Vinasses, residue of fermentation containing biomass of bakers yeast (Saccharomyces cerevisiae)

Endpoint summary

Description of key information

Skin irritation / skin corrosion in vitro (OECD 439): not irritating

Eye irritation in vitro (OECD 437): not irritating

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:

25 Jul - 24 Aug 2018

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 Jul 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

adopted 06 Jul 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: sterilization at 121 °C for 20 min to avoid contamination of the cell culture to avoid contamination of the cell culture

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDermTM reconstituted three-dimensional human epidermis (EPI-200)

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200 SIT kit (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 28646

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 min in the incubator followed by 25 min at room temperature under the sterile flow
- Temperature of post-treatment incubation: 37 °C for 42 h

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 Dulbecco's phosphate buffered saline (DPBS) using a constant stream in about 1.5 cm distance from the tissue surface. This process also occured sequentially, e.g. in 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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min at 37 ± 1 °C
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm without reference wavelength
- Filter bandpass: ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed via MTT cytotoxicity assay. The determined OD (540-570 nm) was 2.216 ± 0.075 and fits to the acceptance criteria of 1.0 - 3.0.
- Barrier function: The barrier function was evaluated by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon treatment with 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.01 h and fits to the acceptance criteria of 4.77 - 8.72 h.
- Contamination: The cells used to produce the EpiDermTM tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected. Tissue sterility was confirmed by long-term incubation with antibiotic and antimycotic free culture.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
Since the test item mixed with aqua dest. showed colouring detectable by unaided eye assessment and absorbed light in the range of 570 ± 30 nm, the test item was checked for its tissue colouring potential.
- Fresh tissues/killed tissues: Living tissues were treated with 30 µL of the test item (TVT). All steps were performed as in the main study except for the MTT staining (Incubation in medium without MTT).
- No. of replicates: 2 living tissues
- Method of calculation used: The non-specific colour of additional viable tissues (NSCliving) was calculated according to the following formular: NSCliving [%] = [ODTVT / ODNK] x 100
NK: negative control, TVT: additional test item treated living tissue without MTT staining.
If NSCliving is ≤ 5% relative to the negative control of living epidermis, no correction of the result is necessary. If NSCliving is > 5% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) was corrected according to the following formular: TODTT = OTM – ODTVT
TM: test item treated with living tissues
If NSCliving is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method.
Since the test substance did not directly reduce MTT, an additional test with freeze-killed tissues to determine the non-specific reduction of MTT was not performed.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 60 min exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 60 min exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL (47 µL/cm2)

NEGATIVE CONTROL
- Amount applied: 30 µL (47 µL/cm2)

POSITIVE CONTROL
- Amount applied: 30 µL (47 µL/cm2)
- Concentration (if solution): 5% (v/v) in deionised water

Duration of treatment / exposure:

35 min at 37 ± 1 °C and 25 min at room temperature

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

triplicates for each treatment and control group

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 3 tissues

Run / experiment:

60 min exposure

Value:

85.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct MTT reduction: The test substance was not considered to be a MTT reducer. Thus, an additional test with freeze-killed tissues was not performed.
- Colour interference: The substance showed colouring upon mixing with water and absorbed light in the relevant range of 570 ± 30 nm, therefore the non-specific color of additional viable tissue was determined. Because the non-specific colour of additional viable tissues was ≤ 5% (NSCliving: 0.3%) relative to the negative control of living epidermis, no correction of the results was necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD 570 nm of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8. The mean OD 570 nm is 1.744 and matches the required acceptability criterion.
- Acceptance criteria met for positive control: Mean relative tissue viability is ≤ 20%. Treatment with the positive control for 1 h induced a decrease in relative absorbance to 4.7% as compared to the negative control.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviation of 3 identical replicates is < 18%. The relative standard deviations between the % variability values of the test item, the positive and negative controls are 5.8% at the highest.

