Dipotassium propanedioate

Endpoint summary

Description of key information

An in vitro skin irritation Epiderm test yielded a relative tissue viability of 94.8%. As this is >50%, dipotassium malonate was concluded to be not irritant to the skin.

An in vitro eye dammage BCOP test yielded an IVIS score of 3.53. As this value is in the range of >3 <= 55, no prediction can be made regarding the classification of the test substance dipotassium malonate according to the evaluation criteria.

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:

18.-20.10.2017

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:

28 July 2015

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

purity: ≥ 95%

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes

Source strain:

other: not applicable

Vehicle:

other: DPBS

Remarks:

to improve the contact between the powder and the epidermis

Details on test system:

- Source: MatTek Corporation (82105 Bratislava, Slovakia).
- The EpiDerm™ tissue: normal, human-derived epidermal keratinocytes which have been cultured to
form a multilayered, highly differentiated model of the human epidermis.
- Surface: 0.63 cm.
- Pre-incubation: 60 ± 5 minutes in the incubator (37 ± 1 °C, 5% CO2) in the upper
wells. Then transferred from upper wells into the lower wells for about 19 hours (37 ± 1 °C, 5 ± 0.5% CO2).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg + 25 μL DPBS

Duration of treatment / exposure:

60 ± 1 minutes

Duration of post-treatment incubation (if applicable):

approx. 42 hours

Number of replicates:

3

Details on study design:

Details of the test procedure used:
- EpiDerm™ tissue of human-derived epidermal keratinocytes was used (EPI-200-SIT)
- Conditions of exposure: 37 ± 1 °C, 5% CO2
- Washing: inserts gently rinsed with DPBS
- Number of tissue replicates used per test chemical and controls: 3
- MTT assay: incubation with 0.3 mL of MTT solution for 60 minutes at 37 ± 1 °C
- Data evaluation: the following was calculated: The mean OD of the three negative control tissues
was calculated after blank correction. The mean of the photometric absorbance of the negative control
is set to 100%.
For each individual tissue treated with the test item or the positive control the individual relative
tissue viability is calculated according to the following formula: Relative viability(%) = (mean OD test i
tem / positive control / mean OD negative control) x 100. For the test item and the positive control the
mean relative viability ± rel. standard deviation of the three individual tissues was calculated

Description of evaluation criteria:
- GHS Cat 2 according to UN GHS is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control
- GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%
- Historical data positive control: Mean Viability: 3.9%; Rel. Standard Deviation: 4.4%
- Historical data negative control: Mean Absorption: 1.831; Rel. Standard Deviation: 0.375;

The test meets acceptance criteria if:
- mean absolute OD570 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%.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Single test with three tissues

Value:

94.8

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:

The controls confirmed the validity of the study:
- the mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8.
- the mean relative tissue viability (% negative control) of the positive control was ≤ 20% (5.0%).
- standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.4% - 10.7%).

Result of the Test Item Dipotassium malonate

Name	NK			PC			TM
Tissue	1	2	3	1	2	3	1	2	3
absolute OD570	1.518	1.533	1.619	0.123	0.119	0.112	1.594	1.569	1.270
	1.554	1.471	1.578	0.124	0.121	0.111	1.458	1.609	1.300
OD570(blank-corrected)	1.475	1.4489	1.576	0.080	0.075	0.068	1.550	1.526	1.227
	1.510	1.428	1.535	0.080	0.078	0..067	1.414	1.566	1.257
mean OD570of the duplicates (blank-corrected)	1.493	1.459	1.555	0.080	0.076	0.068	1.482	1.546	1.242
total mean OD570of 3 replicate tissues (blank-corrected)	1.502*			0.075			1.423
SD OD570	0.049			0.006			0.160
relative tissue viability [%]	99.4	97.1	103.5	5.3	5.1	4.5	98.7	102.9	82.7
mean relative tissue viability [%]	100.0			5.0**			94.8
SD tissue viability [%]***	3.3			0.4			10.7
CV [% viabilities]	3.3			8.4			11.3

* Blank-corrected mean OD570 nm of the negative control corresponds to 100% absolute tissue viability.

