Absolute of Jasmine (Jasminum grandiflorum) obtained from Jasmine concrete by ethanol extraction

Endpoint summary

Description of key information

The potential of Jasmine absolute to induce skin irritation (OECD 439) and eye irritation (OECD 437) was tested in suitable in vitro test methods.

Based on the results, Jasmine absolute is irritating to the skin in accordance with UN GHS "Category 1 or 2". As no further data is available yet, which would allow to distinguish between skin irritation and skin corrosion, the substance will be classified as corrosive to the skin (Skin Corr. 1, H314) as a worst-case assumption. Based on the results from an in vitro eye irritation test, the substance can be considered as non-irritating to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-06-06 to 2018-08-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was applied undiluted. 30 µL of the test item were dispensed directly atop the EpiDerm tissue

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ reconstructed human epidermis model (MatTek)
- Tissue batch number(s): 28623

EpiDerm Kit:
The EpiDerm™ tissues were provided as kits (e.g. EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
- 1x sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm²); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot No.: 28623)
- 2x 24-well plates
- 8x 6-well plates
- 1x bottle of assay medium (DMEM-based medium, Lot No.: 060718MSB)
- 1x bottle of DPBS Rinse Solution (Lot No.: 051518ISA)
- 1x 1 vial 5% SDS Solution (TC-SDS-5%)
- 25 pieces Nylon Mesh circles (8 mm diameter, 200 μm pore)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 +/- 1 °C for the first 35 +/- 1 min, afterwards the plates were placed under the sterile flow until 60 +/- 1 min incubation time of the first dosed tissue was over.
- Temperature of post-treatment incubation (if applicable): 37 +/- 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, staggered again in e.g. one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h +/- 5 min
- Spectrophotometer: Yes
- Wavelength: 570 nm
- Filter bandwidth: +/- 30 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL (undiluted)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 µL DPBS (Gibco, Cat. No.14040-091, Lot No.: 1838067)

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 µL 5% sodium dodecyl sulfate (MatTek, CAS No.: 151-21-3, Lot No.: 040418SVA)

Duration of treatment / exposure:

60 ± 1 min

Duration of post-treatment incubation (if applicable):

42 h post-incubation

Number of replicates:

3 tissues per dose group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three tissues

Value:

40.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, OD570 nm ≥ 0.8 and ≤ 2.8 (1.806)
- Acceptance criteria met for positive control: Yes, mean relative tissue viability of the three positive control tissues was ≤ 20% (3.7%)
- Acceptance criteria met for variability between replicate measurements: Yes, standard deviation (SD) of viability of replicate tissues of all dose groups was ≤ 18%. (0.2-6.0%).

For detailed results see Table 1 in box "Any other information on results incl. tables".

Results of the Pre-Experiments:

The mixture of 30 µL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT was determined to be 0%.

The mixture of 30 µL of the test item per 300 μL isopropanol showed colouring detectable by unaided eye-assessment. Therefore, the absorption of the chemical in isopropanol was measured in the range of 570 ± 30 nm.

Results of the main experiment:

Table 1: Result of the Test Item

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.830	1.783	1.804	0.103	0.103	0.108	0.678	0.879	0.703
	1.827	1.772	1.822	0.113	0.110	0.119	0.687	0.877	0.717
OD570(Blank Corrected)	1.786	1.739	1.760	0.059	0.059	0.064	0.634	0.835	0.659
	1.783	1.728	1.778	0.067	0.066	0.075	0.643	0.834	0.666
Mean OD570of the Duplicates (Blank Corrected)	1.785	1.734	1.769	0.063	0.062	0.070	0.638	0.834	0.66
Total Mean OD570of 3 Replicate Tissues (Blank Corrected)	1.762*			0.065			0.713
SD OD570	0.026			0.004			0.106
Relative Tissue Viability [%]	101.3	98.4	100.4	3.6	3.5	4.0	36.2	47.3	37.8
Mean Relative Tissue Viability [%]	100.0			3.7**			40.4
SD Tissue Viability [%]***	1.5			0.2			6.0
CV [% Viabilities]	1.5			6.4			14.9

* Blank-corrected mean OD_{570 nm}of the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viability of the three positive control tissues is≤ 20%.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

Interpretation of results:

other: The test item is identified as requiring classification and labelling according to UN GHS (“Category 2” or “Category 1”).

Conclusions:

In conclusion, in this in vitro skin irritation study (OECD 439), Jasmine absolute is considered to be irritant to the skin (UN GHS category 1 or 2).