Table 1: Results of the test item

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.765	1.845	1.763	0.119	0.126	0.125	1.395	1.577	1.607
	1.773	1.807	1.778	0.123	0.133	0.134	1.442	1.530	1.628
Mean Absolute OD570	1.789			0.127			1.530
OD570 (Blank Corrected)	1.720	1.800	1.718	0.074	0.081	0.081	1.350	1.532	1.562
	1.729	1.762	1.734	0.078	0.089	0.089	1.398	1.485	1.583
Mean OD570 of the Duplicates(Blank Corrected)	1.725	1.781	1.726	0.076	0.085	0.085	1.374	1.509	1.573
Total Mean OD570 of the 3 Replicate Tissues (Blank Corrected)	1.744*			0.082			1.485
SD of Mean OD570 of the Duplicates (BlankCorrected)	0.032			0.005			0.101
Relative Tissue Viability [%]	98.9	102.1	99.0	4.4	4.9	4.9	78.8	86.5	90.2
Mean Relative Tissue Viability [%]	100.0			4.7			85.2
SD of Relative Tissue Viability [%]	1.8			0.3			5.8
CV [% Viabilities]	1.8			6.2			6.8

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

Table 2: Results of the NSCliving control

NSCliving	TVT		Negative Control
Tissue	1	2	1	2	3
Absolute OD570	0.049	0.048	1.765	1.845	1.763
	0.052	0.050	1.773	1.807	1.778
OD570 (Blank Corrected)	0.004	0.003	1.720	1.800	1.718
	0.007	0.005	1.729	1.762	1.734
Mean OD570 of the Duplicates (Blank Corrected)	0.006	0.004	1.725	1.781	1.726
Total Mean OD570 of the 2 or 3 Replicate Tissues (Blank Corrected)	0.005		1.744
SD of Mean OD570 of the Duplicates (Blank Corrected)	0.001		0.032
NSCliving [%]	0.3		-
Relative Tissue Viability [%]	-		98.9	102.1	99.0
Mean Relative Tissue Viability [%]	-		100.0
SD of Relative Tissue Viability [%]	-		1.8
CV [% Viabilities]	-		1.8

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.

Conclusions:

CLP: not irritating

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01Aug 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:

adopted 09 Oct 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Details on test animals:
SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported on ice in Hank's Buffered Salt Solution (HBSS) containing penicillin/streptomycin.
- Time interval prior to initiating testing: On the test day, fresh eyes were collected from the slaughter house. The corneae were prepared immediately after delivery.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and defective eyes were discarded.
- Indication of any antibiotics used: penicillin/streptomycin containing HBSS was used for transport.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL

NEGATIVE CONTROL:
- Amount applied: 750 µL
- Concentration: 0.9% NaCl solution in deionised water
- Lot no: 18095403

POSITIVE CONTROL:
- Amount applied: 750 µL
- Concentration: 100%
- Lot no.: K50117583

Duration of treatment / exposure:

10 ± 1 min at 32 ± 1 °C in a vertical position

Duration of post- treatment incubation (in vitro):

2 h at 32 ± 1 °C

Number of animals or in vitro replicates:

triplicates for each treatment and control groups

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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 Hanks’ balanced salt solution (HBSS). A specifically designed corneal holder was used to mount each cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS
After an equilibration period, the initial opacity was determined. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay.

TREATMENT METHOD: Closed chamber method
The corneas were mounted in a corneal holder with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and tightened with screws. Starting with the posterior chamber, both chambers of the corneal holder were then filled with complete RPMI 1640 medium and the corneas were incubated for 1 h at 32 ± 1 °C. After the equilibration period an initial illuminescence reading the test item, the positive or the negative control was introduced into the anterior chamber (closed chamber method). The corneas were incubated at 32 ± 1 °C and exposed to the appropriate test substance for 10 ± 1 min.

REMOVAL OF TEST SUBSTANCE
After the incubation either the test item or the control substance was removed and the epithelium was washed at least three times with minimum essential medium (MEM) (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI medium (without phenol red).