** Mean relative tissue viability of the three positive control tissues is ≤ 20%.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%

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”.

Executive summary:

The potential of the test item Dipotassium malonate to induce skin irritation was analysed by using the three-dimensional human epidermis model EpiDerm (MatTek) comprising a reconstructed epidermis with a functional stratum corneum. The test was performed according to OECD TG 439 and in compliance to GLP.

In the present study Dipotassium malonate was applied topically to the EpiDerm tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The mixture of 30 μL 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 equaled 0%. The mixture of 30 μL of the test item per 300 μl aqua dest. or per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equaled 0%.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (94.8%) after 60 min treatment and 42 h post-incubation.

The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8. The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (5.0%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.4% - 10.7%).

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

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:

26 July 2013

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Purity: >=95%, color: white

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test system: Freshly isolated bovine cornea obtained as a by-product from animals freshly
slaughtered.
- Source: A. Moksel AG, Buchloe, Germany.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Anterior compartment received the test item, the negative or positive control at a volume of 0.75 mL
on the surface of the cornea.

Duration of treatment / exposure:

- 4 hours ± 5 min incubation at 32 ± 1 °C, washed at least three times with MEM (containing phenol red).

Duration of post- treatment incubation (in vitro):

- After the illuminance measurement the corneas were incubated for 90 minutes with RPMI and sodium fluorescein solution at 32 ± 1 °C for the optical density determination

Number of animals or in vitro replicates:

3 corneas/group

Details on study design:

PREPARATION OF THE CORNEAS:
The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. 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. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

TREATMENT OF THE CORNEAS:
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control. 0.750 mL of the test substance or the control substance was introduced into the anterior chamber. After 4 h incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded.

After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

TEST GROUPS:
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl

VALIDITY OF THE ASSAY:
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.

EVALUATION OF RESULTS:
The following formula was used to calculate the opacity, whereas the values a and b are equipment specific variables empirically determined by the manufacturer: Opacity= ( I0/I-b)/a; with a = 0.025 and b = 0.9894.

The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment. The mean OD490 for the blank cuvettes was calculated. The mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490). Any dilutions that were made to bring the OD490
values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average corrected
OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:

Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490.

The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

The following formula was used to determine the in vitro irritation score (IVIS):

IVIS = mean opacity value + (15 x mean permeability OD490 value)

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of 3 corneas/4 h incubation time

Value:

3.53

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Other effects / acceptance of results:

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

In Vitro Irritation Score after 4 hours incubation time

Cornea no.	Test item	Corrected opacity	Corrected OD490 value	IVIS
1	Negative control	0.63	0.012	0.6
2		0.1	0.01
3		0.66	0.006
MV		0.46	0.009
4	Positive control	96.59	2.101	119.17
5		83.34	1.736
6		98.54	1.434
MV		92.82	1.757
7	Test item	3.32	-0.006	3.53
8		3.94	-0.004
9		3.57	-0.005
MV		3.61	-0.005

MV = mean value

Interpretation of results:

study cannot be used for classification

Conclusions:

The BCOP study yielded an IVIS score of 3.53. As this value is in the range of >3 <= 55, no prediction can be made regarding the classification of the test substance dipotassium malonate according to the evaluation criteria.

Executive summary:

The eye irritancy potential of dipotassium malonate was investigated in the bovine corneal opacity and permeability assay according to OECD 437 and in compliance to GLP.

The test item was suspended with physiological saline 0.9% NaCl to obtain a 20% concentration. All 3 corneas treated with dipotassium malonate showed slight opacity of the tissue.

The following mean in vitro irritation score (IVIS) was calculated: 3.53.

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. 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.

No prediction can be made regarding the classification of the test substance dipotassium malonate according to the evaluation criteria.

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

An in vitro skin irritation Epiderm test yielded a relative tissue viability of 94.8%. As this is >50%, dipotassium malonate was concluded to be not irritant to the skin.

An in vitro eye dammage BCOP test yielded an IVIS score of 3.53. As this value is in the range of >3 <= 55, no prediction can be made regarding the classification of the test substance dipotassium malonate according to the evaluation criteria.