N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid

Endpoint summary

Description of key information

skin irritation

- study conducted according to OECD guideline 439, GLP, in vitro humn skin model Epiderm™ was exposed to 25 mg of the test item + 25 µL of DPBS for 95 min and the viability of the cells was determined 24 h after incubation using MTT dye, tissue viability after incubation with the test item was 92.8%, not irritating

eye irritation

- study conducted acvcording to OECD guideline 437, GLP, three bovine corneae were exposed to a 20% solution of the test item for 4 h, the corneae were examined for opacity and permeability, IVIS = 1.05, not irritating

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:

2017-09-07 to 2018-01-16

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:

July 28, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 06, 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

recommended by guideline

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstituted three-dimensional human skin model EpiDerm™ (MatTek)
- Tissue batch number(s): Lot No.: 25849, 25864
- Date of initiation of testing: 2017-10-06

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C for 35 ± 1 min followed by room temperature for 60 ± 1min.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, staggered again in e.g. one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3h ± 5 min
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: specification: MTT QC assay, 4 h, n=3, acceptance: OD (540-570 nm) [1.0-3.0], result: 1.572 ± 0.078
- Barrier function: specification: ET 50 assay 100 µL 1% Triton X-100, 4 time-points, n=3, MTT assay; acceptance: ET-50 [4.77-8.72 h]; result: 4.62 h
- Contamination:
HIV-1 virus - Oligonucleotide-directed amplification - Not detected
Hepatitis B virus - OligonucIeotide- 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
- Reproducibility: In accordance to the test guideline the assay is considered to be of sufficient reproducibility if the
- mean absolute OD 570 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8
- mean relative tissue viability of the three positive control tissues is ≤ 20%
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.
The respective results and historical control data are shown in table 1.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : Fresh and killed tissues were used for determination of non-specific MTT-reduction capability, fresh tissue was used to determine the colouring potential of the test substance and killed tissue was used if the substance acts as non-specific MTT reducer and shows non-specific colouring in order to avoid a possible double correction.
- N. of replicates : 2
- Method of calculation used: To check the non-specific MTT-reducing capability of the test the non-specific reduction of MTT (NSMTT) was determined by using killed tissues. For quantitative correction of results, two killed tissues were treated with 25 mg of the test item (KT) and two killed tissues were left untreated as a control (KU), respectively. NSMTT was then calculated relative to the negative control of living tissues (NK) according to the following formula:
NSMTT [%] = [(OD KT - OD KU )/OD NK ] * 100
If the test item is classified as non-irritant and if non-specific MTT reduction is ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT ) of the test item treated living tissues TM was corrected according to the following formula:
TODTT = OD TM – (OD KT – OD KU )
If non-specific MTT reduction is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method.
To check the colouring potential of the test item 25 mg of the test item were mixed per 300 µL aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min. If the test item is classified as non-irritant and colouring is detected by unaided eye-assessment, and the chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential. For quantitative correction of results, the test was performed using two additional living tissues treated with 25 mg of the test item (TVT). The MTT-staining was performed with the test item treated with tissues, which were incubated in medium without MTT. The non-specific colour of additional viable tissues (NSC living ) was then calculated according to the following formula:
NSC living [%] = [OD TVT /OD NK ]*100
If NSC living is ≤ 5% relative to the negative control of living epidermis, no correction of the results is
necessary.
If NSC living is > 5% and ≤ 30% relative to the negative control of living epidermis, the true MTT
metabolic conversion (TOD TT ) was corrected according to the following formula:
TOD TT = OD TM – OD TVT
If NSC living is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method. For test items which are classified as non-irritant and which act as non-specific MTT-reducers and show non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSC killed ) was performed to avoid a possible double-correction for colour interference. Therefore, two killed tissues were treated with 25 mg of the test item (TKT). The MTT-staining was performed with the test item treated with tissues, which was incubated in medium without MTT. The non-specific colour of additional viable tissues (NSC killed ) was then calculated according to the following formula:
NSC killed [%] = [OD TKT /OD NK ]*100
The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSC living plus NSC killed.
If the coloured test material or MTT reducing substance is classified as “irritant” i.e. mean tissue viability is < 50%, the correction procedures are not necessary.


