Extract of fava d'anta, obtained from the fruits of Dimorphandra mollis (Leguminosae) by solvent extraction

Endpoint summary

Description of key information

Skin irritation (in vitro): Key study. Test method according to OECD 439, GLP study. The test item can be considered as not irritant to skin as the mean percent viability of the treated tissues was found 91.8% in the in vitro RHE test.

 

Eye irritation (in vitro): Key study. Test method according to the OECD Guideline 438 with GLP. No prediction could be made for the test item in the ICE test since the combination of the 3 endpoints was 2 x III, 1 x II. Thus, additional testing was conducted in order to establish a definitive classification of the test substance.

 

Eye irritation (in vitro): Key study. Test method according to the OECD Guideline 492 with GLP. Under the conditions of the test, no prediction can be made as the test substance is considered either eye irritant or inducing serious eye damage in the EpiOcular™ Eye Irritation Test. Thus, additional testing was conducted in order to establish a definitive classification of the test substance.

 

Eye irritation (in vivo): Key study. Test method according to the OECD 405 Guideline with GLP. Based on the results of an acute eye irritation study performed on New Zealand White rabbits, the test substance does not meet the classification criteria and hence the test item is not an eye irritant.

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:

04.03.2021-25.03.20221

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 (incl. QA statement)

Test system:

human skin model

Remarks:

(SkinEthic RHE® model)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Justification for test system used:

The SkinEthic RHE® model has been validated for irritation testing and its use is recommended by
the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be
suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 21-RHE-035
- Production date: N/A
- Shipping date: 23.03.2021
- Delivery date: 23.03.2021
- Date of initiation of testing: 23.03.2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature.
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS.
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 μL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours and 5 minutes
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.4 (Acceptance criterion: >0.7). Historical negative control mean OD range = 0.787-1.130 (OD measured after 1:2 dilution in isopropanol; acceptability criteria should be in the range ≥ 0.4 and ≤1.5)
- Barrier function: 6.5 h (Acceptance criterion: 4.0h ≤ ET50 ≤10.0h)
- Morphology: 5.5 Cell layers (specification ≥ 4). Multi-layered, highly differenciated epidermis consisting of organized basal, spinous and granular layers and a multilayered stratum corneum.
- Contamination: No

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The direct interaction of MTT with the test item was checked by adding 16 mg of the test item to 300 μL of the solution of MTT at 1 mg/mL. A purple solution was observed after 3 hours of incubation between 37.1°C and 37.5°C, 5% CO2.
Therefore, the test item was identified as a direct MTT reducer: two killed control tissue models were added to the study which underwent the entire testing procedure to generate a non-specific MTT reduction control.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test item is considered as non-irritant to skin if the mean percent viability after 42 minutes
exposure and 42 hours of post-treatment incubation is > 50%.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2) if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is “non corrosive”.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after 42 minutes exposure and 42 hours of post treatment incubation is ≤ 50% and in absence of information on a skin corrosion test.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg (32 mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

42 min at room temperature.

Duration of post-treatment incubation (if applicable):

41-hour and 36 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

91.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

distilled water

Positive controls validity:

valid

Remarks:

5% SDS

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes.
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A full demonstration of proficiency was
performed for the EpiSkin model, plus a reduced validation with the SkinEthic RHE model, having
into account that both models are very similar. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, mean OD = 0.950 (OD measured after 1:2 dilution
in isopropanol; criterion for acceptability should be in the range ≥ 0.4 and ≤1.5).
- Acceptance criteria met for positive control: yes, mean viability = 1.3% (criterion for acceptability
should be < 40%).
- Acceptance criteria met for variability between replicate measurements: yes. SD of negative, positive and test item replicates were 10.4, 0.1 and 15.3% respectively (criterion for acceptability, SD ≤ 18%).

