Vinasses, residue of fermentation, salt-enriched

Endpoint summary

Description of key information

Skin irritation / skin corrosion in vivo, rabbit (OECD 404): not irritating (read-across from structural analogue source substance Vinasses, residues of fermentation (EC 932-215-9))

Eye irritation in vitro (OECD 492): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

09 Aug - 19 Aug 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Kissleg, Germany
- Age at study initiation: at least 6 weeks old
- Weight at study initiation: at least 1.0 kg
- Housing: controlled environment, individually in labelled cages with perflorated floors
- Diet (e.g. ad libitum): standard laboratory rabbit diet (Charles River Breeding and Maintenance Diet for Rabbits, Altromin, Lage, Germany), approx. 100 g per day. Hay (BMI, Helmond, the netherlands) was provided at least three times a week.
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 5 days before start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 +/- 3.0 (actual range: 20.5-21.6)
- Humidity (%): 30-70 (actual range: 44-78)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity.

IN-LIFE DATES: 09 Aug - 19 Aug 2005

Type of coverage:

semiocclusive

Preparation of test site:

other: clipped

Vehicle:

unchanged (no vehicle)

Controls:

other: adjacant areas of untreated skin of each animal were served as controls

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

3 min, 1 and 4 h (1 animal)
4 h ( 2 further animals)

Observation period:

72 h

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 2x3 cm, flank
- Type of wrap if used: metalline patch was mounted on micropore tape , which was wrapped with Coban elastic bandage (Lohmann GmbH, Neuwied, Germany (Metalline) and 3M, St. Paul, Minnesota, USA (Micropore and Coban)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After removal of a dressing, the treated skin was cleaned of residual test substance using water
- Time after start of exposure: 3 min, 1hour and 4 hours (one rabbit); 4 hours (two further rabbits)

SCORING SYSTEM: Erythema and eschar formation:
no erythema: 0; very slight erythema: 1; well-defined erythema: 2; moderate to severe erythema: 3; severe erythema: 4
Oedema formation:
no oedema: 0; very slight oedema: 1; slight oedema: 2; moderate (raised approx. 1 mm) oedema: 3; severe oedema:4

Irritation parameter:

erythema score

Basis:

animal: #1, #2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicable

Irritation parameter:

edema score

Basis:

animal: #1, #2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicable

Irritant / corrosive response data:

Four-hour exposure to 0.5 mL of NATU-C resulted in very slight erythema in the treated skin areas of the three rabbits. The skin irritation had resolved within 24 hours after exposure in all animals. No oedema was observed.
There was no evidence of a corrosive effect on the skin.

Other effects:

No staining of the treated skin by the test substance was observed and no test substance remnants were seen.
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Base on these results NATU-C is considered to be non-irritating.

Body weights

animal 730, 10 -12 weeks old: prior to application 2274 g; at termination 2399g

animal 739, 7 -9 weeks old: prior to application 1745 g; at termination 1911 g

animal 742, 7 -9 weeks old: prior to application 1757 g; at termination 1639 g

Interpretation of results:

not irritating

Remarks:

Migrated information

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

04 Mar - 07 Mar 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP- guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS (SPF-quality)
- Source: Broekman Institute, Someren, The Netherlands
- Age at study initiation: approx. 14 weeks
- Weight at study initiation: 2150 - 2537 grams
- Housing: Individually in labelled cages with perforated floors and equipped with an automatic drinking system
- Diet (ad libitum): standard laboratory rabbit diet (LKK-20, pellet diameter 4mm, Hope Farms, Woerden, The Netherlands) approx. 100 gram per day.
- Water (ad libitum): tap-water diluted with decalcified water
- Acclimation period: at least 5 days before start of treatment under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 55
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

other: the contralateral flank was similarly prepared (but without test substance) to act as a procedural control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration (if solution): 100%

Duration of treatment / exposure:

4 h

Observation period:

72 h

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: flank, 2x3 cm
- Type of wrap if used: surgical gauze patch mounted on Micropore tape (3M, St. Paul, USA)
The dressing was wrapped around the abdomen and secured with an elastic bandage (Coban, 3M, St. Paul, USA)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): using a tissue moistened with tap-water and subsequently a dry trissue
- Time after start of exposure: 4 hours

SCORING SYSTEM: according to Draize et al., 1944

Irritation parameter:

erythema score

Basis:

animal: #1 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicable

Remarks on result:

other: brown staining of the treated skin by the test substance

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

edema score

Basis:

animal: #1, #2 and #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: reversibility not applicable

Irritant / corrosive response data:

The observed skin irritation consisted of very slight oedema (score 1) in two animals and very slight erythema (score 1) in all three animals. The skin irritation had resolved within 24 hours after exposure in two animals and within 48 hours in the third animal.
There was no evidence of a corrosive effect on the skin.

