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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:
2015-11-18 - 2015-12-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented GLP OECD guideline study without deviations on the registered substance itself.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Version / remarks:
OECD: Guideline for the Testing of Chemicals; Section 4: Health Effects: 442B Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA. Adopted: 22 July 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Fürstenwall 25, 40219 Düsseldorf, Germany
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
3-cyclohexylaminopropane-1-sulphonic acid
EC Number:
214-492-1
EC Name:
3-cyclohexylaminopropane-1-sulphonic acid
Cas Number:
1135-40-6
Molecular formula:
C9H19NO3S
IUPAC Name:
3-cyclohexylaminopropane-1-sulphonic acid
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): 3-(Cyclohexylamino)-propane sulfonic acid, 3-CAPS
- Substance type: pure substance
- Storage condition of test material: Room temperature (20 ± 5°C)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo, 5961 NM Horst, NL
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 17-22 g
- Housing: OP3 (SPF-barrier), in groups of 4 animals in open Eurostandard type III macrolon cages, with Lignocel hygienic animal bedding (J. Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg), heat-treated, Specified pathogen free (SPF) housing with permanent health monitoring by room specific sentinel animals (bedding sentinels) in accordance with FELASA recommendations, with Wooden gnawing blocks, size medium 10 x 2 x 2 cm, debarked, aspen wood, NGM E-022 (ABEDD LAB & VET Service GmbH), heat-treated
- Diet (e.g. ad libitum): Maintenance diet rat/mouse, pellets, No. 1324 TPF (Altromin GmbH & Co. KG, 32791 Lage), ad libitum
- Water (e.g. ad libitum): Sterilized community tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): artificial lighting, 12 h light/12 h dark

IN-LIFE DATES: From: 2015-11-11 To: 2015-11-26

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
CHROMASOLV® (Acetone) SIGMA #34850 mixed with olive oil, highly refined, low acidity: SIGMA #O1514 (4:1 v/v)
Concentration:
13.4%, 5% and 2.5%
No. of animals per dose:
4 females
Details on study design:
RANGE FINDING TESTS:
In a solubility pre-test, the highest achievable concentration in suspension of the test item in Acetone: olive oil (4:1,vv) was found to be 110 g/L. Acetone: olive oil (4:1,vv) is a highly recommended vehicle for the local lymph node assay (OECD442B).
In a pre-screen test the application of 110 g/L test item in vehicle showed no clinical signs indicating systemic toxication and showed no skin reaction indicating an excessive local skin irritation. Therefore 110 g/L (13.4%) test item in vehicle was selected as high dose.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA: BrdU-ELISA
- Criteria used to consider a positive response: Evaluation criteria according to OECD 442B
For assay validity the following system suitability criteria should be fulfilled:
1) ODVC MEAN: 0.1 ≤ ODVC ≤ 0.2
2) SIVC AVERAGE =~1.0
3) SIPC ≥ 1.6 over SIVC*
Classification of SI values is as follows:
SI indicates a positive result when:
1.6 ≤ SI ≤ 1.9 = borderline positive**
2.0 ≤ SI = positive
*No excessive skin irritation or systemic toxicity should be observed in parallel.
**Additional information (such as dose-response relationship, evidence of systemic toxicity or excessive irritation) should be considered to confirm that such results are positives.

TREATMENT PREPARATION AND ADMINISTRATION:
Application
25 μL of the extracted test substance, the vehicle alone or the positive control (PC), were applied to the dorsum of each ear (50 μL per animal) using a tipped pipette. Animals were fixed until evaporation of acetone. In brief, the complete experimental schedule of the assay was as follows:
• Day 1: Individually identify and record the weight of each animal and any clinical observation. Apply 25 μL of the extracted test substance, the vehicle alone or the PC, to the dorsum of each ear.
• Day 2: Repeat the application procedure carried out on day 1.
• Day 3: Repeat the application procedure carried out on day 1.
• Day 4: No treatment.
• Day 5: Inject 0.5 mL (5 mg/mouse) of BrdU2 (10 mg/mL) solution intraperitoneally.
• Day 6: Record the weight of each animal and any clinical observation. To further monitor the local skin response, score an occurring ear erythema approximately 24 h after BrdU injection and humanely kill the animals. Punch both ears and weigh ear punch-pair. Excise the draining auricular lymph nodes from each mouse ear and process separately in phosphate buffered saline for each animal.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Calculation of results
For each experimental group, a stimulation index (SI) was calculated by dividing the mean BrdU labeling index for each individual animal by the mean BrdU labeling index for thevehicle group (the average SI for the vehicle group is 1).
The BrdU labeling index is defined as:
BrdU labelling index = (OD370nm – OD blank370nm) – (OD492nm – OD blank492nm)

Results and discussion

Positive control results:
Group SI = 3.9 (Medium of 3 replicates, 4.1±0.3, 4.2 ±0.6, 3.5±0.4)

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.8
Variability:
±0.3
Test group / Remarks:
2.5 % test group
Key result
Parameter:
SI
Value:
0.9
Variability:
±0.3
Test group / Remarks:
5 % test group
Key result
Parameter:
SI
Value:
0.5
Variability:
±0.1
Test group / Remarks:
13.4 % test group

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
The study was conducted under GLP according to OECD guideline 442B on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the sensitizing potential of the test substance in mice. 3-(Cyclohexylamino)-propane sulfonic acid did not induce signs of general toxicity. No significantly increased ear thickness, in terms of ear punch weight, was detected. Together with the negligible proliferative response of the LNCs, the test item 3-(Cyclohexylamino)-propane sulfonic acid was identified as a non-sensitizing agent in the Local Lymph Node Assay. Hence, no classification as skin sensitizer is triggered.
Executive summary:

A dermal sensitization study was carried out according to guideline OECD 442B (Guideline for the Testing of Chemicals; Section 4: Health Effects: 442B Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA) in CBA/Ca mice using 25 % α-Hexylcinnamaldehyde (HCA) as positive control and acetone: olive oil (4:1 v/v) as vehicle. The test item 3-(Cyclohexylamino)-propane sulfonic acid was diluted in vehicle solution to receive a 13.4% suspension (high dose). In a preliminary screen test the 13,4% test item suspension did not show any signs of systemic intoxication or excessive skin irritation. Therefore the 13.4% test item in suspension was found to be the feasible high dose for this LLNA. In this study the high dose and two subsequent dilutions of 5% and 2.5%, medium and low dose respectively, of the test item were applied to the dorsal side of the ears. Animals were monitored daily. Determination of cellular proliferation was performed using the BrdU method in reference to the OECD guideline 442B. Additionally, ear thickness was determined through ear punch weight on day 6 of the study.

3-(Cyclohexylamino)-propane sulfonic acid did not induce signs of general toxicity. No significantly increased ear thickness, in terms of ear punch weight, was detected. Together with the negligible proliferative response of the lymph node cells (LNCs), the test item 3-(Cyclohexylamino)-propane sulfonic acid was identified as a non-sensitizing agent in the Local Lymph Node Assay.

The study was classified as acceptable, and no classification of the test substance as skin sensitizer is triggered.

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