Table 1. Table of results

	Well ID	OD	Mean OD/disc (#)	Mean OD/product	Viability %	Mean viability %	SD viability	Conclusion
Negative control	SPL-1	1.061 1.073 1.046	1.060	0.950	111.6	100.0	10.4
	SPL-2	0.779 0.886 0.945	0.870		91.6
	SPL-3	0.948 0.891 0.920	0.920		96.8
Positive control	SPL-4	0.012 0.012 0.013	0.013	0.012	1.4	1.3	0.1	Irritant
	SPL-5	0.011 0.010 0.011	0.011		1.2
	SPL-6	0.012 0.012 0.012	0.012		1.3
Test item PH-21/0194	SPL-7	1.070 1.074 1.098	1.081	0.913	113.8	96.1	15.3
	SPL-8	0.848 0.828 0.845	0.841		88.5
	SPL-9	0.821 0.811 0.820	0.818		86.1
PH-21/0194 NSMTT	SPL-10	0.042 0.042 0.042	0.042	0.041	4.4	4.3	0.1
	SPL-11	0.040 0.040 0.040	0.040		4.2
PH-21/0194 corrected						91.8		Non-irritant

#: mean of 3 values (triplicate of the same extract)
SPL: sample
OD: optical density
SD: standard deviation

Interpretation of results:

other: Not classified (CLP Regulation EC no. 1272/2008)

Conclusions:

The test item can be considered as not irritant to skin as the mean percent viability of the treated tissues was found 91.8% in the in vitro RHE test.

Executive summary:

An in vitro skin irritation test was conducted for the test item in a reconstructed human epidermis model ( SkinEthic™) according to OECD TG 439 (GLP study). Three epidermis units, previously moistened with 10 μL of distilled water, were treated with 16 mg test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated for the post-treatment incubation period for 41-hour and 36 minutes in fresh medium at 37ºC, 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol during 2 hours under gentle agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol. Under the test conditions, the mean percent viability of the treated tissues was 91.8%, versus 1.3% in the positive control (5% SDS). Therefore, the test item is considered as not 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:

21 June 2021 to 24 June 2021

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)

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ model and the corresponding test method have been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline (OECD No. 492), and the method is applicable to mixtures, solids, liquids, semi-solids and waxes. Therefore, it was considered to be suitable for this study.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The test was performed using EpiOcular™ Reconstructed Human Corneum Epithelium (RhCE) from MatTek Corporation. The system consists of normal, human-derived corneal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2. The certificate of Analysis of the test system is included in the report. The tissue viability and barrier function tests are within the acceptable ranges and indicate the appropiate formation of the mucosal barrier and a viable basal cell layer. Tissues were used within 48 h of their delivery.
The cells used to produce EpiOcular (TM) tissue are screened for potential biological contaminants:
- HIV-1 virus - oligonucleotide-directed amplification: Not detected.
- Hepatitis B virus - oligonucleotide-directed amplification: Not detected.
- Hepatitis C virus - oligonucleotide-directed amplification: Not detected.
- Bacteria, yeast and other fungi - long term antibiotic, antimycotic free culture: Not detected.

- RhCE tissue or hCE cell construct used, including batch number: 2 living RhCE tissue replicates (EpiOcularTM OCL-200, supplied by MatTek Corporation, batch No. 34916) and 2 additional killed Human skin models (EpiOcularTM OCL-200, supplied by MatTek Corporation, batch No. 34911).

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 for each replicate.

Duration of treatment / exposure:

5 hours and 47 minutes

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2 tissues for the test substance, 2 additional tissues for NSMTT and 2 tissues for each control.

Details on study design:

- Details of the test procedure used: Two tissues (0.6 cm2) were pre-wetted with 20 µL DPBS buffer, incubated for 32 min, then dosed topically with 50 mg test item and exposed for 5 hours and 47 min at 37ºC, 5% CO2, ≥ 95% RH. After exposure, the tissues were rinsed with DPBS, transferred to fresh assay medium, and incubated for 25 min. Then, they were transferred to fresh medium again and incubated for 18 hours at 37ºC, 5% CO2, ≥ 95% RH.
The MTT assay for cell viability evaluation was performed on the tissues, by incubating them for 3 hours with MTT solution at 37ºC, 5% CO2, ≥ 95% RH. The precipitated formazan crystals were then extracted using isopropanol and quantified spectrophotometrically.
- Doses of test chemical and control substances used: 50 mg test item, 50 µL in controls.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: exposure 5 h and 47 min at 37ºC, 5% CO2; post-exposure immersion 25 min at room temperature; post-exposure incubation 18 h at 37ºC, 5% CO2.
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): the test item was identified as a direct MTT reducer. Thus, 2 killed control tissue models were added to the study which were treated in the same manner that the living ones in order to generate non-specific MTT reduction.
- Number of tissue replicates used per test chemical and controls: 2 tissues for the test substance, 2 additional tissues for NSMTT and 2 tissues for each control.
- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device: Wavelength: 570 nm.
- Description of the method used to quantify MTT formazan: Optical density by microtiter plate photometer.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: According to OECD 492, the percentage tissue viability cut-off value distinguishing classified from non-classified test chemicals is 60%. Therefore, the test item is identified as not requiring classification if the mean percent tissue viability after exposure and post-exposure incubation is > 60%. Otherwise, the test item is identified as potentially requiring classification and labelling.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: yes, included in the study report.
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: The validity of the RhCE EpiOcular™ test at laboratory facility was demonstrated in a proficiency study. For this purpose 15 proficiency chemicals (indicated by the OECD 492 guideline) were tested. All of the 15 proficiency chemicals were correctly categorized.
- Positive and negative control means and acceptance ranges based on historical data: yes, see table below.
- Acceptable variability between tissue replicates for positive and negative controls: yes, 2.8% (negative control) and 0.9% (positive control). The values for negative and positive controls were below 20% (OECD Guideline 492).
- Acceptable variability between tissue replicates for the test chemical: yes, 1.5% and 13% (NSMTT control), below 20% (OECD Guideline 492).

