D-ribose

Endpoint summary

Description of key information

In vitro irritation assays were performed for skin and eye irritation.

As results were negative for both skin and eye irritation, corosion assys were considered not relevant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June-September 2018

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

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Riboxyl TM
Appearance: White to slightly yellow powder

Test system:

human skin model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize 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.: 18-EKIN-027)
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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The skin was moistened with 5 μL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and the solid test item (28.1 to 39.9 mg) was added into 12-well plates on top of the skin tissues.
Three tissues were treated, with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively.

Duration of treatment / exposure:

The positive control was re-spread after 7 minutes contact time.
After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item.

Duration of post-treatment incubation (if applicable):

After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Number of replicates:

The test was performed on a total of 3 tissues per test item together with negative and positive
controls.

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 111

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 7.8%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (See Appendix 3). The standard deviation value of the percentage viability of three tissues treated identically was < 10%, indicating that the test system functioned properly.

Table 2

Mean Tissue Viability in the In Vitro Skin Irritation Test with D-Ribose      

	Mean tissue viability (% control)	Standard deviation (%)
Negative control	100	9.9
D-Ribose	111	6.2
Positive control	7.8	3.0

Table1. Mean absorption in the in vitro skin irritation test with D-Ribose

OD570	A	B	C	Mean		SD
Negative Control	1.293	1.234	1.065	1.198	+/-	0.118
D-Ribose	1.417	1.278	1.301	1.332	+/-	0.074
Positive Control	0.061	0.087	0.133	0.093	+/-	0.036

Interpretation of results:

GHS criteria not met

Conclusions:

D-Ribose is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

The objective of this study was to evaluate D-Ribose for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)).

The possible skin irritation potential of

D-Ribose

was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch Q0926501 of D-Ribose was a white to slightly yellow powder. Skin tissue was moistened with 5 μL of Milli-Q water and at least 10 mg of D-Ribose was applied directly on top of the skin tissue for 15 ± 0.5 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 item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with D-Ribose compared to the negative control tissues was 111%. Since the mean relative tissue viability for D-Ribose was above 50% after 15 ± 0.5 minutes treatment Riboxyl TM is considered to be non-irritant.

The positive control had a mean cell viability of 7.8% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 10%, indicating that the test system functioned properly.

In conclusion, D-Ribose is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

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:

July - October 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:

Identification: Riboxyl TM
Batch: Q0926501

Species:

human

Strain:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

At least 50 mg solid was added into the 6-well plates on top of the tissues.
Two tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues with 50 µL Methyl Acetate (positive control) respectively.

Duration of treatment / exposure:

After the exposure period with D-Ribose (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item.

Duration of post- treatment incubation (in vitro):

After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre- labeled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post- Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.

Number of animals or in vitro replicates:

2

Details on study design:

4.6. Experimental Design
4.6.1. Test for the Interference of the Test Item with the MTT Endpoint
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.
4.6.1.1. Test for Color Interference by the Test Item
D-Ribose was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the tissues during the exposure. To assess the color interference, approximately 50 mg of D-Ribose or 50 µL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark.
Furthermore, approximately 50 mg of D-Ribose or 50 µL sterile Milli-Q water as a negative control was added to 2.0 mL isopropanol. The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking. After incubation approximately 1 mL of the mixture was centrifuged for 30 seconds at 16000 g.
At the end of the exposure time, the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. If after subtraction of the negative control, the OD for the test item solution is >0.08, the test item is considered as possibly interacting with the MTT measurement.
4.6.1.2. Test for Reduction of MTT by the Test Item
D-Ribose was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, approximately 50 mg of D-Ribose was added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline). The mixture was incubated for approximately 3 hours at 37.0 ± 1.0°C in the dark. A negative control, 50 µL sterile Milli-Q water was tested concurrently. If the MTT solution color turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT. Only test items which bind to the tissue after rinsing can interact with MTT in the main assay.

4.6.2. Test System Set Up
On the day of receipt the tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for
20 ± 4 hours at 37°C in 1.0 mL fresh pre-warmed Assay Medium. Assay Medium was supplied by MatTek Corporation, Ashland, USA.
DMEM (Dulbecco’s Modified Eagle’s Medium)
Supplemented DMEM medium, serum-free supplied by MatTek Corporation. MTT medium
MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent. Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 52 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.6 - 37.4°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.
4.6.3. Test Item Preparation
No correction was made for the purity/composition of the test item.
The solid test item (55.7 to 58.2 mg) was applied directly on top of the skin tissue. Any residual volumes were discarded.
4.6.4. Application/Treatment of the Test Item
The test was performed on a total of 2 tissues per test item together with a negative control and positive control.
Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free- DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.
Two tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues with 50 µL Methyl Acetate (positive control) respectively.
At least 50 mg solid was added into the 6-well plates on top of the tissues. After the exposure period with D-Ribose (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item.

After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre- labeled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post- Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.
4.6.5. Cell Viability Measurement
After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37°C.
After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 mL isopropanol refrigerated overnight in the dark.
The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.
5. ACCEPTABILITY CRITERIA
The in vitro eye irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be
> 0.8 and < 2.5.
b) The mean relative tissue viability of the positive control should be <50% relative to the negative control.
c) The difference between the % tissue viabilities of the two identically treated replicates should be <20.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
7.1. Calculation of Cell Viability
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl

The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) * 100
8. COMPUTERIZED SYSTEMS
Critical computerized systems used in the study are listed below. All computerized systems used in the conduct of this study have been validated; when a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.

Irritation parameter:

other: tissue viability (%)

Value:

ca. 84

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Table 2

Mean Tissue Viability in the EpiOcular™ Test with D-Ribose

 

	Mean tissue viability (percentage of control)	Difference between two tissues (percentage)
Negative control	100	1.5
D-Ribose	84	9.7
Positive control	15	1.0

Individual OD Measurements at 570 Nm

 

	A (OD570)	B (OD570)
Negative control
OD570 measurement 1	2.0268	1.9934
OD570 measurement 2	1.9704	1.9449
D-Ribose
OD570 measurement 1	1.7854	1.5943
OD570 measurement 2	1.7494	1.5654
Positive control
OD570 measurement 1	0.3211	0.3407
OD570 measurement 2	0.3150	0.3351

OD = Optical density

Duplicate exposures are indicated by A and B.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, D-Ribose is non-irritant in the EpiOcular™ test under the experimental conditions decribed here.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of D-Ribose. For this purpose D-Ribose was topically applied on the Reconstructed Human EpiOcular™ Model.

The possible eye hazard potential of D-Ribose was tested through topical application for 6 hours.

The study procedures described in this report were based on the most recent OECD guideline. Batch Q0926501 of D-Ribose was a white to slightly yellow powder. D-Ribose (55.7 to

58.2 mg) was applied directly on top of the tissue for 6 hours ± 15 minutes.

After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

The positive control had a mean cell viability of 15% after 6 hours ± 15 minutes exposure.The absolute mean OD570(optical density at 570 nm) of the negative control tissues waswithin the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 10%, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with D-Ribose compared to the negative control tissues was 84%. Since the mean relative tissue viability for D-Ribose was above 60% after 6 hours ± 15 minutes treatment D-Ribose is considered to be non-irritant.

In conclusion, D-Ribose is non-irritant in the EpiOcular™ test under the experimental conditions described in this report.

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

As results were negative for both skin and eye irritation in guideline assays performed under GLP conditions, criteria for skin and eye irritation and corrosion are not met.