Registration Dossier

Administrative data

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

Referenceopen allclose all

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
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
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: Dec 2018

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 blue/purple, 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:
91
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

Name

Negative Control

Positive Control

Test item

Tissue

1

2

1

2

1

2

OD570 values

1.950

1.957

0.553

0.737

1.781

1.826

1.944

1.995

0.543

0.732

1.803

1.815

OD570 values (blank-corrected)

1.905

1.911

0.508

0.692

1.736

1.781

1.899

1.950

0.498

0.687

1.757

1.770

mean of the duplicates

1.902

1.931

0.503

0.689

1.747

1.775

mean OD

1.916*

0.596

1.761

TODTT- NSMTT

-

-

1.748

TODTTNSMTT and NSCliving

-

-

1.743

tissue viability [%]

99.2

100.8

26.3

36.0

91.1

92.6

relative tissue viability difference [%]

1.5

9.7

1.5

mean tissue viability [%]

100.0

31.1

91.9

mean tissue viability [%]

- NSMTT corrected

-

-

91.2

mean tissue viability [%]

- NSMTT and NSCliving corrected

 

-

 

-

 

90.9

True Tissue Viability

-

-

91.0

*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 skin irritation induced by Vinasses, residue of fermentation containing biomass of Corynebacterium glutamicum. 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).

Vinasses, residues 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 Corynebacterium glutamincum have no skin irritating potential.

Eye irritation

In vitro

The eye irritating potential of Vinasses, residue of fermentation containing biomass of Corynebacterium glutamicum was analysed in vitro by using the reconstituted three-dimensional human corneal epithelium model EpiOcularTM according to OECD 492 and in compliance with GLP (The Ethanol REACH consortium, 2018).

Each 30 μL of the undiluted test item, the negative control (distilled water) or the positive control (methyl acetate) were applied topically to the EpiOcularTM  tissues for an incubation period of 30 minutes. After the exposure period and removal of the test substances, the tissues were immersed for 12 minutes in medium and post-incubated for 120 minutes. Afterwards, cytotoxic effects were determined via 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.

In pre-tests the substance turned out to reduce MTT and to color-interfere with water. The amount of color interference and unspecific MTT-reduction was quantified by using additional killed and living control tissues and the final results on tissue viability were quantitatively corrected. Compared to the mean tissue viability of the negative control, the test item revealed a tissue viability of 91% and is thus not considered irritating to the eyes.

In vivo

Furthermore, in vivo data on eye irritation with the structurally related analogue substances Vinasses, residue of fermentation were considered by read-across for the hazard assessment of Vinasses, residue of fermentation containing biomass of Corynebacterium glutamicum. 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 Corynebacterium glutamicum have no eye irritating potential.

Conclusion eye irritation:

Based on the results of one in vitro study with Vinasses, residue of fermentation containing biomass of Corynebacterium glutamicum 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 Corynebacterium glutamicum have no eye irritating potential.

Justification for classification or non-classification

Based on experimental data with the test material 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.