Irritation parameter:

mean percent tissue viability 

Run / experiment:

test item

Value:

99.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

mean percent tissue viability 

Run / experiment:

NSMTT

Value:

40.13

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Test item corrected = test item viability - NSMTT viability

Value:

59.35

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (mean OD = 0.866; As the extract was diluted at 50% just before the OD measurement, the acceptability criteria should be in the range > 0.4 and < 1.4 for the negative control)
- Acceptance criteria met for positive control: yes (mean cell viability = 30.1%; demanded < 50% of negative control)

Table 1: Assessment of the eye irritation potential individual and average values of OD

	Well ID	OD	Mean OD  / disc ( #)	Mean OD / product	Viability %	Mean viability %	Difference of viability	Conclusion
Negative control	SPL1	0,678 1,089 0,794	0,854	0,866	98,6	100,0	2,8
	SPL2	0,820 0,892 0,922	0,878		101,4
Positive control	SPL3	0,241 0,260 0,269	0,257	0,261	29,7	30,1	0,9	UN GHS Category 2 or 1
	SPL4	0,247 0,273 0,273	0,265		30,6
Test item PH-21/0194	SPL7	0,810 0,855 0,900	0,855	0,862	98,7	99,5	1,5	UN GHS Category 2 or 1
	SPL8	0,765 0,904 0,933	0,868		100,2
Test item PH-21/0194 NSMTT	SPL9	0,271 0,296 0,305	0,291	0,348	33,6	40,13	13,0
	SPL10	0,375 0,418 0,419	0,404		46,7
Test item PH-21/0194 corrected						59,35		UN GHS Category 2 or 1

#: mean of 3 values (triplicate of the same extract)

OD: optical density

SPL: sample

Acceptability criteria:

Tissues treated with the positive control substance should show a mean tissue viability < 50%.

The difference of viability between two tissue replicates should be less than 20%.

Negative control: OD values of the two replicates should be in the range > 0.8 and < 8. As the extract was diluted at 50% just before the OD measurement, the acceptability criteria should be in the range > 0.4 and < 1.4 for the negative control

 

Interpretation of results:

other: the results obtained under these experimental conditions lead to the category “no prediction can be made” and further testing is required to establish a definitive classification

Conclusions:

Under the conditions of the test, no prediction can be made as the test substance is considered either eye irritant or inducing serious eye damage in the EpiOcular™ Eye Irritation Test.

Executive summary:

