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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:
2018-02-14 to 2018-03-219
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Paris Cedex, July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Official Journal of the European Union No. L142, May 2008, including most recent amendments.
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 reports): JNJ-39722280-AAA (4-Nitrobenzene-1-sulfonyl chloride)
- Physical state: solid (powder)
- Appearance: Slightly yellow to yellowish brown crystalline powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M16LB4523
- Expiration date of the lot/batch: 2018-06-23 (retest date)
- Purity/composition correction factor: 1
- Purity: 99.9% w/w (calculated as is) - Chromatographic purity GC

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: There was no information available regarding the solubility or stability in vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item preparations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was assessed by visual inspection of the solutions. Correction of the purity/composition of the test item was not applicable, since the test method required a logical concentration range rather than specific dose levels to be dosed.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Remarks:
inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/J strain, inbred, SPF-Quality
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Approximately 8 weeks old
- Weight at study initiation: 17.8 - 22.7 grams
- Housing: Group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. On Day 6 of the main study, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet (e.g. ad libitum): ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 18 to 24°C; actual daily mean temperature during the study period: 22°C
- Humidity (%): 40 to 70%; actual main relative humidity during the study period: 40 to 45%
- Air changes (per hr): At least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
PreScreen Test: 25 and 50% w/w
Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (5% and 10%) at a later stage.

Main Study: 0, 1, 2 and 5% w/w
No. of animals per dose:
Five females per group; 4 groups (including control group)
Details on study design:
Rationale vehicle selection:
The vehicle was selected on the basis of maximizing the solubility using the test item data provided by the Sponsor. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methyl ethyl ketone, propylene glycol and dimethylsulfoxide. Formulation in Methyl ethyl ketone has proven to be stable in a separate study.

PRE-SCREEN TESTS:
- At a 10, 25 and 50% test item concentration variations in ear thickness during the observation period exceeded 25% from Day 1 pre-dose values and therefore these concentrations did not meet the selection criteria.
- Compound solubility: no data
- Irritation: At a 5% test item concentration no to very slight irritation was observed
- Systemic toxicity: At a 5% test item concentration no signs of systemic toxicity were noted
- Ear thickness measurements: At a 5% test item concentration 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 item concentration selected for the main study was a 5% concentration.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response:
If the results indicate a SI ≥ 3, the test item 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 (2015) (including all amendments) 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, including all amendments. Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3).

Classification of results:
SI value UN-GHS 2015; EC-CLP 2008 EC Hazard statement
SI < 3 No sensitizer -
SI ≥ 3 Cat 1 Skin sensitizer H317: May cause an allergic skinreaction
EC3 value ≤ 2%: sub-category 1A
EC3 value > 2%: sub-category 1B
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No data

Results and discussion

Positive control results:
At concentrations 5%, 10% and 25% SI values of the positive control item were 1.4, 2.2, and 3.5 respectively. An EC3 value of 19.2% was calculated using linear interpolation.
The calculated EC3 value was in the accepable range of 4.8 and 19.5%. The results of the 6 monthly reliability checks of the recent years were 13.2, 14.1, 17.3, 9.8, 17.8, 18.0, 14.7 and 13.2%
Based on the results, it was concluded that the Local Lymph Node Assay as performed in the laboratory is an appropriate model for testing contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
12.6
Variability:
+/- 2.1
Test group / Remarks:
Based on 5 animals in 1% w/w in methyl ethyl ketone group
Parameter:
SI
Value:
18.1
Variability:
+/- 1.6
Test group / Remarks:
Based on 5 animals in 2% w/w in methyl ethyl ketone group
Parameter:
SI
Value:
20.2
Variability:
+/- 1.0
Test group / Remarks:
Based on 5 animals in 5% w/w in methyl ethyl ketone group
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA:
mean DPM ± SEM:
0% w/w group: mean DPM ± SEM: 821 ± 118
1% w/w group: mean DPM ± SEM: 10345 ± 1743
2% w/w group: mean DPM ± SEM: 14891 ± 1311
5 %w/w group: mean DPM ± SEM: 16562 ± 817
SEM = Standard Error of the Mean

EC3 CALCULATION
The EC3 value (the estimated test item concentration that will give a SI =3) was established to be between 0 and 1%.

CLINICAL OBSERVATIONS:
- Skin reactions/irritation: The scaliness as shown by one animal treated at a concentration of 2% and all animals treated at a concentration of 5% on days 4 and 6 were considered not to have a toxicologically significant effect on the activity of the nodes.
- Systemic toxicity: No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.
- Macroscopy of the auricular lymph nodes and surrounding area: All auricular lymph nodes of the animals of the experimental groups were considered enlarged compared to the control group. The largest auricular lymph nodes were found in the higher dose groups. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

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

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
These results show that the test item elicits a SI ≥ 3. The EC3 value (the estimated test item concentration that will give a SI =3) was established to be between 0 and 1%.
Based on these results:
- according to the recommendations made in the test guidelines (including all amendments), 4-Nitrobenzene-1-sulfonyl chloride would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), 4-Nitrobenzene-1-sulfonyl chloride should be classified as skin sensitizer (Category 1A).
- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), 4-Nitrobenzene-1-sulfonyl chloride should be classified as skin sensitizer (Category 1A) and labeled as H317: May cause an allergic skin reaction.