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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
457-280-6
EC Name:
-
Cas Number:
126803-19-8
Molecular formula:
C16H20F6NOSb
IUPAC Name:
λ⁵-antimony(5+) N-[(4-methoxyphenyl)methyl]-N,N-dimethylanilinium hexafluoride
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: King Industries, Inc.
- Purity, including information on contaminants, isomers, etc.: Not indicated by the sponsor; treated as 100% pure

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In freezer (S -15°C) in the dark
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: soluble, stability not indicated

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Remarks:
inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L'Arbresle Cedex, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean
- Housing: Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Utalabo, S.P.P.S., Argenteuil, France)
- 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: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0°C (actual range: 21.5 - 23.4°C)
- Humidity (%): 30-70% (actual range: 41 - 76%)
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day

- IN-LIFE DATES: From: June 20, 2007To: July 9, 2007

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
10%, 25%, 50%.
50% is the maximum concentration that produces a solution suitable for application according to the guidelines.
No. of animals per dose:
Five
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The vehicle was selected based on trial formulations and on test substance data supplied by the sponsor. The maximum soluble concentration that produces a solution suitable for application according to the
guidelines is 50%.
- Irritation: 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. Two test substance concentrations were tested, 25% and 50%. Two young adult animals were selected (5-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 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

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: Disintegrations Per Minute (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. The results were evaluated according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2003) and the EC criteria for classification and labeling of dangerous substances and preparations (Council Directive 67/548/EEC and all adaptations to technical progress and amendments of this Directive published in the Official Journal of the European Communities). Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3).

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. Homogeneity was obtained to visually acceptable levels.
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.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was epidermally treated (251J1/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.

Treatment - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (GE Health care, Buckinghamshire, UK). After approximately five hours, all animals were killed by intraperitoneal injection with pentobarbital Euthesate® (0.2 ml/animal) (Ceva Sante Animale BV, Naaldwijk, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever comes first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body weights: On Days 1 (pre-treatment) and 6.
Irritation: On Day 3 (3-4 hours after treatment), the skin reactions were assessed. Skin reactions were graded according to the following numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.

Grading Irritation Reactions:
Erythema and eschar formation:
No erythema 0
Slight erythema (barely perceptible) 1
Well-defined erythema 2
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 3
Oedema formation:
No oedema 0
Slight oedema (barely perceptible) 1
Moderate oedema 2
Severe oedema 3

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
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.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks:
Mean - 5 animals
Value:
1
Variability:
± 0.3 (SEM)
Test group / Remarks:
Group 1 - 0% (vehicle)
Parameter:
SI
Remarks:
Mean - 5 animals
Value:
1.1
Variability:
± 0.3 (SEM)
Test group / Remarks:
Group 2 - 10% (test substance in vehicle)
Parameter:
SI
Remarks:
Mean - 5 animals
Value:
1.3
Variability:
± 0.4 (SEM)
Test group / Remarks:
Group 3 - 25% (test substance in vehicle)
Parameter:
SI
Remarks:
Mean - 5 animals
Value:
0.9
Variability:
± 0.3 (SEM)
Test group / Remarks:
Group 4 - 50% (test substance in vehicle)
Parameter:
other: Mean DPM - 5 animals
Value:
416
Variability:
± 88 (SEM)
Test group / Remarks:
Group 1 - 0% (vehicle)
Parameter:
other: Mean DPM - 5 animals
Value:
460
Variability:
± 49 (SEM)
Test group / Remarks:
Group 2 - 10% (test substance in vehicle)
Parameter:
other: Mean DPM - 5 animals
Value:
534
Variability:
± 104 (SEM)
Test group / Remarks:
Group 3 - 25% (test substance in vehicle)
Parameter:
other: Mean DPM - 5 animals
Value:
376
Variability:
± 70 (SEM)
Test group / Remarks:
Group 4 - 50% (test substance in vehicle)

Any other information on results incl. tables

Preliminary irritation study (Table 1)


No irritation was observed in any of the animals examined.


The results of the epidermal exposures for the selection of highest test substance concentration to be tested in the main study are described in the table.


Based on the results, the highest test substance concentration selected for the main study was a 50% concentration.


 


Body weights (Table 2)


Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.


 


Radioactivity measurements (Tables 3 and 4)


Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 460,534 and 376 respectively.


The mean DPM/animal value for the vehicle control group was 416.