An in vitro eye irritation test (EIT) according to OECD TG 492 was conducted under GLP conditions to assess the irritation potential of the test item in reconstructed human cornea-like epithelium (RhCE) tissues (EpiOcular^TM tissue model). Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. The test item was identified as a direct MTT reducer, hence 2 additional killed control tissues were added to the study and treated in the same manner that the living tissues. 2 tissues (0.6 cm2) were pre-wetted with 20 µL DPBS buffer, incubated for 32 min, then dosed topically with 50 mg test item and exposed for 5 hours and 47 min at 37ºC, 5% CO2, ≥ 95% RH. After exposure, the tissues were rinsed with DPBS, transferred to fresh assay medium, and incubated for 25 min. Then, they were transferred to fresh medium again and incubated for 18 hours at 37ºC, 5% CO2, ≥ 95% RH. The MTT assay for cell viability evaluation was performed on the tissues, by incubating them for 3 hours with MTT solution at 37ºC, 5% CO2, ≥ 95% RH. The precipitated formazan crystals were then extracted using isopropanol and quantified spectrophotometrically (OD at 570 nm) in order to determine the cell viability. Concurrent positive and negative controls were run in parallel. All validity criteria were met. The relative true viability (that is, mean test item viability % - mean NSMTT control viability %) of the tissues treated with the test item was 59.35%. This value is below the threshold for eye irritation potential (≤ 60%) which means that the test item is considered either eye irritant or inducing serious eye damage. Thus, no prediction can be made.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 August 2021 - 19 August 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to OECD 405 with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Adita Biosys Private Limited. Plot No.: SPL-26, 2nd Phase, KSSIDC Industrial Estate, Madhugiri Road, Antharasanahalli, Tumakuru-572106, Karnataka, India. CPCSEA Registration No.: 1868/PO/RcBt/16/CPCSEA.
- Age at study initiation:4 months old
- Weight at study initiation: 2.42 - 2.56 kg.
- Housing: The animals were housed individually in stainless steel wire mesh cage (Size: L 24 x B 18 x H 18 inches) having facilities for holding pelleted feed and drinking water.
- Diet (e.g. ad libitum): ad libitum. Altromin Maintenance Diet for rabbits (manufactured by Altromin Spezialfutter GmbH & Co. KG).
- Water (e.g. ad libitum): ad libitum. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: 5 days and 12 days for initial and confirmatory test respectively

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7°C to 22.7°C
- Humidity (%): 49% to 65%.
- Air changes (per hr): 12-15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): initial test: 81.1 mg and confirmatory test: 79.8 mg and 80.9 mg.
- Concentration (if solution): N/A

Duration of treatment / exposure:

The eye was rinsed using 0.9% w/v normal saline after 1 hour treatment.

Observation period (in vivo):

Ocular examinations were performed on both eyes (treated and control) 1 hour, 24, 48 and 72 hours following treatment.

Number of animals or in vitro replicates:

3 (1 for initial test and 2 for confirmatory test)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, with physiological saline because residual test item remained in the eye after one hour.
- Time after start of exposure: 1 h

SCORING SYSTEM: please see table below

TOOL USED TO ASSESS SCORE: Slit lamp examination was carried out using fluorescein strips at 24 hours approximately for both initial and confirmatory test.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 24 h

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 24 h

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritant / corrosive response data:

In both initial and confirmatory test, treated eye (left) revealed ocular lesions like redness at 1 hour observation and the observed lesion reversed back to normal by 24 hours observation. In both initial and confirmatory test, no ocular lesions were observed at 24 hours during slit lamp examination.

For initial test and confirmatory test, the mean score calculated across 3 scoring times (approximately 24, 48 and 72 hours after test item instillation) for cornea, iris, conjunctival redness and conjunctival chemosis were 0, 0, 0 and 0 respectively.

Table 1. Individual animal clinical signs of toxicity and mortality record

Phase of the Experiment	Dose (mL/animal)	Animal No.	Sex	Clinical Signs of Toxicity and Mortality on Day
				1		2		3		4
				#	*	#	*	#	*	#
Initial Test	0.1	Nb8821	F	N	N	N	N	N	N	N
Confirmatory Test	0.1	Nb8822	F	N	N	N	N	N	N	N
	0.1	Nb8823	F	N	N	N	N	N	N	N

M: Male; N: Normal; #: First observation; *: Second observation

 

Table 2. Individual animal eye irritation/corrosion scoring record

Initial Test	Dose: 0.1 mL/animal
Animal No.	Observation Period	Ocular Lesions
		Conjunctiva				Iris		Cornea
		Redness		Chemosis				Opacity		Area
Nb8821	Eyes	LE	RE	LE	RE	LE	RE	LE	RE	LE	RE
	1 hr	1	0	0	0	0	0	0	0	-	-
	24 hrs	0	0	0	0	0	0	0	0	-	-
	48 hrs	0	0	0	0	0	0	0	0	-	-
	72 hrs	0	0	0	0	0	0	0	0	-	-
	Mean Tissue Score	0	0	0	0	0	0	0	0	-	-

 