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (BASF-OP3.0, Duratec GmbH). The light was measured as illuminance (I = luminous flux per area, unit: lux).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a spectrophotometer (Jenway 6405 UV/VIS) at 490 nm (OD 490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA
Test substance with an IVIS > 55 was considered as severe irritant/corrosive and labelled Category 1 according to CLP/EPA/GHS.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 corneae

Run / experiment:

10 min exposure

Value:

1.22

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:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Technical proficiency of the test system was demonstrated with 12 substances according to OECD 437.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria met for positive control: Yes, the positive control resulted in an IVIS value which fell within two standard deviations of the current historical mean.

Table 1: Results on opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Corrected Opacity Value Opacity Value
1	Negative	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
4	Positive	2.86	26.48	23.62	23.35
5		2.9	29.68	26.78	26.52
6		3.42	31.81	28.39	28.13
MV		3.06	29.32	26.26	26
7	Test Item	2.82	2.79	0.15	-0.11
8		2.75	4.16	1.41	1.14
9		2.57	3.61	1.05	0.78
MV		2.71	3.58	0.87	0.61

Table 2: Results on permeability

Cornea No.	Test Item	OD490 Corrected OD490 Value
1	Negative	0.004
2		0.019
3		0.015
MV		0.013
4	Positive	1.262	1.249
5		1.585	1.572
6		1.289	1.276
MV		1.379	1.366
7	Test Item	0.013	0
8		0.009	-0.004
9		0.138	0.125
MV		0.053	0.041

Table 3: Results in vitro irritation score (IVIS)

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative	0.32	0.004
2		0.18	0.019
3		0.29	0.015
MV		0.26	0.013	0.45
4	Positive	23.35	1.249
5		26.52	1.572
6		28.13	1.276
MV		26	1.366	46.49
7	Test Item	-0.11	0
8		1.14	-0.004
9		0.78	0.125
MV		0.61	0.041	1.22

Table 4: Historical mean In Vitro Irritation Score of the positive control

	IVIS Positive Control - Ethanol 100 %
Mean Value (MV)	48.41
Standard Deviation (SD)	9.25
MV- 2xSD	29.91
MV+2xSD	66.91
Number of Replicates providing Historical Mean: 56

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) 1272/2008.

Conclusions:

CLP: not classified

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Aug 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:

adopted 09 Oct 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Details on test animals:
SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported on ice in Hank's Buffered Salt Solution (HBSS) containing penicillin/streptomycin.
- Time interval prior to initiating testing: On the test day, fresh eyes were collected from the slaughter house. The corneae were prepared immediately after delivery.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and defective eyes were discarded.
- Indication of any antibiotics used: penicillin/streptomycin containing HBSS was used for transport.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL

NEGATIVE CONTROL:
- Amount applied: 750 µL
- Concentration: 0.9% NaCl solution in deionised water
- Lot no: 18095403

POSITIVE CONTROL:
- Amount applied: 750 µL
- Concentration: 100%
- Lot no.: K50117583

Duration of treatment / exposure:

10 ± 1 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:

triplicates for each treatment and control groups

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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 Hanks’ balanced salt solution (HBSS). A specifically designed corneal holder was used to mount each cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS
After an equilibration period, the initial opacity was determined. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay.

TREATMENT METHOD: Closed chamber method
The corneas were mounted in a corneal holder with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and tightened with screws. Starting with the posterior chamber, both chambers of the corneal holder were then filled with complete RPMI 1640 medium and the corneas were incubated for 1 h at 32 ± 1 °C. After the equilibration period an initial illuminescence reading the test item, the positive or the negative control was introduced into the anterior chamber (closed chamber method). The corneas were incubated at 32 ± 1 °C and exposed to the appropriate test substance for 10 ± 1 min.

REMOVAL OF TEST SUBSTANCE
After the incubation either the test item or the control substance was removed and the epithelium was washed at least three times with minimum essential medium (MEM) (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI medium (without phenol red).

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (BASF-OP3.0, Duratec GmbH). The light was measured as illuminance (I = luminous flux per area, unit: lux).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a spectrophotometer (Jenway 6405 UV/VIS) at 490 nm (OD 490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA
Test substance with an IVIS > 55 was considered as severe irritant/corrosive and labelled Category 1 according to CLP/EPA/GHS.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 corneae

Run / experiment:

10 min exposure

Value:

1.55

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:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Technical proficiency of the test system was demonstrated with 12 substances according to OECD 437.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria met for positive control: Yes, the positive control resulted in an IVIS value which fell within two standard deviations of the current historical mean.