 


Toxicity and Mortality


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


 


 





























































Table 1: Skin reactions after epidermal exposure and body weights



 



 



Day 1



Day 3



 



 



 



 



Skin reactions dorsal surface ear



 



 



 



 



Left



Right



 



Animal number



% test substance



Body weight (g)



Erythema



Oedema



Erythema



Oedema



Body weight (g)



1



25



23



0



0



0



0



23



2



50



22



0



0



0



0



22



 


 



































































































































































































































































































Table 2: Skin reactions, body weights and relative size auricular lymph nodes



 



 



 



Day 1



Day 3



Day 6



 



 



 



 



Skin reactions dorsal surface ear



 



Size nodes4



 



 



 



 



Left



Right



 



Left



Right



Group



Animal number



% test substance1



BW (g)3



Erythema



Oedema



Erythema



Oedema



BW (g)3



 



 



1



1



0%



23



0



0



0



0



24



n



n



2



25



0



0



0



0



25



n



n



3



26



0



0



0



0



25



n



n



4



25



0



0



0



0



25



n



n



5



20



0



0



0



0



20



n



n



2



6



10%



24



0



0



0



0



24



n



n



7



26



0



0



0



0



24



n



n



8



23



0



0



0



0



23



n



n



9



24



0



0



0



0



23



n



n



10



21



0



0



0



0



21



n



n



3



11



25%



24



0



0



0



0



23



n



n



12



22



0



0



0



0



21



n



n



13



24



0



0



0



0



24



n



n



14



25



0



0



0



0



25



n



n



15



24



0



0



0



0



23



n



n



4



16



50%



22



0



0



0



0



23



n



n



17



25



0



0



0



0



24



n



n



18



23



0



0



0



0



24



n



n



19



21



0



0



0



0



21



n



n



20



23



0



0



0



0



22



n



n


           


  1. Vehicle: Dimethyl formamide

  2. Animal number

  3. Body weight (grams)

  4. Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n: considered to be normal)


 


 







































































































Table 3: Radioactivity measurements (individual animals)



Group



Animal number



% test substance



DPM / animal



1



1



0%
(vehicle)



383



2



345



3



739



4



208



5



405



2



6



10%



394



7



385



8



620



9



370



10



529



3



11



25%



437



12



565



13



293



14



464



15



910



4



16



50%



415



17



330



18



297



19



623



20



214



 


 







































Table 4: Disintegrations Per Minute (DPM) and Stimulation Index (S1)



Group



% test substance



Mean
DPM ± SEM



Mean
SI ± SEM



1



0%
(vehicle)



416 ± 88



1.0 ± 0.3



2



10%



460 ± 49



1.1 ± 0.3



3



25%



534 ± 104



1.3 ± 0.4



4



50%



376 ± 70



0.9 ± 0.3



 


 


 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The SI values calculated for the substance concentrations 10, 25 and 50% were 1.1, 1.3 and 0.9 respectively. Since there was no indication that the test substance could elicit an SI ≥ 3 when tested up to 50%, it was established that the EC3 value (if any) exceeds 50%.
Based on these results and according to the recommendations made in the test guidelines, the test substance would not be regarded as skin sensitizer.
Moreover, the test substance does not meet the GHS/CLH criteria to be classified for skin for sensitisation.
Executive summary:

The skin sensitization potential of N-(4-(methoxybenzyl))-N,N-dimethylanilinium hexafluoroantirronate in mice was assessed based on the guidelines described in:OECD, Section 4, Health Effects, No.429 (2002); EC, Council Directive 67/548/EEC, Annex V, B.42 (2004); EPA, OPPTS 870.2600 (2003) "Skin Sensitisation".


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 10%, 25% or 50% 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 with 3H-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.


No skin reactions were observed in any of the animals examined. All 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 10, 25 and 50% were 460, 534 and 376 respectively. The mean DPM/animal value for the vehicle control group was 416. The SI values calculated for the substance concentrations 10, 25 and 50% were 1.1, 1.3 and 0.9 respectively. Since there was no indication that the test substance could elicit an SI ≥ 3 when tested up to 50%, it was established that the EC3 value (if any) exceeds 50%.


Based on these results and according to the recommendations made in the test guidelines, the test substance would not be regarded as skin sensitizer. The test substance does not meet the GHS/CLH criteria to be classified for skin for sensitisation.