Confirmatory Test	Dose: 0.1 mL/animal
Animal No.	Observation Period	Ocular Lesions
		Conjunctiva				Iris		Cornea
		Redness		Chemosis				Opacity		Area
Nb8822	Eyes	LE	RE	LE	RE	LE	RE	LE	RE	LE	RE
	1 hr	1	0	0	0	0	0	0	0	-	-
	24 hrs	0	0	0	0	0	0	0	0	-	-
	48 hrs	0	0	0	0	0	0	0	0	-	-
	72 hrs	0	0	0	0	0	0	0	0	-	-
	Mean Tissue Score	0	0	0	0	0	0	0	0	-	-
Nb8823	1 hr	1	0	0	0	0	0	0	0	-	-
	24 hrs	0	0	0	0	0	0	0	0	-	-
	48 hrs	0	0	0	0	0	0	0	0	-	-
	72 hrs	0	0	0	0	0	0	0	0	-	-
	Mean Tissue Score	0	0	0	0	0	0	0	0	-	-

LE: Left Eye (Treated Eye); RE: Right Eye (Untreated control Eye)

Mean Tissue Score = (24 hr+48 hr+72 hr)/3

Redness:  0: Normal, 1: Some blood vessels hyperaemic (injected)

Chemosis: 0: Normal

Iris: 0: Normal                

Opacity: 0: No ulceration or opacity

 

Table 3. Individual animal body weight (kg) and percent change in body weight with respect to day 1

Phase of the Experiment	Dose (mL/animal)	Animal No.	Sex	Body Weight (kg) on Day		Percent Change in Body Weight with Respect to Day
				1	4	1 to 4
Initial Test	0.1	Nb8821	Female	2.42119	2.47002	2.01678
Confirmatory Test	0.1	Nb8822	Female	2.35091	2.39115	1.71168
	0.1	Nb8823	Female	2.56012	2.61196	2.02491
			Mean	2.45552	2.50156	1.86829
			±SD	0.14793	0.15614	0.22149
			n	2	2	2

 

Interpretation of results:

other: Not classified (CLP Regulation EC no. 1272/2008)

Conclusions:

Based on the results of an acute eye irritation study performed on New Zealand White rabbits, the test substance does not meet the classification criteria and hence the test item is not an eye irritant.

Executive summary:

The eye irritation potential of the test substance was determined in accordance with the OECD guideline 405 with GLP. The study was performed in New Zealand White rabbits in two phases: an initial test using single female rabbit and a confirmatory test using two female rabbits. 0.1 mL (initial test: 81.1 mg and confirmatory test: 79.8 mg and 80.9 mg) of test item was instilled into the conjunctival sac of the left eye while right eye served as control. The eyes were scored at 1, 24, 48 and 72 hours. Slit lamp examination was carried out using fluorescein strips at 24 hours for both initial and confirmatory test. The corneal opacity, iris and the conjunctivae (redness and chemosis) scores were recorded. All animals were observed twice daily for clinical signs of toxicity and mortality. The body weights were recorded on the day of receipt, on the day 1 and on day 4 of the experiment. No treatment related clinical signs of toxicity and mortality were observed in any animal. No changes were noted in body weight and percent change in body weight with respect to day 1. In both initial and confirmatory test, treated eye (left) revealed ocular lesions like redness at 1 hour observation and the observed lesion reversed back to normal by 24 hours observation. In both initial and confirmatory test, no ocular lesions were observed at 24 hours during slit lamp examination. For initial test and confirmatory test, the mean score calculated across 3 scoring times (24, 48 and 72 hours after test item instillation) for cornea, iris, conjunctival redness and conjunctival chemosis were 0, 0, 0 and 0 respectively. Based on these results, the test substance does not meet the classification criteria and hence the test item can be considered as non irritant to the eye.

 

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22.03.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughterhouse (Etablissement Brun, 33820 Etauliers, France)
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): approximately 7 weeks old, 1.5 - 2.5 kg
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 22 March 2021 at 8:25 am. Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline
- Time interval prior to initiating testing: 1 hour and 37 minutes (22 March 2021 at 10:02 am)
- Indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No
- Selection and preparation of corneas: The enucleated eye was mounted in a stainless-steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 31.8°C and 32.1°C.
- Quality check of the isolated corneas: the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

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): 30 mg

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

Number of animals or in vitro replicates:

Triplicates (3 eyes) were used for the treated and positive control. A single measure (1 eye) was used for the negative control.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The enucleated eye was mounted in a stainless-steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 31.8°C and 32.1°C.