Table 1: Results on opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Corrected Opacity Value Opacity Value
1	Negative	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
4	Positive	2.86	26.48	23.62	23.35
5		2.9	29.68	26.78	26.52
6		3.42	31.81	28.39	28.13
MV		3.06	29.32	26.26	26
7	Test Item	2.86	3.77	0.91	0.64
8		2.75	3.84	1.09	0.83
9		2.31	3.42	1.11	0.85
MV		2.64	3.68	1.04	0.78

Table 2: Results on permeability

Cornea No.	Test Item	OD490 Corrected OD490 Value
1	Negative	0.004
2		0.019
3		0.015
MV		0.013
4	Positive	1.262	1.249
5		1.585	1.572
6		1.289	1.276
MV		1.379	1.366
7	Test Item	0.062	0.049
8		0.022	0.009
9		0.109	0.096
MV		0.064	0.052

Table 3: Results in vitro irritation score (IVIS)

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative	0.32	0.004
2		0.18	0.019
3		0.29	0.015
MV		0.26	0.013	0.45
4	Positive	23.35	1.249
5		26.52	1.572
6		28.13	1.276
MV		26	1.366	46.49
7	Test Item	0.64	0.049
8		0.83	0.009
9		0.85	0.096
MV		0.78	0.052	1.55

Table 4: Historical mean In Vitro Irritation Score of the positive control

	IVIS Positive Control - Ethanol 100 %
Mean Value (MV)	48.41
Standard Deviation (SD)	9.25
MV- 2xSD	29.91
MV+2xSD	66.91
Number of Replicates providing Historical Mean: 56

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) 1272/2008.

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

In vitro

The skin irritation potential of Vinasses, residue of fermentation containing biomass of baker’s yeast (Saccharomyces cerevisiae) was determined in vitro by using the skin irritation test with reconstituted human epidermis according to OECD Guideline 439 and in compliance with GLP (The Ethanol REACH Association, 2018). Pre-tests revealed that the test item was non-MTT-reducing but colour-interfering with water. However, the non-specific colour of additional viable tissues was ≤ 5% relative to the negative control of living epidermis, therefore no correction of the results was necessary.

In the main study 30 µL each of the undiluted test substance, the negative control (DPBS) and the positive control (5% SDS) were applied topically to the surface of the EpiDermTM tissues for 60 min (35 min at 37 °C, 25 min at room temperature). After the exposure and 42 h post-incubation period, cytotoxicity was evaluated by MTT reduction.

Compared to the relative absorbance value of the negative controls, the mean relative absorbance value of the test item was reduced to 85.2% after 60 min of exposure. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is considered as non-irritant. The controls confirmed the validity of the study.

In vivo

Within the Vinasses category, in vivo data on skin irritation are available for Vinasses, residue of fermentation. This data was used to support the negative skin irritation properties of Vinasses, residue of fermentation containing biomass of baker’s yeast (Saccharomyces cerevisiae) by read-across. All Vinasses subgroups share a common origin and are therefore constituted of the same components determining their toxicological properties. A detailed justification for category approach is attached in IUCLID section 13.

Vinasses, residue of fermentation were tested for acute dermal irritation/corrosion in two studies according to OECD guideline 404 and complying with GLP (Beerens-Heijnen, 2005; Pels Rijcken, 1992). In each study, 3 albino rabbits were exposed to 0.5 mL of the undiluted test material, applied onto the clipped or shaved skin for 4 h using a semi-occlusive dressing. The treated skin was observed for reactions after patch removal and evaluations were made at 1, 24, 48 and 72 h post-application.

In one study, very slight erythema was observed at the treated skin areas of all 3 animals at 1 h post-application. This effect was fully reversible within 24 hours post-application in all animals. No oedema was observed (Beerens-Heijnen, 2005).

