Reaction mass of rel-{(1R,2S)-1-methyl-2-[(2R)-5-methylhex-4-en-2-yl]cyclopropyl}methanol and rel-{(1S,2R)-1-methyl-2-[(2R)-5-methylhex-4-en-2-yl]cyclopropyl}methanol

Endpoint summary

Description of key information

An in-vitro study in order to evaluate the registration substance for its ability to induce skin irritation, in line with the OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (adopted 26 July 2013), was performed. It was concluded that the registration substance is irritant.

Furthermore, in line with the OECD Guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test, the EpiDerm™ Skin Model (MatTek Corporation, MA, USA) an assessment on the potential skin corrosivity of the registration substance was carried out. The test article was predicted to be non-corrosive.

Screening for the eye irritancy potential of the registration substance in line with OECD Guideline no. 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”(adopted July 26, 2013), was perfomed. Rosyfolia induced an IVIS > 3 ≤ 55, and based on the prediction model outline by the Guideline, no prediction on the classification can be made. However, the data does indicate the test substance is not a severe irritant with a mean IVIS of 14.1.

Furthermore the Short Time Exposure (STE) Test Using Statens Seruminstitut Rabbit Cornea (SIRC) Cells with the test article, ROSYFOLIA, was conducted in compliance with the OECD Guideline 491: Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (2018). According to the prediction model presented in the Guideline, the results suggest that additional testing would be required for a definitive classification of eye irritation potential per GHS labeling.  However, the data indicates a lack of irritation at the lowest concentration tested (0.05%).

.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

GLP compliance:

yes

Remarks:

See statement of conmpliance in study report < IN VITRO SKIN CORROSION ASSAYUSING THE EPIDERMTM SKIN MODEL (EPI-200): 3- AND 60-MINUTE EXPOSURE PROTOCOL >

Specific details on test material used for the study:

GR-50-1408 is the Givaudan identification code which was employed for ROSYFOLIA during the early, developmental and testing period.

Test system:

human skin model

Details on animal used as source of test system:

N/A - in-vtiro study

Details on test system:

The EpiDerm™ Skin Model (MatTek Corporation, MA, USA) was used to assess the potential skin corrosivity of the test article. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after test article exposure1. The protocol is consistent with the OECD Test Guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test”

The EpiDerm™ Model (EPI-200) (MatTek Corporation, Ashland, USA) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of basal, spinouts and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in-vivo. The model incorporates several features which make it advantageous in the study of potential dermal corrosively. Firstly, the test system uses a serum-free medium which eliminates the possibility of serum protein and test article interaction (Shopsis and Eng. 1988). Secondly, the target cells are epitherlial, derived from human skin (Cannot et al., 1994). Third, since the tissue has a functional stratum corneum, the test materials are applied directly to the tissue surface, at air interface, so that undiluted and/or end use dilutions can be tested directly (Harbell et al., 1994).

Species:

other: N/A in-vitro study

Strain:

other: EpiDerm™ Skin Model

Type of coverage:

other: apical side of the tissue

Preparation of test site:

other: stratum corneum

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

The test article was administered to the test system without dilution (neat).

Details on study design:

The experimental design of this study consists of the determination of the direct MTT reduction potential, assessment of colorant potential, a pH determination, if possible, and a definitive corrosion assay. The in-vitro skin corrosion assay is used to determine the potential skin corrosively of test substances. The method consists of exposing stratum corneum surface (apical side) of the tissue to the test substance. After a 3- or 60minute exposure (2 tissues per exposure), the test substance is removed from the tissue by rinsing with calcium and magnesium-free Dulbecco’s phosphate buffered saline (CMF-DPBS). The rinsed tissue is then incubated for 3 ± 0.1 hours in an MTT dye solution to determine the degree of cytotoxicity (cell death) caused by exposure to the test substance. Viable cells reduce the yellow, soluble oxidised form of the MTT to the blue-black insoluble from. The reduced dye is extracted from the tissue with isopropanol and the amount of reduced dye is determined spectrophotometrically. The relative viability of the treated tissues is calculated relative to the negative control viability. Test substances that reduce tissue viability to <50%, after a 3-minute exposure, are classified corrosive by this method. In addition, test substances which result in tissue viability ≥50% after a 3-minute exposure and <15% after a 60-minute exposure are also classified corrosive. Test substances which result in tissue viability ≥50% after a 3-minute exposure and ≥15% after a 60-minute exposure are classified non-corrosive.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60/3 minutes