Other effects:

Brown staining of the treated skin by the test substance was observed in two animals on day 1.
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occured.

Interpretation of results:

not irritating

Remarks:

Migrated information

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:

25 Jul - 06 Sep 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 25 Jun 2018

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:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 28 Jun 2019

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 contaminations of the cell culture

Species:

human

Strain:

other: EpiOcularTM, reconstructed three-dimensional human corneal epithelium

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL (83.3 µL / cm²)

NEGATIVE CONTROL:
- Amount applied: 50 µL
- Lot No.: RNBG3520

POSITIVE CONTROL:
- Amount applied: 50 µL
- Lot No.: S6943111

Duration of treatment / exposure:

30 ± 2 min at 37 ± 2 °C

Duration of post- treatment incubation (in vitro):

120 ± 15 min at 37 ± 2 °C

Number of animals or in vitro replicates:

2 tissues for each treatment and control group

Details on study design:

DETAILS OF THE TEST PROCEDURE USED
The EpiOcular™ Eye Irritation Test (EIT) consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

RHCE TISSUE CONSTRUCT
- Model used: OCL-200-EIT (MatTek Corporation, Bratislava, Slovakia)
- Tissue Batch number: 27062 (main experiment), 27066 (viable tissue controls), 27027 (killed tissue controls)

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiOcular™ tissues was assessed via MTT cytotoxicity assay. The determined OD (540-570 nm) was in the accepted range 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 0.3% Triton X-100. The ET-50 value fits to the acceptance criteria of 12.2 – 37.5 min.
- Contamination: The cells used to produce the EpiOcularTM 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.

DURATION AND TEMPERATURE
- EXPOSURE: 30 ± 2 min at 37 ± 2 °C
- POST-EXPOSURE IMMERSION: 12 ± 2 min at room temperature
- POST-EXPOSURE INCUBATION: 120 ± 15 min at 37 ± 2 °C

REMOVAL OF TEST MATERIAL AND CONTROLS: The tissues were extensively rinsed with Dulbecco's phosphate buffered saline (DPBS).

INDICATION OF CONTROLS USED FOR DIRECT MTT-REDUCERS AND/OR COLOURING TEST CHEMICALS:
The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a pre-experiment. Since the MTT solution color turned black, the test substance is presumed to have reduced the MTT. Therefore, an additional test using freeze-killed tissues was performed. A second pre-test revealed that the test item was also colour interfering upon mixing with water. Therefore coloured tissue controls were performed using two additional viable tissues. Moreover, since the test item showed non-specific colouring of living tissues, a third control for non-specific tissue colour in killed tissues was performed to avoid a possible double-correction for colour interference. The true tissue viability was then calculated as the percent living tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled.
True Tissue Viability = [%] mean Tissue viability - NSMTT - NSCliving + NSCkilled
NSMTT: non-specific reduction of MTT
NSCliving: non-specific colour of additional viable tissues
NSCkilled: non-specific colour of additional killed tissues
- No. of replicates: 2 tissues (killed/viable) for each test item and controls for determination of NSMTT, NSCliving and NSCkilled.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 15 min at 37 ± 2 °C
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter band pass: ± 30nm

EVALUATION CRITERIA
The test substance was considered to be not irritating to eye if the tissue viability after 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation is > 60% relative to the negative control treated tissue.

TEST ACCEPTANCE CRITERIA
- Negative control: The mean absolute OD 570 nm is between 0.8 and 2.5.
- Positive control: The mean relative tissue viability is < 50% of the negative control viability.
- Relative tissue viability difference of replicate tissues is < 20%.

REFERENCE TO HISTORICAL DATA OF THE RHCE TISSUE CONSTRUCT: Please refer to Table 1

Irritation parameter:

other: % tissue viability mean value of 2 tissues

Run / experiment:

30 min exposure

Value:

85.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: tissue viability was corrected for non-specific reduction of MTT and non-specific colour interference

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 (1.961).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control was <50% compared to the negative control (31.1%).
- Relative tissue viability difference of replicate tissues was < 20% (values between 1.5 and 9.7).

