Alfalfa, ext.

Endpoint summary

Description of key information

Skin irritation: not irritating (OECD TG 439)

Eye irritation: irritating (OECD TG 437 and OECD TG 493)

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

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)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AL01
- Expiration date of the lot/batch: 10.09.2019
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: water solubility 5.35 mg/L at 25C
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none, tested neat
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

OTHER SPECIFICS: none

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Initially, the predictive capacity of the modified EpiDerm Skin Irritation Test (SIT) test method using MatTek EpiDermTM tissue model EPI-200 underwent full prospective validation from 2003-2007. The test method components of this method were used to define the essential test methods components of the original and updated ECVAM Performance Standards (PS).
A modification of the original EpiDerm SIT was validated using the original ECVAM PS in 2008. In 2008, ESAC concluded that the Modified EpiDerm SIT has sufficient accuracy and reliability for prediction of R38 skin irritating and no-label (non-skin irritating) test substances.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm
- Tissue batch number(s): 28672
- Production date: not available
- Shipping date: not available
- Delivery date: 04.12.2018
- Date of initiation of testing: 26.11.2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 mg neat test material, 30 µL positive and negative control
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: not specified
- Incubation time: not specified
- Spectrophotometer:
- Wavelength: 570 nm
- Filter: none
- Filter bandwidth: not applicable
- Linear OD range of spectrophotometer: not available

NUMBER OF REPLICATE TISSUES: 3 per condition

PREDICTION MODEL / DECISION CRITERIA
- A test item is considered an irritant (I) to skin in accordance with UN GHS Category 2 or EU R38 if the skin model viability after exposure and post-treatment incubation is =50%.
- A test item may be considered as a non-irritant (NI) if the skin model viability after exposure and post-treatment incubation is >50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg

Duration of treatment / exposure:

60 ± 1 minutes

Duration of post-treatment incubation (if applicable):

42 ± 4 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

95.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

See attached additional information on results.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results from the Reconstructed Human Epidermis test, conducted according to OECD test guideline 439 and to GLP, Alfalfa, ext.was classified as non-irritant to the skin.

Executive summary:

The Reconstructed Human Epidermis test, conducted according to OECD test guideline 439 and to GLP, was performed to evaluate skin irritation potential of Alfalfa, ext.

After 60-minute exposure on the surface of the EpiDerm constructed epidermis, and 42 ± 4 hours post exposure incubation time, viability of the tissue was assessed and compared to the negative (DPBS) and positive control (SDS). The percentage of viability obtained was 95.3% and therefore, Alfalfa, ext. was classified as non-irritant to the skin.

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:

10 April 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:

Guideline version from 9 October 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AL01
- Expiration date of the lot/batch: 10.09.2019
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approximately at 4 °C in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: water solubility 5.35 mg/L at 25C
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not available
- Preliminary purification step (if any): not available
- Final dilution of a dissolved solid, stock liquid or gel: not available
- Final preparation of a solid: not available

OTHER SPECIFICS: none

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): adult cattle, 12-60 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): transported to the test facility over ice packs on the same day of slaughter
- Time interval prior to initiating testing: corneas were prepared immediately on arrival from abattoir
- Indication of any existing defects or lesions in ocular tissue samples: only damage free corneas were used
- Indication of any antibiotics used: 100 IU/mL penicillin and 100 IU/mL streptomycin

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): 0.75 mL
- Concentration (if solution): not applicable; applied undiluted

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Triplicate

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing Hanks’ Balanced Salt Solution (HBSS) until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects.

QUALITY CHECK OF THE ISOLATED CORNEAS
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer.

NUMBER OF REPLICATES
Three corneas were randomly allocated to the test item, negative and positive control.

NEGATIVE CONTROL USED
Sodium chloride, 0.9 % w/v.

SOLVENT CONTROL USED (if applicable)
Not applicable

POSITIVE CONTROL USED
Ethanol, >99.8%

APPLICATION DOSE AND EXPOSURE TIME
0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1ºC for 10 minutes.

TREATMENT METHOD: closed chamber.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

POST-EXPOSURE INCUBATION: 32 ± 1°C for 120 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader] (OD492)
- Others (e.g, pertinent visual observations, histopathology): (please specify):
The condition of the cornea was visually assessed post treatment and post incubation. No histopathology was performed.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

DECISION CRITERIA:
For an acceptable test the following positive control criterion should be achieved:
Neat ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during 2016 for this testing facility. Therefore, the In Vitro Irritancy Score should fall within the range of 31.6 to 58.7.

For an acceptable test the following negative control criteria should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2016 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be =3.0 and for permeability =0.077.

The test item was classified according to the following prediction model:
IVIS Classification
= 3: No category. Not requiring classification to UN GHS or EU CLP;
> 3: =55 No prediction of eye irritation can be made;
> 55: Category 1. UN GHS or EU CLP Causes serious eye damage.

