1,3,5-triazine-2,4,6-triamine phosphate

Endpoint summary

Description of key information

Skin: Melamine phosphate was found to be non-irritating in a GLP-compliant in vitro study with human reconstructed epidermis equivalent to OECD TG 439.

Eye: In a screening test equivalent to OECD TG 405 with one rabbit, no adverse effects were observed on iris and on cornea.

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:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen, Germany

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00228CN9
- Expiration date of the lot/batch: January 2, 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable

OTHER SPECIFICS: The substance was homogenous by visual inspection

Test system:

human skin model

Remarks:

EpiDermTM model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPIDERM(TM) 200 kit from MaTek Corp, Ashland MA, USA

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: tissues were incubated in the incubator at 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: washed with sterile PBS once
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Wavelength: 570 nm
- spectrometer: SunriseTM Absorbance Reader

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: freeze-killed tissue
- N. of replicates : 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability is greater than 50 %
- The test substance is considered to be non-irritant to skin if the viability is lower or equal than 50 %


25 μL sterile PBS was applied first. Thereafter, a bulk volume of 25 μL of the test material was applied with a sharp spoon and homogeneously distributed together with the fluid. Control tissues are concurrently applied with 30 μL sterile PBS or 5% SDS (positive control).

The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. After all tissues had been rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After that the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.

After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in 2 mL isopropanol for at least 2 hours at room temperature on a plate shaker (ca. 120 rpm). After shaking the isopropanol extract and piercing the tissues, 2 aliquots of each extract per tissue were transferred to a 96-well microtiter plate. The optical density of the extracted formazan complex was determined spectrophotometrically using a filter with a wavelength of 570 nm.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

25 μL applied to each tissue, spread to match tissue size.

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

ca. 46 h

Number of replicates:

3

Type of coverage:

open

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1st

Value:

90

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

7%

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2nd

Value:

99

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

5%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

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

first experiment		Tissue 1	Tissue 2	Tissue 3	mean	SD
Negative control	mean OD570 nm	2.2577	2.1342	2.1507	2.1808
	viability [% of negative control]	103.5	97.9	98.6	100	3.07
melamine phosphate	mean OD570 nm	1.9412	*	2.0047	1.9729
	viability [% of negative control]	89	*	91.9	90	2.06
5% (w/v) sodium dodecyl sulfate	mean OD570 nm	0.1302	0.1507	0.1487	0.1432
	viability [% of negative control]	6	6.9	6.8	7	0.52
"invalid tissue
second experiment		Tissue 1	Tissue 2	Tissue 3	mean	SD
Negative control	mean OD570 nm	1.8535	2.3	*	2.0768
	viability [% of negative control]	89.3	110.7	*	100	15.2
melamine phosphate	mean OD570 nm	2.152	1.924	2.1105	2.0622
	viability [% of negative control]	103.6	92.6	101.6	99	5.85
5% (w/v) sodium dodecyl sulfate	mean OD570 nm	0.1005	0.114	0.1	0.1048
	viability [% of negative control]	4.8	5.5	4.8	5	0.38

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

only one animal used

GLP compliance:

no

Specific details on test material used for the study:

Purity >98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark
- Stability under test conditions: Stable

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

no details reported

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.1 mL ( 42 mg)

Duration of treatment / exposure:

single exposure, no rinsing

Observation period (in vivo):

14 days; values are given for 1h, 24h, 48h, 72h and 7 days.

Number of animals or in vitro replicates:

one

Details on study design:

A sample of 41.8 mg (a volume of approx. 0.1 ml) was instilled in the conjunctival sac of one eye of an albino rabbit (New Zealand White, SPF quality) after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control.
Immediately after the 24 hour observation, a solution of 2% fluorescein in water was instilled into both eyes to determine corneal epithelial damage.

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48h

Other effects:

At the one hour reading, the scores were 1 for iris and redness of conjunctive and 2 for chemosis.

Instillation of approximately 42 mg (a volume of approx. 0.1 mL) into an eye of a rabbit resulted in effects on the iris and conjunctivae. The iridic irritation (grade 1) was noted on day 1 only. The irritation of the conjunctivae, which consisted of redness, chemosis and discharge, had completely resolved within 7 days in the animal.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

No indication of skin irritation was observed in the in-vitro skin irritation study similar to OECD TG 439. All acceptability criteria were met during performance of the assay. Due to absence of any reduction in viability by the test item, no further in vivo testing is considered necessary (BASF SE 61V0034/10A007; 2010).

Eye

Reversible effects on conjunctivae were observed in a screening study with one rabbit. This might be due to the slightly acid properties, as a saturated solution of 3.9 g/L shows a pH of 3.5. Conjuncitvae scores for redness were 1, 2, 1 and 1 at the 1h, 24h, 48h and 72h readings. Conjunctivae scores for chemosis were 2, 1, 0 and 0 at the 1h, 24h, 48h and 72h readings. No findings were observed at the 7 day reading. These results are below the threshold for classification as an irritant to eyes, therefore follow up testing with the three animals required by the OECD testing guideline is considered not justifiable (BASF SE 232357; 1998).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008:

The available experimental test data are reliable and suitable for classification purposes. As a result the substance is not considered to be classified for skin or eye irritation under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EU) No. 2020/217.