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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:
30 March 2011 to 11 April 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
(2003)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): FDCA
- Substance type: White to off-white powder

In vivo test system

Test animals

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. 9 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- 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 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 - 23.4
- Humidity (%): 38 - 65
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

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

IN-LIFE DATES: From 30 March 2011 to 11 April 2011

Study design: in vivo (LLNA)

Vehicle:
other: Water (Elix, Millipore S.A.S., Molsheim, France) with 1% pluronic L92 (BASF, New Jersey, USA)
Concentration:
0, 10, 25 and 50%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
A weight of evidence analysis was performed prior to start of this study. All available information was evaluated

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, based on the test guideline and recommendations done by ICCVAM. Consideration was given to the EC3 value (the estimated test substance concentration that will give an SI =3; Basketter et al., J Appl Toxicol 1999;19:261-266).

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle and another group of five animals was treated with the positive control substance.

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. 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. Trial formulation results showed that no suitable formulation could be prepared using the commonly used vehicles Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol or dimethylsulfoxide. Therefore, 1% watery pluronic L92 was selected as suitable vehicle based on trial formulations performed at NOTOX. L92 provides good skin wetting properties for prolonged dermal contact and has been shown to yield positive LLNA results using a number of watersoluble dermal sensitizers (Basketter et al., J Appl Toxicol 1999;19:261-266).

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 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing) according to the guideline. Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: All animals were sacrificed at day 6 by intra-peritoneal injection with Euthasol® 20% (0.2 mL/animal).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed.

Results and discussion

Positive control results:
The reliability check with Alpha-hexylcinnamicaldehyde (performed in March 2011) indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.9
Test group / Remarks:
Concentration: 10%
Parameter:
SI
Value:
0.8
Test group / Remarks:
Concentration: 25%
Parameter:
SI
Value:
0.7
Test group / Remarks:
Concentration: 50%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 406, 340 and 309 DPM respectively. The mean DPM/animal value for the vehicle control group was 433.

Any other information on results incl. tables

Results Pre-screen test:

No irritation and no signs of systemic toxicity were observed in any of the animals examined. 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 substance concentration selected for the main study was a 50% concentration.

Other results - main study:

No irritation of the ears was observed in any of the animals examined. White staining of the ears was noted, not hampering the scoring of the ears.

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 in any of the animals.

 

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant.

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
According to Regulation (EC) No 1272/2008
Conclusions:
In an LLNA skin sensitisation study, performed according to OECD 429 test guideline and GLP principles, FDCA was found not be a skin sensitiser, as the SI was not ≥ 3 when tested up to 50%.
Executive summary:

An LLNA skin sensitisation study was performed according to OECD 429 test guideline and GLP principles. No irritation of the ears was observed in any of the animals examined. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 406, 340 and 309 DPM respectively. The mean DPM/animal value for the vehicle control group was 433. The SI values calculated for the substance concentrations 10, 25 and 50% were 0.9, 0.8 and 0.7% respectively. Based on these results FDCA is not regarded as a skin sensitizer and is not classified.