Irritation parameter:

in vitro irritation score

Value:

5.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

The In Vitro irritancy scores:

Treatment	In Vitro Irritancy Score
Test Item	5.9
Negative Control	0.0
Positive Control	45.4

Interpretation of results:

other: No prediction can be made

Conclusions:

Based on the results from the Bovine Corneal Opacity and Permeability (BCOP) test, conducted according to OECD test guideline 437 and to GLP on Alfalfa, ext. no prediction of eye irritation can be made.

Executive summary:

The Bovine Corneal Opacity and Permeability (BCOP) test, conducted according to OECD test guideline 437 and to GLP, was performed to evaluate the eye hazard potential of Alfalfa, ext.

The undiluted test item was applied for 10 minutes then rinsed followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

The In Vitro Irritancy Score for Alfalfa, ext. was determined at 5.9; therefore, no prediction of eye irritation can be made.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 31 July to 02 August 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AL01
- Expiration date of the lot/batch: 10.09.2019
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: water solubility 5.35 mg/L at 25C
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none, tested neat
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

OTHER SPECIFICS: none

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcularTM Eye Irritation Test (ElT), using the MatTek EpiOcularTM tissue model COL-200, was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcularTM ElT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose, as an alternative to Draize Rabbit Eye Test with excellent correlation - in vivo to in vitro test results.

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): 50 mg
- Concentration (if solution): neat

Duration of treatment / exposure:

6 hours ± 15 minutes

Duration of post- treatment incubation (in vitro):

18 hours ± 15 minutes

Number of animals or in vitro replicates:

3

Details on study design:

- Details of the test procedure used: After pre-wetting tissues with 20m PBS (sterile Dulbecco’s Phosphate Buffered Saline) for 30 minutes ± 2 minutes, a single, topical application of a nominal 50 mg of neat test item was applied to the surface of the tissue using a push-pin applicator, or 50 µl of reference items was applied to the surface of the EpiOcular model for 6 hours ± 15 minutes, followed by a 25 minutes ± 2 minutes post-treatment immersion, and 18 hours ± 15 minutes post-treatment incubation, prior to the MTT endpoint; three tissues per condition (n=3).
- RhCE tissue construct used, including batch number: 27061
- Doses of test chemical and control substances used: 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): exposure for 6 hours ± 15 minutes, followed by a 25 minutes ± 2 minutes post-treatment immersion, and 18 hours ± 15 minutes post-treatment incubation, at 37ºC
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 3 for test item, positive control and negative control
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm

Irritation parameter:

other: % of vialability

Value:

6.525

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean OD570 of negative control = 1.839 (acceptance criteria >0.8 and < 2.5
- Acceptance criteria met for positive control: the mean of the positive control relative percentage viability was below 50% of negative control viability after 6 hours exposure

See attached additional information on results.

Interpretation of results:

other: no prediction can be made

Conclusions:

Based on the results from the Reconstructed human Cornea-like Epithelium test, conducted according to OECD test guideline 492 and to GLP on Alfalfa, ext. no prediction of eye irritation can be made.

Executive summary:

The Reconstructed human Cornea-like Epithelium test, conducted according to OECD test guideline 492 and to GLP, was performed to evaluate the eye hazard potential of Alfalfa, ext.

The undiluted test item was applied for 6 hours to the surface of EpiOcular reconstructed ocular epithelium. Followed by 18 hours post-incubation time, the viability of the tissues was assessed and compared to a negative control. The percentage viability obtained was 6.525%.

No prediction of eye irritation can be made for Alfalfa, ext. as the viability was below 60%.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the Reconstructed Human Epidermis test, conducted according to OECD test guideline 439 and to GLP, Alfalfa, ext. was considered not irritating to the skin based on the tissue viability determined at 95.2%.

In the Bovine Corneal Opacity and Permeability (BCOP) test, conducted according to OECD test guideline 437 and to GLP, the In Vitro Irritancy Score for Alfalfa, ext. was determined at 5.9; therefore, no prediction of eye irritation could be made.

In the Reconstructed human Cornea-like Epithelium test, conducted according to OECD test guideline 492 and to GLP, no prediction of eye irritation could be made for Alfalfa, ext. as the viability was below 60%.

Based on the results from these two in vitro studies, it is concluded that Alfalfa, ext. is an eye irritant (Category 2) and therefore, classified as such according to the criteria outlined in Annex I of CLP Regulation (EC) 1272/2008.

Justification for classification or non-classification

Based on the available information, Alfalfa, ext. does not meet the classification criteria for skin irritation, however, it is considered an eye irritant (Category 2) according to the criteria outlined in Annex I of CLP Regulation (EC) 1272/2008.