Registration Dossier

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Administrative data

Description of key information

According to OECD 431


According to OECD 438


According to OECD 439


 


 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 25 JUNE 2018 to 31 AUGUST 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) 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:
foreskin from multiple donors
Source strain:
not specified
Details on animal used as source of test system:
No animals were used
Justification for test system used:
The objective of this study was to determine the potential skin corrosivity of a test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile with the in vitro skin corrosion test using reconstructed human epidermis (RhE; EpiSkinTM-SM) following OECD test guideline 431. This method allows classification of the test item in non-corrosive or corrosive substances and mixtures according to the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS). It also allows a partial sub-categorization of corrosives.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermis (RhE; EpiSkinTM-SM).
- Tissue batch number(s): 18-EKIN-030
- Production date:
- Shipping date:
- Delivery date:
- Date of initiation of testing: 24.07.18

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 +/- 1ºC
- Temperature of post-treatment incubation (if applicable):

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml
- Incubation time: 3h +/- 15 min
- Spectrophotometer:
- Wavelength: between 540 to 600 nm
- Filter: no reference filter was used
- Filter bandwidth:
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function:
- Morphology:
- Contamination:
- Reproducibility:

NUMBER OF REPLICATE TISSUES:

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20g
- Concentration (if solution):

VEHICLE
- Amount(s) applied (volume or weight with unit): 100%
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL of negative control (0.9% NaCl)
- Concentration (if solution): 0.9% NaCl

POSITIVE CONTROL
- Amount(s) applied (volume or weight):50 µL of positive control (glacial acetic acid)
- Concentration (if solution): 100%
Duration of treatment / exposure:
3 min, 1h (±5 min) and 4h (±10 min) at room temperature
Duration of post-treatment incubation (if applicable):
incubated for 3h (± 15 min) under culture conditions
Number of replicates:
A single testing run was sufficient becuuse was unequivocal.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1 (duration exposure time 3 minutes), Run 2 (duration exposure time 1 hour) and run 3 (duration exposure time 4 hours)
Value:
>= 35
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
Taking into account the results obtained in the current study, it is concluded that, under the assayed experimental conditions and according to the OECD guideline for the Testing of Chemicals Nº. 431, the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile can be categorised as Non-Corrosive according to the United Nations Globally Harmonized System of Classification and Labelling of Chemicals.
Interpretation of results:
GHS criteria not met
Conclusions:
Taking into account the results obtained in the current study, it is concluded that, under the assayed experimental conditions and according to the OECD guideline for the Testing of Chemicals Nº. 431, the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile can be categorised as Non-Corrosive according to the United Nations Globally Harmonized System of Classification and Labelling of Chemicals.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 25 JUNE 2018 to30 AUGUST 2018
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)
Deviations:
yes
Remarks:
Detected mistake when was added much more tested item in a plate. The mistake was detected and fixed removing the excess. At last, was used 0.0061 g of test item instead 0.01 g, but was considered without impact because of the absorbance values were simil
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Source strain:
not specified
Justification for test system used:
The objective of this study was to determine the potential skin irritation of the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile with the in vitro skin irritation test using reconstructed human epidermis (RhE; EpiSkinTM-SM) following OECD test guideline 439. This method allows classification of the test item in irritant (UN GHS Category 2) or non-irritant (Un GHS No Category) substances and mixtures according to the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS). A limitation of this method is that it does not allow the classification of substances to the optional UN GHS Category 3 (mild irritants).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermis (RhE; EpiSkinTM-SM).
- Tissue batch number(s): 18-EKIN-029
- Production date:
- Shipping date:
- Delivery date:
- Date of initiation of testing: 17.07.2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
- Temperature of post-treatment incubation (if applicable):

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
- Incubation time:
- Spectrophotometer:
- Wavelength:
- Filter:
- Filter bandwidth:
- Linear OD range of spectrophotometer:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function:
- Morphology:
- Contamination:
- Reproducibility:

NUMBER OF REPLICATE TISSUES:

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg
- Concentration (if solution):