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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after exposure and post-treatment incubation is less than 50%.
- The test substance is considered to be non-irritant to skin if the viability after exposure and post-treatment incubation is greater than or equal to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of the test item + 25 µL of DPBS

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL DPBS
- Lot/batch no. (if required): 1838067

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL 5% SDS
- Concentration (if solution): 5% solution

Duration of treatment / exposure:

95 min ± 1 min

Duration of post-treatment incubation (if applicable):

24 h ± 2 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

92.8

Vehicle controls validity:

valid

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: no
- Direct-MTT reduction: The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.
- Colour interference with MTT: The mixture of 25 mg of the test item per 300 μl aqua dest. showed colouring detectable by unaided eye-assessment. Therefore, the absorption of the chemical in water was measured in the range of 570 ± 30 nm.
The test item in water absorbed light in the relevant range. For quantitative correction of results, the non-specific colour of additional viable tissues (NSCliving) was determined by using additional viable tissues without MTT-staining and calculated according to the following formula:
NSCliving [%] = [ODTVT/ODNK]*100 = 0.25%
NSCliving was ≤ 5% relative to the negative control of living epidermis, therefore no correction of the results was necessary.

ACCEPTANCE OF RESULTS:
Acceptance criteria: The test is is considered to be valid if
- mean absolute OD 570 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8
- mean relative tissue viability of the three positive control tissues is ≤ 20%
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.

Table 2: Result of the NSC_livingcontrol

NSCliving	TVT		Negative Control
Tissue	1	2	1	2	3
absolute OD570 -values	0.046	0.050	1.911	1.656	1.728
	0.048	0.047	1.911	1.672	1.722
absolute OD570 -  Blank corrected values	0.0027	0.0064	1.8676	1.6125	1.6844
	0.0045	0.0037	1.8674	1.6286	1.6789
mean OD570  (mean of 3 aliquots)	0.004	0.005	1.868	1.621	1.682
total mean OD570 (mean of replicate tissues)	0.004		1.723*
SD OD570  (of the 2 replicate tissues)	0.001		0.129
NSCliving[%]	0.25		-
Relative Tissue Viability [%]	-		108.4	94.0	97.6
Mean Relative Tissue Viability [%]	-		100.0
SD Tissue Viability [%]	-		7.5
CV [% Viabilities]	-		7.5

Table 3: Result of the Test Item Tetrahydrofolic acid (THFA)

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.518 1.554	1.533 1.471	1.619 1.578	0.123 0.124	0.119 0.121	0.112 0.111	1.344 1.394	1.515 1.520	1.427 1.421
OD570(Blank Corrected)	1.475 1.510	1.489 1.428	1.576 1.535	0.080 0.080	0.075 0.078	0.068 0.067	1.3001 1.351	1.471 1.476	1.384 1.377
OD570(Blank Corrected) - NSClivingCorrected	-			-			1.300 1.351	1.471 1.476	1.384 1.377
Mean OD570of the Duplicates (Blank Corrected)	1.493	1.459	1.555	0.080	0.076	0.068	1.326	1.474	1.381
Total Mean OD570of 3 Replicate Tissues (Blank Corrected)	1.502*			0.075			1.393
TODTT	-			-			1.393
SD OD570	0.049			0.006			0.075
Relative Tissue Viability [%]	99.4	97.1	103.5	5.3	5.1	4.5	88.2	98.1	91.9
Mean Relative Tissue Viability [%]	100.0			5.0**			92.8
Mean Relative Tissue Viability [%] - NSClivingCorrected	-			-			92.8
SD Tissue Viability [%]***	3.3			0.4			5.0
CV [% Viabilities]	3.3			8.4			5.4

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

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:

2017-08-03 to 2018-01-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Number of animals: not applicable
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): transported in HBSS + Pen/Strep on ice
- Time interval prior to initiating testing: 1h at 32 ± 1°C

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% solution in physiological saline

VEHICLE
- Concentration (if solution): 0.9% NaCl solution
- Lot/batch no. (if required): 17296405

Duration of treatment / exposure:

4h ± 5 min

Observation period (in vivo):

90 min

Number of animals or in vitro replicates:

3 corneae per treatment

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Immediately after arrival of the eyes, cornea preparation was initiated. The eyes were carefully examined for defects and any defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded.

