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Description of key information

Two in vitro tests are available to evaluation the irritancy potential of Nitrosodiphenylamine.

The Episkin irritation test show no skin irritation with 105% of viability cells. And BCOP test show no eye irritation with an IVIS of 0. Therefore Nitrosodiphenylamine is considered to be not skin or eye 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:
28 June 2017 - 04 September 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
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiSkin Laboratories, Lyon, France
Details on animal used as source of test system:
see below
Justification for test system used:
The EpiskinTM model is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum.
Based on the peer review of the results of an inter-laboratory study with the EpiskinTM model, the ECVAM Scientific Advisory Committee (ESAC) endorsed the conclusion that the EpiskinTM model can be used for distinguishing between skin irritant and non-irritant chemicals. The EpiskinTM model is used for hazard identification and the classification of irritation potential within the context of the sequential skin irritation testing strategy (OECD Test Guideline No. 404, 24th April 2002 and Commission Regulation (EC), No. 440/2008, Part B.4, 30 May 2008).
Vehicle:
unchanged (no vehicle)
Details on test system:
The EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra structure and is functionally equivalent to human in vivo epidermis.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Concentration: neat (as supplied)
- Amount applied (volume or weight with unit): As the test item was a solid, 5 µL of water for injections was first applied to the surface of each epidermis to improve further contact between the test item and the tissue. Then, 10 mg (± 2 mg) of the test item were applied evenly to the surface of each tissue, taking care to spread it over the whole tissue area without damaging the tissue sample.
Duration of treatment / exposure:
Exposure period of 15 minutes, followed by rinsing.
Duration of post-treatment incubation (if applicable):
42-hour recovery period.
Number of replicates:
Three tissues were used for each item.
At the end of the viability assay, formazan level in tissues were assessed in duplicate for each tissue.
The mean OD values (mean OD) were then calculated from the six replicates on each plate.
Details on study design:
PRELIMINARY TESTS
* Test for direct MTT reduction with the test item
As a test substance may directly reduce MTT, thus mimicking mitochondrial succinate dehydrogenase activity, it is necessary to test the ability of the test item to directly reduce MTT before performing the main test.
To identify any test item interference with the MTT endpoint, the following preliminary test was performed:
- 11 mg (± 1 mg) of the test item were added to 2 mL of a 0.3 mg/mL freshly prepared MTT solution,
- a negative control was tested concurrently by adding 10 µL of water for injections to 2 mL of a 0.3 mg/mL freshly prepared MTT solution,
- both mixtures were incubated in darkness at +37°C for 3 hours (± 5 minutes).
Then the colour of the solutions obtained was evaluated.

* Test for the detection of the colouring potential of the test item
The intrinsic colour or the ability of the test item to become coloured in contact with water (simulating a tissue humid environment) was evaluated by adding 11 mg (± 1 mg) of test item to 90 µL of water for injections in a transparent recipient. After 1 hour (± 3 minutes) of mixing, the presence and intensity of the colouration was evaluated.

MAIN TEST
One 12-well plate was used for each test and control items: one plate for the test item, one plate for the negative control and one plate for the positive control. Each item was applied onto triplicate tissues.

PRE-INCUBATION OF THE TISSUES ON THEIR DAY OF ARRIVAL (Day 0)
A volume of 2 mL of pre-warmed (at +37°C) maintenance medium was added to 3 wells on a 12 well plate (one plate per item). Each Episkin TM tissue was then transferred into the maintenance medium pre-filled wells (three tissues per plate). The plates were incubated at +37°C, 5% CO2 in a humidified incubator for at least 24 hours.

TREATMENT OF TISSUES (Day 1)
A volume of 2 mL of pre-warmed maintenance medium was added into 3 wells on the 12 well plate for each item, respectively.
Each test or control item was then applied on three tissues for an exposure period of 15 minutes (± 1 minute) at room temperature. The items were applied topically to the corresponding tissues and gently spread over the epidermal surfaces to ensure uniform covering of the tissues. The tissues were processed (treatment and rinsing) in the same order and at regular time intervals to ensure each tissue received an equal exposure period.

For the positive control only, the SDS solution was re-spread after 7 minutes (± 1 minute) contact time with a curved spatula to improve the distribution of the SDS for the remainder of the contact period.

RINSING OF TISSUES AND INCUBATION FOR 42 HOURS (Day 1)
At the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS. Rinsing was achieved by gently filling and emptying several times each tissue with D PBS to gently remove any residual test or control items. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well and the plates were incubated at +37°C, 5% CO2 in a humidified incubator for 42 hours.