EQUILIBRATION AND BASELINE RECORDINGS
Once all eyes had been examined and approved, they were incubated between 45 and 65 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero-reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
physiological saline – Dutscher Batch No. C0314. One eye was treated with the negative control

POSITIVE CONTROL USED
sodium hydroxide – Fisher Scientific, Batch No. 0000080257. Three eyes were treated with the positive control

APPLICATION DOSE AND EXPOSURE TIME
Immediately following the zero reference measurements, three eyes (in their holder) were removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied, as supplied, during 10 seconds to the cornea such that the entire surface of the cornea was evenly covered with the test item.

OBSERVATION PERIOD
Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The eyes were rinsed twice with 10 mL of physiological saline at ambient temperature.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: It was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points.
- Damage to epithelium based on fluorescein retention: The mean fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: For the measurement of corneal thickness, the slit-width was set at 9½, equalling 0.095 mm.
- Macroscopic morphological damage to the surface: include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.

SCORING SYSTEM:
- Mean corneal swelling (%): Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item.
- Mean maximum opacity score: Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item
- Mean fluorescein retention score at 30 minutes post-treatment: It was used for the overall category score given for each test or control item

DECISION CRITERIA: Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test item.
Once each endpoint was evaluated, ICE classes were assigned based on a predetermined range. Interpretation of corneal thickness (Table 3), opacity (Table 4), and fluorescein retention (Table 5) using four ICE classes was done. The in vitro classification for a test item was assessed by reading the GHS classification that corresponds to the combination of categories obtained for corneal swelling, corneal opacity, and fluorescein retention and applying the scheme presented in Table 6

Irritation parameter:

cornea opacity score

Run / experiment:

maximal mean

Value:

1.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class III

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class III

Irritation parameter:

corneal swelling 

Run / experiment:

Maximal mean

Value:

8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:no. No morphological effects were noted, whatever the examination
time.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: no

Table 7. Test item results (30 mg).

Endpoint measured	Eye  No.	Time (min)
		-45	30	75	120	180	240
Corneal opacity	10	0	1	1	1	1	1
	11	0	1	1	1	2	2
	12	0	1	1	1	2	2
Mean		0.0	1.0	1.0	1.0	1.7	1.7
ICE class			III
Fluorescein retention	10	0.5	2	-	-	-	-
	11	0.5	2	-	-	-	-
	12	0.5	2	-	-	-	-
Mean		0.5	2.0	-	-	-	-
ICE class			III	-	-	-	-
Corneal thickness	10	0.55	0.55	0.60	0.60	0.60	0.60
	11	0.51	0.54	0.57	0.57	0.57	0.57
	12	0.52	0.52	0.52	0.52	0.53	0.53
Corneal swelling (%)	10	-	0	9	9	9	9
	11	-	6	12	12	12	12
	12	-	0	0	0	2	2
Mean			2	7	7	8	8
ICE class			II
Combination of the 3 Endpoints		2 x III; 1 x II
CLASSIFICATION		No prediction can be made

Note: No morphological effects were noted, whatever the examination time.

Interpretation of results:

other: the results obtained under these experimental conditions lead to the category “no prediction can be made” and further testing is required to establish a definitive classification

Conclusions:

In the ICE test, the test item lead to the combinations of the 3 endpoints for the test item were 2 x III, 1 x II. Therefore, no prediction can be made.

Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 under GLP conditions. The test item was applied at the dose of 30 mg, to 3 enucleated chicken eyes, for 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at -45, 30, 75, 120, 180- and 240-minutes post-dose. The ocular reactions observed in eyes treated with the test item were:
- maximal mean score of corneal opacity: 1.7, corresponding to ICE class III; - mean score of fluorescein retention: 2.0, corresponding to ICE class III; - maximal mean corneal swelling: 8%, corresponding to ICE class II. The combination of the three endpoints for the test item was 2 x III; 1 x II. Therefore, these experimental conditions lead to the category “no prediction can be made” and further testing is required to establish a definitive classification. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information, the substance is not classified for skin irritation/corrosion in accordance with CLP, Regulation (EC) No. 1272/2008.

Based on the available information, the substance is not classified for serious eye damage/eye irritation in accordance with CLP, Regulation (EC) No. 1272/2008.