2-amino-5-(2-hydroxyethyl)-6-methyl-4(3H)-pyrimidinone

Endpoint summary

Description of key information

The test item SP02 (trade name: 5OHMlC) is considered as non-irritant to skin and eyes.

Skin irritation:

The performed vitro study evaluates the potential of SP02 (trade name: 5OHMlC) to evoke skin irritation in a reconstructed human epidermis (RhE) test method. It consists of a topical exposure of the neat test item to a RhE model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid, present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The skin irritation potential is determined from the percentage reduction of cell viability in comparison to untreated negative controls.

Three tissues of the human skin model EpiDermTM were treated with SP02 for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier). DPBS-buffer was used as negative control and 5% SDS solution was used as positive control. After treatment with the negative control, the mean absorbance value was 1.9 (mean OD), falling within the required acceptability criterion: 0.8 ≤ mean OD ≤ 2.8. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 1.7% (required: ≤ 20%). The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%). After the treatment with the test item, the mean value of relative tissue viability was reduced to 96.0 %. This value is larger than the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin. Therefore, SP02 (trade name: 5OHMlC) is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

Eye irritation:

The test item SP02 (trade name: 5OHMlC) was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. The solid test item was applied to two tissue replicates. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

Demineralised water was used as negative control and methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and <2.5, OD was 1.7. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 38.8 % (< 50%). Variation within tissue replicates was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 99.5 %. This value is above the threshold for eye irritation potential (≤ 60%). Under the conditions of the test, SP02 (trade name: 5OHMlC) is considered non- eye irritant in the EpiOcularTM Eye Irritation Test.

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:

9 October 2017 - 12 October 2017

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)

Version / remarks:

EU-Method B.46 resp.

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: 103901
- Expiration date of the lot/batch: 1 February 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in the test facility in a closed vessel at room temperature (20 ±5°C).
- Stability under test conditions: Stable, solid

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Tissue Replicate: 3
- Amount(s) applied (volume or weight with unit):
1: 25.6 mg
2: 24.7 mg
3: 25.8 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30E-6 L

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30E-6 L
- Concentration (if solution): 5%

Duration of treatment / exposure:

~ 25 hours

Duration of post-treatment incubation (if applicable):

19 hours

Number of replicates:

3

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):

Tissue Replicate: test item Amount
1: 25.6 mg
2: 24.7 mg
3: 25.8 mg

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
30E-6 L
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
30E-6 L
- Concentration (if solution):

Duration of treatment / exposure:

~ 25 hours

Irritation / corrosion parameter:

% tissue viability

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Based on visual inspection it was consluded that there was no damage on the test system.
- Direct-MTT reduction: In a pre-test it was demonstrated that no direct MTT reduction by the test item occurred; hence no data correction was necessary.
- Colour interference with MTT: In a pre-test it was demonstrated that the test item does not develop a colour without MTT addition

DEMONSTRATION OF TECHNICAL PROFICIENCY: Technical proficiency was demonstrated in Annex V.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: n.a.

Designation	SP02 (trade name: 5OHMlC)	Positive Control
% Tissue viability (tissue 1)	97.0%	1.6%
% Tissue viability (tissue 2)	93.3%	1.6%
% Tissue viability (tissue 3)	97.7%	1.8%
% Tissue viability (mean)	96.0%	1.7%
± SD of mean tissue viability (%)	2.3%	0.1%

Interpretation of results:

GHS criteria not met

Conclusions:

The test item SP02 (trade name: 5OHMlC) is considered as non-irritant to skin. After the treatment, the mean value of relative tissue viability was reduced to 96.0%. This value is above the threshold for skin irritation (50%). The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8. The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system. Variation within replicates was within the accepted range for negative control, positive control and test item (required: ≤ 18%). For these reasons, the result of the test is considered valid.

Executive summary:

This in vitro study evaluates the potential of SP02 (trade name: 5OHMlC) to evoke skin irritation in a reconstructed human epidermis (RhE) test method. It consists of a topical exposure of the neat test item to a RhE model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid, present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The skin irritation potential is determined from the percentage reduction of cell viability in comparison to untreated negative controls.

Three tissues of the human skin model EpiDermTM were treated with SP02 for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier). DPBS-buffer was used as negative control and 5% SDS solution was used as positive control. After treatment with the negative control, the mean absorbance value was 1.9 (mean OD), falling within the required acceptability criterion: 0.8 ≤ mean OD ≤ 2.8. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 1.7% (required: ≤ 20%). The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%). After the treatment with the test item, the mean value of relative tissue viability was reduced to 96.0 %. This value is larger than the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin. Therefore, SP02 (trade name: 5OHMlC) is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

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:

Nov 2017 - Mar 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: 103901
- Expiration date of the lot/batch: 01. Feb. 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (20 ± 5°C)
- Stability under test conditions: Stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none

Species:

human

Details on test animals or tissues and environmental conditions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37 ± 1 °C,
- Humidity (%): 80 - 100
-CO2: 5 ± 1 %

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): 52.8 an 53.1 mg

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
- RhCE tissue construct used, including batch number : EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia. Designation of the kit: OCL-200-EIT. Day of delivery: 12. Dec. 2017. Batch no.: 27017
- Doses of test chemical and control substances used : 52.8 an 53.1 mg of test chemical , 50 μL of the controls

Irritation parameter:

other: % cell viability as measured by dehydrogenase conversion of MTT.

Value:

>= 96.4 - <= 102.5

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: no eye irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None

DEMONSTRATION OF TECHNICAL PROFICIENCY: Technical proficiency is demonstrated in Annex 4

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

For the test item and the positive control, the following percentage values of tissue viability were calculated in comparison to the negative control:

Designation	Positive Control	SP02 (trade name: 5OHMlC)
% Viability (Tissue 1)	37.8%	96.4%
% Viability (Tissue 2)	39.8%	102.5%
% Viability Mean	38.8%	99.5%

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the test, SP02 (trade name: 5OHMlC) is considered non- eye irritant in the EpiOcularTM Eye Irritation Test.

Executive summary:

The test item SP02 (trade name: 5OHMlC) was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. The solid test item was applied to two tissue replicates. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

Demineralised water was used as negative control and methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and <2.5, OD was 1.7. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 38.8 % (< 50%). Variation within tissue replicates was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 99.5 %. This value is above the threshold for eye irritation potential (≤ 60%).

Under the conditions of the test, SP02 (trade name: 5OHMlC) is considered non- eye irritant in the EpiOcularTM Eye Irritation Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

The GHS criteria for classification were not met.