Cymbopogon nardus, ext.

Endpoint summary

Description of key information

Skin Corrosion (OECD TG 431): Not corrosive

Eye Irritation (OECD TG 437): IVIS ≤ 3, not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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 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.

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.

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)

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Feb 2018 - 14 Mar 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: cattle
- Characteristics of donor animals (e.g. age, sex, weight): 6 - 60 months of age
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline without antibiotics in a suitable container under cooled conditions.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- Indication of any antibiotics used: no antibiotics used

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

10 +/- 1 minutes

Duration of post- treatment incubation (in vitro):

120 +/- 10 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32°C. The corneas were incubated for the minimum of 1 hour at 32°C.

QUALITY CHECK OF THE ISOLATED CORNEAS
The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
Yes

SOLVENT CONTROL USED (if applicable)
No

POSITIVE CONTROL USED
Yes

APPLICATION DOSE AND EXPOSURE TIME
Undiluted 750µL, 10 minutes

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: yes 120 +/- 10 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: untill all substance removed

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter (measured with the device OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

DECISION CRITERIA: according to Test Guideline

Irritation parameter:

in vitro irritation score

Run / experiment:

Main

Value:

2.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:

DEMONSTRATION OF TECHNICAL PROFICIENCY: Historical Control Data for the BCOP Studies Feb 2015-2018

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline:

Negative control
- Opacity -2.9 – 3.0 (mean 0.18, SD 1.10, N=113)
- Permeability -0.034 – 0.100 (mean 0.00, SD 0.01, N=113)
- In vitro Irritancy Score -2.8 – 3.0 (mean 0.23, SD 1.13, N=113)

Positive control
- In vitro Irritancy Score 28.0 – 110.9 (mean 55.28, SD 15.14, N=88)

Interpretation of results:

other: Not irritating to eyes

Remarks:

in accordance with Annex I of the CLP Regulation (1272/2008/EC).

Conclusions:

Citronella nardus oil induced an IVIS ≤ 3. Based on these results, the test substance does not need to be classified as eye irritant according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Executive summary:

The eye irritation potential of Citronella nardus oil was measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test), OECD test guideline 437 under GLP conditions.The negative cotrol, positive control or test item was applied as received, directly on top of the corneas (750 µL) for 10 minutes. Thereafter the eyes were washed and incubated for 120 minutes before evaluating the effects. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 39 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Citronella nardus oil did not induce ocular irritation through both endpoints, resulting in a meanin vitro irritancy score of 2.6 after 10 minutes of treatment. In conclusion, since Citronella nardus oil induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin corrosion

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

Acute dermal toxicity

The acute dermal toxicity of Citronella nardus oil was examined in albino rabbits (method similar to OECD 402, pre-GLP). The undiluted substance was applied at 0.5, 1.0, 2.0, 4.0 or 8.0 mL/kg onto the intact or abraded skin of the animals (2 animals per condition). Based on the results, the LD50 was estimated 4.7 mL/kg bw. However, the report also mentions the appearance of severe burns (intact or abraded skin was not specified). Therefore, though the substance is considered to be not acute toxic via the dermal route, it is likely to be a skin irritant.

Eye irritation

The eye irritation potential of Citronella nardus oil was measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test), OECD test guideline 437 under GLP conditions. The negative cotrol, positive control or test item was applied as received, directly on top of the corneas (750 µL) for 10 minutes. Thereafter the eyes were washed and incubated for 120 minutes before evaluating the effects. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 39 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Citronella nardus oil did not induce ocular irritation through both endpoints, resulting in a meanin vitro irritancy score of 2.6 after 10 minutes of treatment. In conclusion, since Citronella nardus oil induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Justification for classification or non-classification

Based on the available endpoint specific skin corrosion information, the test substance 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). However, as in vivo data (acute dermal toxicity study in rabbits) has shown that the substance is a potential skin irritant, Citronella nardus oil will be classified as Skin Irrit. 2, H315 Causes skin irritation.

In conclusion, since Citronella nardus oil induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).