Value:

ca. 104.67 - ca. 120.52

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

no indication of corrosion

Other effects / acceptance of results:

The assay was accepted since:
1. the positive control resulted in a corrosive classification (i.e., < 50% cell viability compared to negative controls, after a 3-minute exposure and/or < 15% cell viability compared to negative controls after a 60-minute exposure)
2. the mean OD550 value of the negative control tissues was ≥ 0.8 and ≤ 2.8. The mean OD550 value of the tissues treated with the negative control at 3- and 60-minutes was 1.790 for the 60 minute exposure and 1.598 for the 3 minute exposure.
3. In the range of 20 to 100% viability, the Coefficient of Variation (CV) between 2 identically treated replicates of the negative and positive control per each exposure time was ≤ 30%.

Sponsor's Designation	Conc	Exposure time			% viability	Prediction	Subcategory	pH
Positive Control	NA	60 mins/3 mins			9.04/18.06	Corrosive	1A	NA
Rosyfolia	Neat	60 mins/3 mins			104.67/120.52	Non-corrosive	NA	5.0

Interpretation of results:

GHS criteria not met

Conclusions:

According to the prediction model presented in the attached study report, the test article was predicted to be non-corrosive.

Executive summary:

The potential of rosyfolia to be corrosive was evaluated in EpiDerm tissue using a protocol consistent with OECD TG 431 In vitro skin corrosion: reconstructed human epidermis (RHE) test method. The apical side of the EpiDerm tissues were exposed to rosyfolia and after 3 or 60minute exposures (2 tissues/exposure) the test substance was removed from the tissue and the tissue incubated for 3 hrs in MTT dye solution to determine the degree of cytotoxicity caused by the test substance. The relative viability of the treated tissues is calculated relative to the negative control viability. Test substances that results in tissue viabililty of greater than or equal to 50% after 3 minute of exposure and 15% after a 60 minute exposure are classified as non-corrosive. The results showed that the viability was greater 50% at 3 minutes and well over 15% at 60 minutes. Based on the results of this test, rosyfolia is not considered corrosive.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

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)

Specific details on test material used for the study:

GR-50-1408 is the Givaudan identification code which was employed for ROSYFOLIA during the early, developmental and testing period.

Test system:

human skin model

Source species:

human

Cell type:

other: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2 , Batch no.: 14-EKIN-045

Cell source:

other: three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes

Details on animal used as source of test system:

N/A - in-vtiro study

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 14-EKIN-045. This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Amount/concentration applied:

The liquid test substance was applied undiluted (25 µl) directly on top of the tissue.

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42hr

Number of replicates:

3 tissues/treatment group

Species:

human

Strain:

other: EPISKIN Small ModelTM

Details on test animals or test system and environmental conditions:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC)
EPISKIN Small ModelTM.
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 75 - 88%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 36.8°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Type of coverage:

other: not applicable, in vitro test (see report)

Preparation of test site:

other: not applicable, in vitro test (see report)

Vehicle:

unchanged (no vehicle)

Remarks:

The liquid test substance was applied undiluted directly on top of the tissue.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The liquid test substance was applied undiluted (25 μl) directly on top of the tissue.

Duration of treatment / exposure:

15 minutes at room temperature

Observation period:

not applicable, in vitro test (see report)

Number of animals:

not applicable, in vitro test (see report)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minutes

Value:

7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.

Table 1 Mean absorption in the in vitro skin irritation test with GR-50-1408
		A (OD570)	B  (OD570)	C (OD570)	Mean                              (OD570)                          SD
Negative control		1.13	1.003	1.258	1.13	±	0.128
GR-50-1408		0.063	0.103	0.077	0.081	±	0.02
Positive control		0.084	0.13	0.104	0.106	±	0.023

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

Mean tissue viability in the in vitro skin irritation test:

	Mean tissue Viability (Percentage of Control)
Negative Control	100
Rosyfolia	7
Positive Control	9

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The relative mean tissue viability obtained after 15 minutes treatment with rosyfolia compared to the negative control tissues was 7%. Since the mean relative tissue viability was below 50% it is considered to be irritant.