NUMBER OF REPLICATES
3 corneae for the test item
3 corneae as negative controls treated with physiological saline 0.9% NaCl
3 corneae as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl


APPLICATION DOSE AND EXPOSURE TIME
750 µl of the test item preparation or the control substance was introduced into the anterior chamber

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes, 90 min

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times with MEM containing phenol red and once rinsed with RPMI without phenol red.

- POST-EXPOSURE INCUBATION: After the washing steps the anterior chamber was filled with RPMI and an illuminance measurement was performed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The corneal opacity was determined by an opacimeter (BASF-OP3.0, Duratec GmbH). Three glass filter were calibrated for the measurement. F2, readout 540-560 lux, F3, readout 300-310 lux and F4 readout 95-105 lux.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (Jenway 6405 UV/VIS; OD490)
- Others: each cornea was observed visually and pertinent observations were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) . The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA: The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Remarks:

20% solution in physiological saline

Value:

1.05

Vehicle controls validity:

valid

Remarks:

IVIS = 0.35

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

IVIS = 102.44

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
- Acceptance criteria met for positive control: yes, the negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Table 2: Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative control	1.26	1.29	0.04
2		1.40	1.43	0.04
3		1.26	1.72	0.47
MV		1.30	1.48	0.18
4	Positive control	2.20	72.73	70.53	70.35
5		2.76	81.45	78.68	78.50
6		2.57	94.26	91.69	91.51
MV		2.51	82.81	80.30	80.12
7	Test item	0.70	1.72	1.03	0.85
8		1.87	2.96	1.09	0.91.
9		1.01	2.46	1.45	1.27
MV		1.19	2.38	1.19	1.01

Table 3: Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1	Negative control	0.010
2		0.013
3		0.012
MV		0.012
4	Positive control	1.296	1.284
5		1.363	1.351
6		1.840	1.828
MV		1.500	1.488
7	Test item	0.013	0.001
8		0.024	0.012
9		0.006	-0.006
MV		0.014	0.003

Table 4: In Vitro Irritation Score

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative control	0.04	0.01	0.35
2		0.04	0.013
3		0.47	0.012
MV		0.18	0.012
4	Positive control	70.35	1.284	102.44
5		78.50	1.351
6		91.51	1.828
MV		80.12	1.488
7	Test item	0.85	0.001	1.05
8		0.91	0.012
9		1.27	-0.006
MV		1.01	0.003

   

Interpretation of results:

GHS criteria not met

Conclusions:

The eye irritancy potential of Tetrahydrofolic acid (THFA) was investigated in the bovine corneal opacity and permeability assay. According to the evaluation criteria the test item Tetrahydrofolic acid (THFA) is classified into UN GHS No Category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

skin irritation:

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (adopted July 28, 2015),Tetrahydrofolic acid was applied to the three-dimensional human epidermis model tissue for an exposure period of 95 minutes in triplicates. 25 μL of DPBS were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 25 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

After 35 minutes exposure at 37 °C ± 5°C and 60 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (DPBS) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 95 minutes treatment with Tetrahydrofolic acid compared to the negative control tissues was 92.8%. Since the mean relative tissue viability for the test substance was above 50%,Tetrahydrofolic acid is identified to be not irritating.

eye irritation:

This in vitro study was performed to assess the corneal irritation and damage potential of Tetrahydrofolic acid by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 7 September 2009.

The corneae were incubated with the test substance and controls for 4 h. After rinsing with saline, the corneae were incubated for another 90 min at 32 ± 1°C. The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

The test item caused no increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was 1.05.

The positive control (Imidazole 20%) increased the opacity and permeability of the corneae (mean in vitro score 122.82) corresponding to a classification as corrosive to the eye. With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro score 0.35). Since the mean in vitro irritancy score of the test substance was < 3, Tetrahydrofolic acid is not considered to be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described.

Justification for classification or non-classification

Based on the available data Tetrahydrofolic acid does not need to be classified according to Regulation (EC) No 1272/2008 (CLP) and the Globally HArmonized System for Classification and Labelling of Chemicals (GHS) with respect to skin and eye irritation.