Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine

Endpoint summary

Description of key information

Skin:

The test item did not reveal a skin irritation potential in vitro using the EPISKIN model.

Eyes:

The test item did not reveal an ocular corrosion or severe irritation potential in vitro in the Isolated Chicken Eye model.

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:

31 August - 10 October, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Details on test system:

Units: EpiSkinTMSM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EpiSkinTMSM biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium” for incubations. (Batch No.: 16 MAIN3 064; Exp. Date: 21 September 2016)
A flask of sterile “Assay Medium” for use in MTT assays.
(Batch No.: 16 ESSC 040; Exp. Date: 21 September 2016)
The EpiSkinTMSM units were kept in their packaging at room temperature until the pre-incubation was started. The maintenance and assay medium were stored at 2-8°C.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

The epidermal surface was first moistened with 5 µL deionised water* in order to improve further contact between powder and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.
Positive and negative control
A volume of 10 µL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Duration of treatment / exposure:

Exposure:
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.
Rinsing
After the incubation time the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL PBS 1x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).

Duration of post-treatment incubation (if applicable):

After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5 % CO2, ≥95% humidified atmosphere.
MTT test after 42 hours incubation
After the 42 hours incubation the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37°C in an incubator with 5 % CO2 protected from light, ≥95% humidified atmosphere.

Number of replicates:

In this assay 3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls, 2 replicates color controls and 2 replicates non-specific colour control were used. Furthermore, 3 killed treated tissues and 3 killed negative control tissues are used for the MTT evaluation.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-3

Value:

>= 67 - <= 92

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean viability: 76%

Other effects / acceptance of results:

The mean OD value of the three negative control tissues was 0.869. The mean OD value obtained for the positive control was 0.136 and this result corresponds to 16 % viability when compared to the results obtained from the negative control. Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

As the test item has an intrinsic colour (ocher clay), the check-method for possible direct MTT reduction with test item was impossible. The direct interaction with MTT was not defined. However, to avoid the effect of possible interactions with the MTT, an additional control was necessary.
The non-specific MTT reduction (NSMTT) was determined (0%)*, the correction of viability percentage was not necessary.
*: The calculated NSMTT was -3.672%. However, for the calculation of non-specific MTT reduction, small negative numbers are counted as zero, because the reason of the small negative number is a slight difference between the used killed epidermis (biological variability).

As the test item has an intrinsic colour (ocher clay), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.022. The Non Specific Colour % (NSC %) was calculated as
2.6 %. Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.

OD values and viability percentages of the controls:

Substance	Optical Density (OD)		Viability (%)
Negative Control: 1x PBS	1	1.018	117
	2	0.823	95
	3	0.765	88
	mean	0.869	100
	standard deviation (SD)		15.22
Positive Control: SDS (5 % aq.)	1	0.167	19
	2	0.168	19
	3	0.072	8
	mean	0.136	16
	standard deviation (SD)		6.34

OD values and viability percentages of the test item:

Test Item	Optical Density (OD)		Viability (%)
	1	0.604	70
	2	0.581	67
	3	0.796	92
	mean	0.660	76
	standard deviation (SD)		13.55

 OD values of additional controls for MTT-interacting test item:

Additional controls	Optical Density (OD)
Negative control killed tissues: 1x PBS	1	0.065
	2	0.078
	3	0.079
	mean	0.074
Test item treated killed tissues	1	0.042
	2	0.037
	3	0.048
	mean	0.042

 

OD values and NSC % of additional control:

Additional colour control	Optical Density (OD)		Non Specific Colour %(NSC %)
test item treated tissueswithout MTT incubation	1	0.025	2.6
	2	0.020
	mean	0.022

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential.

Executive summary:

The EpiSkinTMSM test has been performed to predict the irritation potential of the test item according to the OECD Test Guideline No. 439. Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure was terminated by rinsing with PBS 1x solution. The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 5% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. The test item has an intrinsic colour (ocher clay), therefore two additional test item treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer, therefore further additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference. In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 76 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

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:

23 August - 28 September, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal
(19.4ºC to 20.3ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4 5 heads/box).
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was applied in its original form, no formulation was required. The test item and the positive control were finely ground before application.
The test item and positive control applied in an amount of 0.03 g/eye.

Duration of treatment / exposure:

The exposure period was 10 seconds.

Number of animals or in vitro replicates:

3 test item treated eyes and 3 positive control eyes and one negative control eye were used in the study.

Details on study design:

isolated chicken eye test (ICET)
Removal of test item: The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.
The Imidazole and test item were stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole treated eyes after the 30, 75, 120 and 180 minutes of observation, but cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse.
The gentle rinsing with 20 mL saline was performed in all (three eyes) test item treated eyes after the 30 and 75 minutes of observation. All test item treated eyes were totally cleared at 120 minutes after the post-treatment rinse.
Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE (Isolated Chicken Eye) class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test substance .

Tool used to assess score: hand-slit lamp/biomicroscope/fluorescein
The appropriate number of eyes was selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm.

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

the test item is categorized as "NO CATEGORY"; ICE classes: 3xI

In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes.

Positive and negative controls showed the expected results. The experiment was considered to be valid

 

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	1%	I
Mean maximum corneal swelling at up to 240 min	1%	I
Mean maximum corneal opacity	0.5	I
Mean fluorescein retention	0.5	I
Other Observations	None
Overall ICE Class^1	3xI

 

 

Positive Control: Imidazole

 

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	25%	III
Mean maximum corneal swelling at up to 240 min	33%	IV
Mean maximum corneal opacity	4.0	IV
Mean fluorescein retention	3.0	IV
Other Observations	Cornea opacity score 4 was observed in two eyes at 30 minutes after the post-treatment rinse.
Overall ICE Class^1	3xIV

 

The positive control Imidazole was classed as corrosive/severely irritating,UNGHS Classification: Category 1

Negative Control: NaCl (9 g/L saline)

 

Observation	Value	ICE Class^1
Mean maximum corneal swelling at up to 75 min	2%	I
Mean maximum corneal swelling at up to 240 min	3%	I
Mean maximum corneal opacity	0.5	I
Mean fluorescein retention	0.0	I
Other Observations	None
Overall ICE Class^1	3xI

The negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model, no ocular corrosion or severe irritation potential was observed.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. Imidazole was used as positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 µL saline solution. In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. Positive and negative controls showed the expected results. In this in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model with the test item, no ocular corrosion or severe irritation potential was observed. According to the guideline OECD 438, the test item is categorized as “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:

The EpiSkinTMSM test has been performed to predict the irritation potential of the test item according to the OECD Test Guideline No. 439. Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure was terminated by rinsing with PBS 1x solution. The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 5% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. The test item has an intrinsic colour (ocher clay), therefore two additional test item treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer, therefore further additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference. In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 76 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Eye irritation:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. Imidazole was used as positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 µL saline solution. In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. Positive and negative controls showed the expected results. In this in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model with the test item, no ocular corrosion or severe irritation potential was observed. According to the guideline OECD 438, the test item is categorized as “No Category”.

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 under Regulation (EC) No 1272/2008. Based on available data on skin irritation/corrosion and eye irritation, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.