Table 2: Results of the test item and controls

Name	Negative Control		Positive Control		Test item
Tissue	1	2	1	2	1	2
OD570 values	1.950	1.957	0.553	0.737	1.689	1.677
	1.944	1.995	0.543	0.732	1.714	1.688
OD570 values (blank-corrected)	1.905	1.911	0.508	0.692	1.644	1.632
	1.899	1.950	0.498	0.687	1.669	1.643
mean of the duplicates	1.902	1.931	0.503	0.689	1.657	1.637
mean OD	1.916*		0.596		1.647
TODTT- NSMTT	-		-		1.644
TODTTNSMTT and NSCliving	-		-		1.636
tissue viability [%]	99.2	100.8	26.3	36.0	86.5	85.4
relative tissue viability difference [%]	1.5		9.7		1.0
mean tissue viability [%]	100.0		31.1		85.9
mean tissue viability [%] - NSMTT corrected	-		-		85.7
mean tissue viability [%] - NSMTT and NSCliving corrected	-		-		85.3
True Tissue Viability	-		-		85.5

*Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

OD: optical density

NSMTT: non-specific reduction of MTT

NSC living: non-specific color of additional viable tissues

Interpretation of results:

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

Conclusions:

CLP: not irritating

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Jul - 06 Sep 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 25 Jun 2018

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:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 07 Mar 2020

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 contaminations of the cell culture

Species:

human

Strain:

other: EpiOcularTM, reconstructed three-dimensional human corneal epithelium

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL (83.3 µL / cm²)

NEGATIVE CONTROL:
- Amount applied: 50 µL
- Lot No.: RNBG3520

POSITIVE CONTROL:
- Amount applied: 50 µL
- Lot No.: S6943111

Duration of treatment / exposure:

30 ± 2 min at 37 ± 2 °C

Duration of post- treatment incubation (in vitro):

120 ± 15 min at 37 ± 2 °C

Number of animals or in vitro replicates:

2 tissues for each treatment and control group

Details on study design:

DETAILS OF THE TEST PROCEDURE USED
The EpiOcular™ Eye Irritation Test (EIT) consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

RHCE TISSUE CONSTRUCT
- Model used: OCL-200-EIT (MatTek Corporation, Bratislava, Slovakia)
- Tissue Batch number: 27062 (main experiment), 27066 (viable tissue controls), 27027 (killed tissue controls)

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiOcular™ tissues was assessed via MTT cytotoxicity assay. The determined OD (540-570 nm) was in the accepted range 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 0.3% Triton X-100. The ET-50 value fits to the acceptance criteria of 12.2 – 37.5 min.
- Contamination: The cells used to produce the EpiOcularTM 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.

DURATION AND TEMPERATURE
- EXPOSURE: 30 ± 2 min at 37 ± 2 °C
- POST-EXPOSURE IMMERSION: 12 ± 2 min at room temperature
- POST-EXPOSURE INCUBATION: 120 ± 15 min at 37 ± 2 °C

REMOVAL OF TEST MATERIAL AND CONTROLS: The tissues were extensively rinsed with Dulbecco's phosphate buffered saline (DPBS).

INDICATION OF CONTROLS USED FOR DIRECT MTT-REDUCERS AND/OR COLOURING TEST CHEMICALS:
The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a pre-experiment. Since the MTT solution colour turned black, the test substance is presumed to have reduced the MTT. Therefore, an additional test using freeze-killed tissues was performed. A second pre-test revealed that the test item was also colour interfering upon mixing with water. Therefore coloured tissue controls were performed using two additional viable tissues. Moreover, since the test item showed non-specific colouring of living tissues, a third control for non-specific tissue colour in killed tissues was performed to avoid a possible double-correction for colour interference. The true tissue viability was then calculated as the percent living tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled.
True Tissue Viability = [%] mean Tissue viability - NSMTT - NSCliving + NSCkilled
NSMTT: non-specific reduction of MTT
NSCliving: non-specific colour of additional viable tissues
NSCkilled: non-specific colour of additional killed tissues
- No. of replicates: 2 tissues (killed/viable) for each test item and controls for determination of NSMTT, NSCliving and NSCkilled.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 15 min at 37 ± 2 °C
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter band pass: ± 30nm

EVALUATION CRITERIA
The test substance was considered to be not irritating to eye if the tissue viability after 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation is > 60% relative to the negative control treated tissue.