In the second study, the observed skin reaction consisted of very slight oedema in 2 animals and very slight erythema in all 3 animals at 1 h post-application. These effects were fully reversible within 24 h post-application in 2 animals and within 48 h in the third animal (Pels Rijcken, 1992).

There was no evidence of an irritating/corrosive effect of the test materials on the skin and no other signs of intoxication were seen. The mean erythema and edema scores over 24, 48 and 72 h were equal to 0 for all 3 animals in both studies.

Based on the results obtained with Vinasses, residue of fermentation, it can be assumed that Vinasses, residue of fermentation containing biomass of baker’s yeast (Saccharomyces cerevisiae) have no skin irritating potential.

Conclusion skin irritation:

Based on the results of one in vitro study with Vinasses, residue of fermentation containing biomass of baker’s yeast (Saccharomyces cerevisiae) and two in vivo studies conducted with the structural analogue substance Vinasses, residue of fermentation , the substances Vinasses, residue of fermentation containing biomass of baker’s yeast (Saccharomyces cerevisiae) have no skin irritating potential.

Eye irritation

In vitro

The eye irritating potential of Vinasses, residues of fermentation containing biomass of baker’s yeast (Saccharomyces cerevisiae) was investigated in vitro by using the bovine corneal opacity and permeability (BCOP) test. The BCOP test was conducted for two different batches of the test item according to OECD guideline 437 and in compliance with GLP (The Ethanol REACH association, 2018). In both experiments, a set of three freshly isolated bovine corneas was used for each dose group. After an initial reading of the basal corneal opacity, 750 µL of undiluted test substance, negative control (physiological saline) and positive control (ethanol) were applied to the epithelial surface of the corneas. After 10 min of incubation the test substance was removed and the corneas were post-incubated for 2 h at 32 ± 1 °C. Afterwards a second opacity reading was performed. In addition, permeability of the corneas were determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation for 90 min at 32 ± 1 °C. The negative control and the positive control confirmed the validity of the test system and were in the range of the historical controls. Relative to the negative control, the mean IVIS values of the two test substance batches were calculated to be 1.22 and 1.55, respectively. Thus, the test item is not considered irritant to the eye.

In vivo

In addition, read across was performed with the structurally related analogue substances Vinasses, residue of fermentation. All Vinasses subgroups share a common origin and are therefore constituted of the same components determining their toxicological properties. A detailed justification for category approach is attached in IUCLID section 13.

Vinasses, residue of fermentation were tested for eye irritation/corrosion in albino rabbits in a study performed according to the OECD guideline 405 and complying with GLP (van Otterdijk, 2010b). The test material (0.1 mL) was applied into the conjunctival sac of one eye, the other eye serving as control. The eyes were examined and scored 1, 24, 48 and 72 h after application. Initially, one animal was tested only, in which no corrosive effects were observed. The negative response was confirmed using two additional animals. Irritation of the conjunctivae, which consisted of redness, chemosis and discharge, was observed at 1 h post-application. The latter two findings were only present in two animals. These effects were fully reversible within 48 h post-instillation in all animals. No iridial irritation or corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 h post-instillation revealed no corneal epithelial damage. The average cornea, iris, and chemosis scores over 24, 48, and 72 h were all 0 for all animals. The mean conjunctivae score over 24, 48 and 72 h was 0.3 for each of the 3 animals.

Based on the results obtained with Vinasses, residue of fermentation, it can be assumed that Vinasses, residue of fermentation containing biomass of baker’s yeast (Saccharomyces cerevisiae) have no eye irritating potential.

Conclusion eye irritation:

Based on the results of two in vitro studies with Vinasses, residue of fermentation containing biomass of baker’s yeast (Saccharomyces cerevisiae) and of one  in vivo study conducted with the structural analogue substance Vinasses, residue of fermentation , the substances Vinasses, residue of fermentation containing biomass of baker’s yeast (Saccharomyces cerevisiae) have no eye irritating potential.

Justification for classification or non-classification

Based on the data obtained with the test substance and based on read-across from structurally similar substances, the available data on skin irritation/corrosionand eye irritation do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.