Executive summary:

An in vitro skin irritation test was conducted using Rosyfolia. In this test, human skin model ( (EPISKIN Small Model (EPISKIN-SMTM) was exposed to rosyfolia, undiluted, through topical application for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment compared to the negative control tissues was 7%. Since the mean relative tissue viability for rosyfolia was below 50% after 15 minutes treatment it is considered to be an irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

2015

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (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:

GR-50-1408 is the Givaudan identification code which was employed for ROSYFOLIA during the early, developmental and testing period.

Species:

other: Bovine eyes

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Bovine eyes were used as soon as possible after slaughter on the same day.
The eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Vehicle:

unchanged (no vehicle)

Remarks:

The test substance was tested neat

Controls:

other: not applicable, in vitro test (see report)

Amount / concentration applied:

750 μl of either the negative control, positive control (10% (w/v) Benzalkonium Chloride) or test substance was introduced onto the epithelium of thecornea.

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.

Observation period (in vivo):

not applicable, in vitro test (see report)

Duration of post- treatment incubation (in vitro):

After exposure the cornea was thoroughly rinsed to remove the test substance and incubated for 2 hours with fresh medium

Number of animals or in vitro replicates:

not applicable, in vitro test (see report)

Details on study design:

The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont-Ferrand, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean after 10 mins of treatment

Value:

ca. 14.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

No prediction on the classification can be made

Other effects / acceptance of results:

Rosyfolia induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of IVIS of 14.1 after 10 minutes of treatment.


IVIS UN GHS
< 3 No Category
> 3; < 55 No prediction can be made
> 55 Category 1



Since rosyfolia induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.


Acceptance criteria:
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within the laboratory historical mean value
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range

All acceptance criteria were met.

The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Additionally the opacity and permeability values were evaluated independently to determine whether the test substance induced irritation through only one of the two endpoints.

Table 1 Summary of opacity, permeability and in vitro scores
Treatment		Mean Opacity1	Mean Permeability1	Mean In vitro Irritation Score1, 2
Negative control		-1	0	-0.7
Positive control (Benzalkonium Chloride)		91	3.571	144.2
Rosyfolia		13	0.051	14.1
1 Calculated using the negative control mean opacity and mean permeability values.
2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

The treated corneas showed opacity values ranging from 12 to 15 and permeability values ranging from 0.033 to 0.067. The corneas were slightly turbid with spots after the 10 minutes of treatment with rosyfolia. No pH effect of the test substance was observed on the rinsing medium.

Rosyfolia induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 14.1 after 10 minutes of treatment.

Since rosyfolia induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Interpretation of results:

other: Rosyfolia mean in vitro irritancy score of 14.1 showed a lack of severe irritation after the exposure period

Remarks:

GHS Category 2

Conclusions:

Rosyfolia induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 14.1 after 10 minutes of treatment. Since rosyfolia induced an IVIS > 3 ≤ 55, no prediction on the classification can be made

Executive summary:

The Bovine Corneal Opacity and Permeability Assay (BCOP) was used to assess the potential ocular irritancy of the test article to isolated bovine corneas. The methods and procedures used in this assay were consistent with OECD Test 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 (2013) and EC Guideline B.47 under GLP. Rosyfolia was topically applied undiluted to the epithelium of the bovine cornea for 10 minutes. After exposure, the cornea was thoroughly rinsed to remove the test substance and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein. A negative control of saline and a positive control of 10% benzalkonium chloride were included concurrently in the experiments. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 144 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Rosyfolia induced ocular irritation through both endpoints, with an IVIS > 3 ≤ 55, indicating that no prediction on the classification can be made. However, the

resulting mean in vitro irritancy score of 14.1 after 10 minutes of treatment does indicate that rosyfolia is not a severe irritant.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 491 (Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes

Remarks:

See statement of conmpliance in study report

Specific details on test material used for the study:

GR-50-1408 is the Givaudan identification code which was employed for ROSYFOLIA during the early, developmental and testing period.

Species:

other: N/A in-vitro study

Vehicle:

other: mineral oil

Controls:

yes, concurrent positive control

Amount / concentration applied:

5-minute exposure to the test article at two concentrations of 5 % and 0.05 %.