MTT VIABILITY ASSAY (Day 3)
Following the 42 hours incubation period, 2mL of a freshly prepared MTT solution (0.3 mg/mL) were added into 3 wells on each 12 well plate. The tissues were then transferred to the MTT filled wells and incubated for 3 hours (± 5 minutes) at +37°C, 5% CO2 in a humidified incubator.

At the end of the MTT incubation period, a total biopsy of the epidermis was made by using the Episkin™ biopsy punch. Each tissue was examined with the naked eye and the degree of MTT staining was evaluated. The epidermis was separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into micro tubes and incubated with 500 µL of acidic isopropanol. Then, to extract the formazan (reduced MTT) from the MTT-loaded tissues, each tube was stored after vortexing at +5°C and protected from light until Day 6 of the experiment.


OPTICAL DENSITY MEASUREMENTS (Day 6)
At the end of the formazan extraction period, each tube was vortexed until the solution colour became homogenous. Each tube was used to fill 2 wells of a 96-well plate with 200 µL of extract per well. One 96 well plate was used for the negative and positive controls (placed at opposite ends of the plate), and a separate 96-well plate was used for the test item.
For each 96-well plate, the average Optical Density value (OD) of 6 wells containing 200 µL of acidified isopropanol only was used as the blank.
The OD was measured at 570 nm using a plate reader.
Irritation / corrosion parameter:
% tissue viability
Remarks:
15 min
Run / experiment:
Mean
Value:
105
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
6%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE CRITERIA FOR NEGATIVE AND POSITIVE CONTROLS
- the mean cOD of the three negative controls should be = 0.6 and the Standard Deviation (SD) value of the % viability should be = 18%,
- the relative mean viability of the positive control should be = 40% of the relative mean viability of the negative control and the SD value of the % viability should be = 18%.


EVALUATION OF THE COLOURATION OF TISSUES AT THE END OF THE MTT INCUBATION PERIOD
The qualitative evaluation of the MTT staining was performed with the naked eye.
All test item and negative-treated tissues appeared blue which was considered indicative for viable tissues.

EVALUATION OF THE MTT RESULTS
All of the acceptance criteria for the negative and positive controls were fulfilled, therefore the study was considered to be valid.
Interpretation of results:
GHS criteria not met
Remarks:
not skin irritating
Conclusions:
Under the experimental conditions of this study, the test item is considered to be non-irritant to skin.
Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item using the EpiskinTM reconstructed human epidermis model.

The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46). The study was conducted in compliance with CiToxLAB France standard operating procedures and the principles of Good Laboratory Practice.

 

Methods

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential.

Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at +, 5% CO2in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay.

Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

 

Results

 

Preliminary tests

In the preliminary tests, the test item was found to have neither direct MTT reducing properties, nor colouring potential.

 

Main test

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 105% with a standard deviation of 4%. As the mean viability was > 50% after the MTT reduction,the results met the criteria for a non-irritant response.

 

Conclusion

Under the experimental conditions of this study, the test item is considered to be non-irritant to skin.

According to the results of this study, the classification of the test item should be No Category (UN GHS and Regulation (EC) No. 1272/2008).

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:
28 June 2017 - 24 August 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
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
- Species: bovine cattle (Bos Taurus).
- Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
- Age: as French Authorities avoid the use of any organs from the head of bovines aged more than 12 months, bovine cattle were up to 12 months old (typically, 5 to 8 months old).
- Reason for choice: bovine corneas are recommended by Regulatory Authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
- Transport from Supplier to CiToxLAB France: the eyes were transported to CiToxLAB France at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.


Upon arrival at CiToxLAB France, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

- Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light.
- Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
- Washing of the corneas: the corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature. The corneas were used immediately.
Vehicle:
other: paraffin oil
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied = 750 µL
- Concentration = 20% (w/v) in vehicle
Duration of treatment / exposure:
Exposure period of 4 hours (± 5 minutes), followed by rinsing
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
Triplicate corneas for each tested substance (test item formulation, vehicle control, positive control)
Details on study design:
EYES SELECTION
The corneas were carefully examined macroscopically before their assembly in the holders, in order to detect the presence of any defects. Any corneas with defects were discarded. The corneas were then mounted in the corneal holders with the endothelial side against the O-ring of the posterior chamber. Each cornea was identified with the corresponding holder number. After pre-incubation, corneas that showed any macroscopic defect or an opacity value over 7 were discarded.