TEST ACCEPTANCE CRITERIA
- Negative control: The mean absolute OD 570 nm is between 0.8 and 2.5.
- Positive control: The mean relative tissue viability is < 50% of the negative control viability.
- Relative tissue viability difference of replicate tissues is < 20%.

REFERENCE TO HISTORICAL DATA OF THE RHCE TISSUE CONSTRUCT: Please refer to Table 1

Irritation parameter:

other: % tissue viability mean value of 2 tissues

Run / experiment:

30 min exposure

Value:

84

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: tissue viability was corrected for non-specific reduction of MTT and non-specific colour interference

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 (1.961).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control was <50% compared to the negative control (31.1%).
- Relative tissue viability difference of replicate tissues was < 20% (values between 1.5 and 9.7).

Table 2: Results of the test item and controls

Name	Negative Control		Positive Control		Test item
Tissue	1	2	1	2	1	2
OD570 values	1.950	1.957	0.553	0.737	1.672	1.644
	1.944	1.995	0.543	0.732	1.722	1.646
OD570 values (blank-corrected)	1.905	1.911	0.508	0.692	1.627	1.599
	1.899	1.950	0.498	0.687	1.677	1.601
mean of the duplicates	1.902	1.931	0.503	0.689	1.652	1.600
mean OD	1.916*		0.596		1.626
TODTT- NSMTT	-		-		1.614
TODTTNSMTT and NSCliving	-		-		1.609
tissue viability [%]	99.2	100.8	26.3	36.0	86.2	83.5
relative tissue viability difference [%]	1.5		9.7		2.7
mean tissue viability [%]	100.0		31.1		84.8
mean tissue viability [%] - NSMTT corrected	-		-		84.1
mean tissue viability [%] - NSMTT and NSCliving corrected	-		-		83.8
True Tissue Viability	-		-		84.0

*Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

OD: optical density

NSMTT: non-specific reduction of MTT

NSC living: non-specific color of additional viable tissues

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)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

There are no data available on the skin irritation potential of Vinasses, residue of fermentation, salt enriched. Within the Vinasses category, data on skin irritation are only available for Vinasses, residue of fermentation. However, all Vinasses subgroups share a common origin and are therefore constituted of the same components determining their toxicological properties. Thus, read-across is performed based on a category approach. (A detailed justification for category approach is attached in IUCLID section 13).

The read across was performed with the structurally related analogue substance Vinasses, residue of fermentation. Vinasses, residue of fermentation were tested for acute dermal irritation/corrosion in two in vivo 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, salt enriched have no skin irritating potential.

Eye irritation

In vitro

The available data on eye irritation of Vinasses, residues of fermentation, salt enriched comprise two in vitro studies using reconstructed human corneal epithelium tissues. Two batches of Vinasse, residues of fermentation, salt enriched were analysed for their eye irritating potential using the EpiOcularTM in vitro test according to OECD guideline 492 and in compliance with GLP (The Ethanol REACH association, 2018).

Each 30 µL of the test item, the negative control (distilled water) and the positive control (methyl acetate) were applied topically to the surface of the EpiOcularTM tissue for an incubation period of 30 min. After the exposure period and removal of the test substances, the tissues were immersed for 12 minutes post-exposur and post-incubated for 120 minutes. Afterwards, cytotoxic effects were determined via the MTT reduction assay. Absorbance values of the negative control (OD ≥ 0.8 and ≤ 2.8) and the positive control (tissue viability < 50%) confirmed the validity of the test system.

Pre-tests revealed a MTT-reducing effect of the test compound, as well as colour-interference with water. The amount of colour interference and MTT-reduction was quantified using additional killed and living control tissues and the results on tissue viability were quantitatively corrected. Compared to the mean tissue viability of the negative control, the two batches of the test item revealed a tissue viability of 85.5% and 84%, respectively and are therefore not considered irritating to the eyes.

In vivo

Furthermore, in vivo data on eye irritation of the structurally related analogue substance Vinasses, residue of fermentation was considered by read-across for the hazard assessment of Vinasses, residue of fermentation, salt enriched. 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 were 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, salt enriched have no eye irritating potential.

Conclusion eye irritation:

Based on the results of two in vitro studies with Vinasses, residue of fermentation, salt enriched and of one in vivo study conducted with the structural analogue substance Vinasses, residue of fermentation the substances Vinasses, residue of fermentation, salt enriched have no eye irritating potential.

Justification for classification or non-classification

Based on experimental data with the test substance and based on read-across with structurally similar substances, the available data on skin irritation/corrosion and 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.