Executive summary:

In a primary dermal irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200-SIT) was topically exposed to Jasmine absolute (100% purity) for 60 min and 42 h post incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability (% negative control) was ≤ 50% (40.4%) after 60 min treatment and 42 h post-incubation. Based on this result, Jasmine absolute is considered to be irritating to the skin in accordance with UN GHS "Category 1 or 2" .

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

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:

2018-07-10 to 2018-10-31

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:

adopted in October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was used as provided by the sponsor.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES:
- Source: Isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany. On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice.

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 µL

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 h at 32 ± 1 °C

Number of animals or in vitro replicates:

3 corneas each for the test item, negative control and positive control

Details on study design:

SELECTION AND PREPARATION OF CORNEAS / QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes were carefully examined for defects and any defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

APPLICATION DOSE AND EXPOSURE TIME
750 μL of the test substance or the control substance was introduced into the anterior chamber. After 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red).

TREATMENT METHOD: closed chamber
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI medium. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control. 750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded. After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA:
- IVIS ≤ 3: UN GHS No Category
- IVIS > 3 to ≤ 55: No prediction can be made
- IVIS > 55: UN GHS Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of three replicates

Value:

2.76

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control. For details on the results see box "Any other information on results incl. tables"

Table 1: In vitro Irritation Score (IVIS)

Cornea No.	Test Item	Corrected Opacity Value	Corrected OD490 Value	IVIS
1	Negative Control Physiological Saline	0.32	0.004	0.45
2		0.18	0.019
3		0.29	0.015
MV		0.26	0.013
1	Positive Control 100% Ethanol	23.35	1.249	46.49
2		26.52	1.572
3		28.13	1.276
MV		26.00	1.366
1	Test Item Jasmine Absolute	3.45	-0.005	2.76
2		1.57	-0.001
3		3.39	-0.003
MV		2.80	-0.003

MV = mean value

Table 2: Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative Control Physiological Saline	1.92	2.24	0.32
2		1.96	2.13	0.18
3		1.96	2.24	0.29
MV		1.94	2.21	0.26
1	Positive Control 100% Ethanol	2.86	26.48	23.62	23.35
2		2.90	29.68	26.78	26.52
3		3.42	31.81	28.39	28.13
MV		3.06	29.32	26.26	26.00
1	Test Item Jasmine absolute	-0.63	3.08	3.72	3.45
2		1.36	3.20	1.83	1.57
3		0.19	3.84	3.65	3.39
MV		0.31	3.37	3.07	2.80

MV = mean value

Table 3: Permeability

Cornea No.	Test Item	OD490	Corrected OD490
1	Negative Control Physiological Saline	0.004
2		0.019
3		0.015
MV		0.013
1	Positive Control 100% Ethanol	1.262	1.249
2		1.585	1.572
3		1.289	1.276
MV		1.379	1.366
1	Test Item Jasmine absolute	0.008	-0.005
2		0.012	-0.001
3		0.010	-0.003
MV		0.010	-0.003

MV = mean value

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, based on the mean in vitro irritation score of 2.76 obtained in the bovine corneal opacity and permeability assay (OECD 437), the test substance Jasmine absolute is classified into UN GHS "No Category".

Executive summary:

The eye irritation potential of Jasmine absolute (100% purity) was investigated in the bovine corneal opacity and permeability assay (OECD 437). The undiluted test item was applied to the corneas. A mean in vitro irritation score of 2.76 was determined. The positive control induced the appropriate responses, indicating the validity of the assay. Based on the obtained mean in vitro irritation score, Jasmine absolute is classified into "No Category" according to the UN GHS classification criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The potential of Jasmine absolute (100% purity) to induce skin irritation (OECD 439) and eye irritation (OECD 437) was tested in suitable in vitro test methods. Based on the results, the substance can be considered as non-irritant to the eye. To assess the skin irritant potential of the substance, the EpiDerm™ reconstructed human epidermis model was used, and the study was conducted according to OECD testing guideline 439. This test can correctly identify chemicals that do not require classification or require labelling for skin irritation (Category 2) or serious skin damage (Category 1). However, it is not possible to distinguish between skin irritation and skin corrosion in this test. Based on the results, Jasmine absolute is irritating to the skin in accordance with UN GHS "Category 1 or 2". As no further data is available yet, which would allow to distinguish between skin irritation and skin corrosion, the substance will be classified as corrosive to the skin (Skin Corr. 1, H314) as a worst-case assumption. At the time, new data will be available the skin irritation/corrosion endpoint and subsequently the classification will be re-evaluated.