TREATMENT
The three corneas from the same series were always processed in the same order at each step.
The medium of the anterior chamber was removed and each item was applied onto the epithelium of the cornea for 4 hours in a water bath at +32°C (± 1°C).

REMOVAL OF TEST SUBSTANCE
The purpose of rinsing was to eliminate as much items as possible, while taking care not to damage the cornea. On completion of the treatment period, the test item formulation was removed from the front opening of the anterior chamber and the epithelium was rinsed as follows:
- the residual test item formulation adhering to the walls of the anterior chamber was first removed with a cotton bud and then using a pipette of heated cMEM (32°C),
- the corneas were rinsed four times with pre-warmed cMEM containing phenol red (i.e. until the test item formulation had been completely removed from the chamber or until the phenol red was not discoloured). Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.

SCORING SYSTEM
- Opacity:
An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. The average change in opacity for the corneas treated with the vehicle control was calculated and this value was subtracted from the change in opacity for each cornea treated with test item formulation or positive control to obtain a corrected opacity value (cOPT). The mean cOPT value of each series of three corneas was calculated from the individual corrected opacity values.

- Permeability:
After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a fluorescein solution (5mg/mL) in a water bath at +32°C (± 1°C) for 90 minutes (± 5 minutes). At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube.
The corrected OD490 nm (cOD490 nm) value (i.e. permeability) for each cornea treated by test item formulation or positive control was calculated by subtracting the average vehicle control cornea OD490 nm value from the original OD490 nm value of each cornea. The mean cOD490 nm value of each series of three corneas was calculated from the individual cOD490 nm values.

- Scoring:
In vitro irritancy score (IVIS) = cOPT + (15 x cOD490 nm)
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
MACROSCOPIC EXAMINATION:
Fluorescein fixation was observed on all corneas treated with the vehicle control and the test item.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.


ACCEPTANCE CRITERIA:
For the validation of an experiment, the following criteria had to be fulfilled:
- the mean In Vitro Irritancy Score (IVIS) of the positive control corneas should fall within two standard deviations of the historical mean,
- the mean opacity and mean OD490 nm of the vehicle control corneas should be less than the established upper limit of historical mean.
Interpretation of results:
GHS criteria not met
Remarks:
not eye irritating
Conclusions:
Under the experimental conditions of this study, the test item was identified as not requiring classification for eye irritation or serious eye damage (UN GHS No Category).
Executive summary:

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item to the eye. The Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage.

The design of this study was based on the OECD Guideline 437 and the study was performed in compliance with CiToxLAB France standard operating proceduresand with the OECD Principles of Good Laboratory Practice.

 

Method

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C.

A single experiment was performed using three corneas for each treated series (test item, positive control and vehicle control).

Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.

The test item was tested at 20% (w/v) in paraffin oil, in a single experiment using a treatment time of 4 hours and the open-chamber treatment method. Vehicle and positive controls were applied using the same treatment time but the closed-chamber treatment method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed.

A second opacity measurement was then performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes(± 5 minutes) at +32°C.

At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

 

Results

 

Macroscopic examination

Fluorescein fixation was observed on the three corneas treated with the test item.

 

In Vitro Irritancy Score

All acceptance criteria were fulfilled. The study was therefore considered as valid.

 The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 0.

 As the mean IVIS was < 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Conclusion

Under the experimental conditions of this study, the test item was identified as not requiring classification for eye irritation or serious eye damage (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

in vitro skin irritation study / OECD 439 (Grignard 2017a)

The objective of this study was to evaluate the skin irritation potential of the test item using the Episkin reconstructed human epidermis model.

Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential.

Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at +, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).

In the preliminary tests, the test item was found to have neither direct MTT reducing properties, nor colouring potential.

Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 105% with a standard deviation of 4%. As the mean viability was > 50% after the MTT reduction,the results met the criteria for a non-irritant response.

Under the experimental conditions of this study, the test item is considered to be non-irritant to skin.

In vitro eye corrosion study / OECD 437 (Grignard 2017b)

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item to the eye. The Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage.

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C. A single experiment was performed using three corneas for each treated series (test item, positive control and vehicle control).

Fluorescein fixation was observed on the three corneas treated with the test item.

 The mean in vitro Irritancy Score (IVIS) of the test item-treated corneas was: 0.

 As the mean IVIS was < 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Justification for classification or non-classification

Based on the in vitro studies, no classification for irritating properties is required for Nitrosodiphenylamine according to the Regulation EC n°1272/2008.

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