Duration of treatment / exposure:

5-minute exposure to the test article at two concentrations of 5 % and 0.05 %.

Details on study design:

The Short Time Exposure (STE) assay was used to evaluate the potential ocular irritancy of a test article by measuring 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye conversion by Statens Seruminstitut Rabbit Cornea (SIRC) cells after a 5-minute exposure to the test article at two concentrations of 5 % and 0.05 %. The protocol meets the requirements of the OECD Test Guideline 491: Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (2018)1.

Irritation parameter:

in vitro irritation score

Run / experiment:

0.05% concentration

Value:

102.6

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Irritation parameter:

in vitro irritation score

Run / experiment:

5% concentration

Value:

26.8

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Other effects / acceptance of results:

IIVS Test Article Sponsor Designation Conc. (w/v) % Relative Viability
Trial 1 Trial 2 Trial 3 Mean ± SD
20AE81 ROSYFOLIA 5% 18.6% 27.8% 34.0% 26.8% ± 7.7%
0.05% 99.7% 101.2% 107.0% 102.6% ± 3.8%
Positive Control SLS 0.01% 27.9% 37.0% 29.6% 31.5% ± 4.8%

Criteria for a valid test:
An individual assay was considered valid when the following criteria were met:
1) The viability of the positive control fell within 2 standard deviations of the historical range. The current historical range at IIVS (updated every three months) is 10.6% to 58.4%. The positive control resulted in viabilities of 27.9%, 37.0%, and 29.6% for the fifth, sixth, and seventh definitive assays.
2) The corrected OD value for the procedural control was ≥ 0.300. The procedural control was >0.300 in all definitive trials.
3) The mean OD values of the solvent controls (saline and mineral oil) were ≥ 80% of the mean OD values of the procedural control. The % comparisons for saline were 89.5%, 97.7%, and 90.3% for saline, and 104.8%, 105.9%, and 94.9% for mineral oil for the fifth, sixth, and seventh definitive assays, respectively.

The study results were considered valid when the standard deviation of cell viabilities for the 3 valid definitive trials was ≤ 15%. The standard deviations for the test article and positive control were <15%.

Interpretation of results:

other: rosyfolia produced a mean viability ± standard deviation of 26.8 ± 7.7% for the 5% concentration, and 102.6% ± 3.8% for the 0.05% concentration. However, the data indicates a lack of irritation at the lowest concentration tested (0.05%).

Remarks:

GHS Category 2

Conclusions:

In the STE assay rosyfolia produced a mean viability ± standard deviation of 26.8 ± 7.7% for the 5% concentration, and 102.6% ± 3.8% for the 0.05% concentration. According to the prediction model presented in the OECD test guideline, when the 5% concentration viability is ≤ 70 % and the 0.05% concentration viability is >70%, additional testing would be required for a definitive classification of eye irritation potential per GHS labeling. However, the data indicates a lack of irritation at the lowest concentration tested (0.05%).

Executive summary:

The Short Time Exposure Assay (STE) was used to evaluate the potential ocular irritancy of Rosyfolia by measuring 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye conversion byStatens Seruminstitut Rabbit Cornea (SIRC) cells after a 5 minute exposure to the test article at two concentrations of 5% and 0.05%. The protocol met the requirements of the OECD Test Guideline 491: Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (2018). Rosyfolia was prepared in mineral oil and two concentrations of the test substance (5% and 0.05%) were exposed to a confluent monolayer of SIRC cells for a 5 minute exposure period. After the 5 minute exposure, the treatment was removed and the cells rinsed and the relative viability was determined using MTT. The average cell viability of three definitive assays used to determine ocular irritation of the test article. The results showed that at 5% the mean viability ± standard deviation was 26.8 ± 7.7%, and 102.6% ± 3.8% for the 0.05% concentration. From these results a UN GHS classification was not predicted; however, the data shows that rosyfolia is a irritant at high concentration (5%) but not a the lower concentration of 0.05%.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Based on the OASIS TIMES eye irritation model ( v.06 -July 2018)), itt predicted the irritating to eye, alert info--alphatic alcohols, alert performance 0.94.

Justification for classification or non-classification

The potential of rosyfolia to be corrosive was evaluated in EpiDerm tissue using a protocol consistent with OECD TG 431