2.5%, and of some animals (3/5) at 0.5%, appeared larger in size as compared to the other treated groups. The largest auricular lymph nodes were found in the higher dose groups. No macroscopic abnormalities of the surrounding area were noted. Mean degradations per minute (DPM)/animal for the experimental groups treated at 0.5, 1 and 2.5% were 3733, 4588 and 6646, respectively. The SI values calculated for the substance concentrations 0.5, 1 and 2.5% were 6.2, 7.6 and 11.1, respectively. These results show that the test substance elicits an SI ≥ 3. The EC3 value (the estimated test substance concentration that will give a SI = 3) was established to be between 0 and 0.5%. Under the study conditions, the test substance was considered to be a strong skin sensitizer (Beerens-Heijnen, 2012).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A study was conducted to assess the contact hypersensitivity of DPHA in the mouse local lymph node assay according to OECD Guideline 429, EU Method B.42 and EPA Guideline OPPTS 870.2600 in compliance with GLP.Test substance 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 concentrations of 0.5, 1 or 2.5% w/w on three consecutive days by application on the ears.Five vehicle control animals were similarly treated, but with vehicle alone (N,N Dimethylformamide). 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.No irritation was observed in any of the animals examined. The auricular lymph nodes of all animals at 1 and 2.5%, and of some animals (3/5) at 0.5%, appeared larger in size as compared to the other treated groups. The largest auricular lymph nodes were found in the higher dose groups. No macroscopic abnormalities of the surrounding area were noted. Mean degradations per minute (DPM)/animal for the experimental groups treated at 0.5, 1 and 2.5% were 3733, 4588 and 6646, respectively. The SI values calculated for the substance concentrations 0.5, 1 and 2.5% were 6.2, 7.6 and 11.1, respectively. These results show that the test substance elicits an SI ≥ 3. The EC3 value (the estimated test substance concentration that will give a SI = 3) was established to be between 0 and 0.5%. Under the study conditions, the test substance was considered to be a strong skin sensitizer (Beerens-Heijnen, 2012).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the results of an in vivo mouse local lymph node assay, the test substance warrants classification as Skin Sens. 1A - H317 (may cause allergic skin reactions) according to CLP (EC 1272/2008) criteria.