Iotroxic acid

Endpoint summary

Description of key information

The potential of Iotroxic acid (99.95% purity) to induce skin irritation (OECD 439) and eye irritation (OECD 437) was tested in suitable in vitro test methods. Based on the results, the target substance can be considered non-irritant to the skin. By assessing the results from an in vitro eye irritation test ,the target substance can be considered non-irritant to the eye.

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:

2018-01-22 to 2018-05-18

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:

adopted 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Firstly, 25 µL of sterile DPBS were applied to the epidermal surface in order to improve the contact between the powder and the epidermis. Afterwards, 25 mg (39 mg/cm2) of the test item were applied directly atop the EpiDerm tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue by using a bulb-headed Pasteur pipette

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™-Standard Model (EPI-200-SIT, MatTek)
- Tissue batch number(s): 25882, 25883

EpiDerm Kit:
The EpiDerm tissues were provided as kits (e.g. EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm2); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot No.: 25882, 25883)
2x 24-well plates
8x 6-well plates
1x bottle of assay medium (DMEM-based medium, Lot No.: 021518TMB, 022218ALE)
1x bottle of DPBS Rinse Solution (Lot No.: 092817MGKA)
1x 1 vial 5% SDS Solution (TC-SDS-5%)
25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for the first 35 +/- 1 min, afterwards the plates were placed under the sterile flow until 60 +/- 1 min incubation time of the first dosed tissue was over.
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C


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. 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: 3 h ± 5 min
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 3


PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after exposure and 42 h post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after exposure and 42 h post-treatment incubation is greater than 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 + 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 µL DPBS (DPBS; Gibco, Cat. No. 14040-091, Lot No.: 1838067)

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 µL 5% SDS solution (TC-SDS 5%, MatTek, CAS No.: 151-21-3, Lot No: 092817MGKA).

Duration of treatment / exposure:

60 min +/ 1 min

Duration of post-treatment incubation (if applicable):

42 h post-incubation

Number of replicates:

3 tissues per dose group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three tissues

Value:

89.5

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:

For detailed results see Table 1 in box "Any other information on results incl. tables".

Results of the  Pre-Experiments:

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 was determined to be 0%.

The mixture of 25 mg of the test item per 300 µL aqua dest. or per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC_livingwas determined to be 0%.

Table 1: Result of the test item Iotroxic acid

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	2.070	1.785	2.187	0.100	0.101	0.100	1.597	1.872	1.900
	2.045	1.789	2.133	0.103	0.107	0.100	1.630	1.902	1.875
OD570(Blank Corrected)	2.027	1.741	2.144	0.057	0.057	0.056	1.554	1.828	1.857
	2.002	1.746	2.090	0.060	0.063	0.057	1.587	1.859	1.832
Mean OD570of the Duplicates (Blank Corrected)	2.014	1.743	2.117	0.058	0.060	0.056	1.570	1.843	1.844
Total Mean OD570of 3 Replicate Tissues (Blank Corrected)	1.958*			0.058			1.753
SD OD570	0.193			0.002			0.158
Relative Tissue Viability [%]	102.9	89.0	108.1	3.0	3.1	2.9	80.2	94.1	94.2
Mean Relative Tissue Viability [%]	100.0			3.0**			89.5
SD Tissue Viability [%]***	9.8			0.1			8.1
CV [% Viabilities]	9.8			3.2			9.0

* Blank-corrected mean OD_{570 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%.

Table 2:  Quality Criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nmNK	1.545	0.8 ≤ NK ≤ 2.8	pass
Relative Viability [%] PC	5.0	≤ 20%	pass
SD Viability[%]	0.4 -8.1	≤ 18%	pass

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions, the test item showed no irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was > 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

 In a primary dermal irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200 -SIT,) was topically exposed to Iotroxic acid for 60 mins and 42 h post incubation period. 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 mean relative tissue viability (% negative control) was > 50% (89.5 %). Based on this result, the test item is classified as a non-irritant according to the UN GHS.

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:

2018-03-13 to 2018-04-19

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

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany
- Number of animals: 3 replicates
- Characteristics of donor animals (e.g. age, sex, weight): No data
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories.
- Time interval prior to initiating testing: Immediately after arrival of the eyes, cornea preparation was initiated.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.
- Indication of any antibiotics used: No data

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20%

VEHICLE
- Concentration (if solution): 0.9%
- Lot/batch no. (if required): 17406416

Duration of treatment / exposure:

4 hours ± 5 minutes at 32 °C

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : physiological saline 0.9% NaCl

POSITIVE CONTROL USED : imidazole 20% in physiological saline 0.9% NaCl

APPLICATION DOSE AND EXPOSURE TIME : 750 µL for 4 h ± 5 min

TREATMENT METHOD: Closed chamber method
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. 750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes 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 5 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).

POST-INCUBATION PERIOD: Yes, 90 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: see Table 1 under "Any other information on materials and methods incl. tables"

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of three replicates

Value:

1.41

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Proper conduct of the BCOP was confirmed via a positive response with imidazole and a negative response with 0.9% NaCl. For detailed results please refer to table 2 in box "Any other information on results incl. tables".

Table 2: In Vitro Irritation Score

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative Control	1.03	0.013	0.94
2		0.60	0.010
3		0.62	0.015
Mean value		0.75	0.013
1	Positive Control	80.24	2.127	120.11
2		77.65	3.057
3		75.51	3.277
Mean value		77.80	2.821
1	Test Item	1.35	-0.002	1.41
2		1.79	0.005
3		1.12	-0.006
Mean value		1.42	-0.001

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions, Iotroxic acid is classified as a non - irritant under the UN GHS "No Category."

Executive summary:

In a primary eye irritation study according to OECD 437, 750 µL of the test substance, Iotroxic acid (Purity 99.95 %) was applied to bovine corneas by closed chamber method for 4 hours. The post incubation period was 90 minutes. The in vitro irritation score for the mean of three replicates was found to be 1.41.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 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. Based on these results, the test item is considered a non – irritant under 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

The potential of Iotroxic acid (99.95% purity) to induce skin irritation (OECD 439) and eye irritation (OECD 437) was tested in suitable in vitro test methods.

In a primary dermal irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200 -SIT) was topically exposed to Iotroxic acid for 60 mins and 42 h post incubation period. 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 mean relative tissue viability (% negative control) was > 50% (89.5 %). Based on this result, the test item is classified as a non-irritant according to the UN GHS.

In a primary eye irritation study according to OECD 437, 750 µL of the test substance, Iotroxic acid (Purity 99.95 %) was applied to bovine corneas by closed chamber method for 4 hours. The post incubation period was 90 minutes. The in vitro irritation score for the mean of three replicates was found to be 1.41. 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 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. Based on these results, the test item is considered a non – irritant under UN GHS “No Category.”

Justification for classification or non-classification

Based on available data, no classification for skin and/or eye irritation is warranted based on the results from suitable in vitro tests (OECD 439, 437).