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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 May 2018 - 31 May 2018
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
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
July 29, 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC method B.40 bis: In vitro Skin Corrosion: Human Skin Test Method.
Version / remarks:
May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
07-07-2016

Test material

Constituent 1
Reference substance name:
Cymbopogon nardus, ext.
EC Number:
289-753-6
EC Name:
Cymbopogon nardus, ext.
Cas Number:
89998-15-2
IUPAC Name:
Essential oil of Citronella obtained from the aerial parts of Cymbopogon nardus, (Poaceae), by steam distillation
Test material form:
liquid
Details on test material:
Citronella nardus oil
CAS no.: 8000-29-1

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek Corporation
Source strain:
not specified
Details on animal used as source of test system:
Human source
Justification for test system used:
According to guideline recommendations
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200-SCT), Lot no. 28620
- Tissue batch number(s): 00267
- Production date: 30 May 2018
- Date of initiation of testing: 30 May 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing steps: After exposure tissues were rinsed, blotted and assay medium was replaced by MTT assay medium

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ mL
- Incubation time: 3h
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS IN REFERENCE TO HISTORICAL DATA
- Viability: Pass
- Barrier function: Pass
- Morphology: Pass
- Contamination: Pass
- Reproducibility: Pass

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Freeze killed tissues
- Procedure used to prepare the killed tissues: frozen tissues were stored in the freezer (-20 ± 5°C).
- N. of replicates: 2
- Method of calculation used:
True viability = Viability of treated tissue – Interference from test chemical = OD tvt – OD kt
where OD kt = (mean OD tkt – mean OD ukt)
tvt = treated viable tissue kt = killed tissues
tkt = treated killed tissue ukt = untreated killed tissue (NC treated tissue)


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- the test item is considered to be corrosive to skin and classified as category 1 (or optional category 1A ), if the viability after 3 minutes exposure is less than 50%;
- the test item is considered to be corrosive to skin and classified as sub-category 1B-and-1C, if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%;
- the test item is considered to be non-corrosive to skin, if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): unchanged

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): 8 N KOH
Duration of treatment / exposure:
3 minutes or 1 hour
Duration of post-treatment incubation (if applicable):
3 hour
Number of replicates:
Two replicate tissues for each treatment (exposure periods) were employed.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure period
Value:
109.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure period
Value:
78.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- The mean optical density (OD) of the negative control of 2 tissues was 2.243 (3 minute exposure) or 2.411 (1-hour exposure) and therefore well within the acceptable range of ≥ 0.8 to ≤ 2.8.
- The viability of cells treated with the positive reference item 8 N KOH was 3.4% or 2.8% (3-minute or 1-hour exposure, respectively) of the negative control and, hence well below the 15% cut-off value at the 1-hour exposure.
- The difference of viability between the two tissue replicates (at 20 - 100% viability) was below the limit of acceptance of 30%. Hence, all acceptance criteria were fulfilled
- Range of historical values if different from the ones specified in the test guideline: see table below in the additional information section.

Any other information on results incl. tables

Material

Average OD

(mean% difference ±SD)

Average viability [%] (mean% difference ±SD)

Range

No. of

unqualified experi-ments

Viability [%]

% difference

Short time incubation – 3‑min

Negative control

(Non-Corrosive)

1.624

(2.5±2.4)

100

(2.5±2.4)

93.7–106.3

0.14–8.6

0#1

8 N KOH

(Corrosive)

0.126

(8.8±7.4)

7.6

(8.8±7.4)

2.0–15.6

<0.01–23.7

0

Long time incubation – 60‑min

Negative control

(Non-Corrosive)

1.650

(4.0±5.0)

100

(4.0±5.0)

85.6–114.4

0.13–18.3

0#1

8 N KOH

(Corrosive)

0.090

(5.7±9.8)

5.9

(5.7±9.8)

2.0–12.6

0.30–38.4

0#2

OD: Optical density. Viability for negative control is set = 100%

SD: Standard deviation

CV: Coefficient of variation

#1       Unqualified results =      if the mean OD of the NC tissues is < 0.8 or > 2.8

                                      if difference in viability for duplicate tissues > 30%

#2       Unqualified results =      8 N KOH: viability > 15% (1-hour exposure)

Applicant's summary and conclusion

Interpretation of results:
other: not corrosive
Remarks:
based on CLP criteria (1272/2008/EC).
Conclusions:
Based on the results of this study, Citronella nardus oil does not need to be classified for skin corrosion in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The purpose of this study was to assess the corrosive properties of Citronella nardus oil to human skin, in an experiment according to OECD TG 431 with an artificial three-dimensional model of human skin. The EpiDerm™ model was employed. Two tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.

Citronella nardus oil was applied topically as supplied (liquid). Sterile deionised water was used as the negative control. 8 N KOH was used as the positive reference item. The test item and the reference items were applied to the skin model surface at two exposure periods of 3 minutes or 1 hour.

In comparison to the negative controls, the mean viability of cells exposed to the test item was 109.9% after a 3-minute exposure period and 78.7% after a 1 hour exposure (corrected viability calculated for MTT reducing test items using freeze-killed control tissues) and hence, the 3-minute and the 1-hour exposure values were above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of ≥ 50% and ≥ 15%, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.All acceptance criteria were fulfilled.

Under the present test conditions Citronella nardus oil tested at two exposure periods of 3 minutes or 1 hour was non-cytotoxic and, hence, predicted to be non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin

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