VEHICLE
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity: 100%

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL of negative control (DPBS)
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL of positive control (5% SLS in purified water)
- Concentration (if solution): 5%
Duration of treatment / exposure:
15 min at room temperature inside the laminar hood
Duration of post-treatment incubation (if applicable):
incubated under culture conditions for 42h (±1h)
Number of replicates:
One single run composed of three replicates tissues.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1 (duration exposure 15 min)
Value:
ca. 98.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
For the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile, the percentage of viability determined was 98.3%. Therefore, the compound can be classified as non-irritant following the criteria of OECD 439, since the viability was >50%.
Interpretation of results:
GHS criteria not met
Conclusions:
The run carried out with the living RhE inserts met the predefined acceptance criteria, i.e., i) mean ODNC of the RhE inserts treated with the negative control was > 0.6 at 540 nm and the standard deviation of the viability was ≤ 18; ii) the relative viability for the positive control expressed as % of the negative control was ≤40% and the standard deviation was ≤ 18.
The NSC percentage obtained was <5%. Therefore, the normal calculation of the viability was made.
For the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile, the percentage of viability determined was 98.3%. Therefore, the compound can be classified as non-irritant following the criteria of OECD 439, since the viability was >50%.
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
Remarks:
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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 9 JULY 2019 to 19 JULY 2018
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)
Deviations:
yes
Remarks:
The eyes were incubated between 45 and 64 minutes instead of between 45 and 60 minutes, as initially scheduled. As the results obtained with the negative control, the deviation is considered as without impact on the conclusion of the study.
GLP compliance:
yes (incl. QA statement)
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: slaughterhouse (Etablissement Brun, 33820 Etauliers, France)
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight): 7 weeks old, 1.5-2.5 Kg
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transport time 75 minutes, at ambient temperature in plastic boxes humidified with towels moistened with physiological saline
- Time interval prior to initiating testing:
- indication of any existing defects or lesions in ocular tissue samples: none
- Indication of any antibiotics used:
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg
- Concentration (if solution):

VEHICLE
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:
Duration of treatment / exposure:
10 seconds
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. when the eye is removed from the orbit, a visible portion of the optic nerve should be attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.

The enucleated eye was mounted in a stainless steel clamp with he cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. Then clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.0ºC and 32.4ºC.

After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or (iii) any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

EQUILIBRATION AND BASELINE RECORDINGS
Once all eyes had been examined and approved (see table in appendix 4), the eyes were incubated between 45 and 64 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES
3 replicates for test item test.

NEGATIVE CONTROL USED
Concurrent negative control (physiological saline - Dutscher Batch No. 3013132). One eye was treated with the negative control, rinsed twice with 10 mL of physiological saline and a humidified cotton swab at ambient temperature (as for the eye treated with the test item).

SOLVENT CONTROL USED (if applicable)
None

POSITIVE CONTROL USED
Positive control (sodium hydroxide - Fisher Scientific, Batch No. 1550248) were included in this experiment. Three eyes were treated with the positive control.

APPLICATION DOSE AND EXPOSURE TIME
Three eyes (in their holder) were removed from the superfusion apparatus, placed in a horitzontal position, and 30 mg of the test item was applied, after being reduced in fine powder, during 10 seconds to the cornea such that the entire surface of the cornea was evenly covered with the test item.

OBSERVATION PERIOD

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The eyes were rinssed twice with 10 mL of physiological saline and humidified cotton swab at ambient temperature.
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Haag-Streit BP900 split-lamp microscope with depth-measuring device no. I.
- Damage to epithelium based on fluorescein retention: Haag-Streit BP900 split-lamp microscope with depth-measuring device no. I.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Haag-Streit BP900 split-lamp microscope with depth-measuring device no. I.
- Macroscopic morphological damage to the surface:Haag-Streit BP900 split-lamp microscope with depth-measuring device no. I.
- Others (e.g, histopathology):

SCORING SYSTEM:
- Mean corneal swelling (%): 0%, corresponding to ICE class I
- Mean maximum opacity score: 0.0, corresponding to ICE class I
- Mean fluorescein retention score at 30 minutes post-treatment: 1.5, corresponding to ICE class II

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
The combination of the three endopoints for the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile was 2 x I, 1 x II.

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihidro-6-hydroxy-4-methyl-2-oxonicotinonitrile does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No Category).
No signal word and hazard statement are required.
Irritation parameter:
percent corneal swelling
Run / experiment:
Run 1 (10 seconds)
Value:
ca. 0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as "No Category", as expected.
Positive controls validity:
valid
Remarks:
The combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe Irritant", as expected.
Irritation parameter:
fluorescein retention score
Run / experiment:
Run 1 (exposure 10 seconds)
Value:
ca. 1.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as "No Category", as expected.
Positive controls validity:
valid
Remarks:
The combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe Irritant", as expected.
Irritation parameter:
cornea opacity score
Run / experiment:
Run 1 (duration of exposure: 10 seconds)
Value:
ca. 0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as "No Category", as expected.
Positive controls validity:
valid
Remarks:
The combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe Irritant", as expected.
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: he combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as "No Category", as expected.
- Acceptance criteria met for positive control:The combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe Irritant", as expected.
- Range of historical values if different from the ones specified in the test guideline:
Irritant / corrosive response data:
See Table 9 (overall remarks, attachments)






















































































































































































Endpoint measured



Eye No.



Time (min)



0



30



75



120



180



240



Corneal opacity



4



0



0



0



0



0



0



5



0



0



0



0



0



0



6



0



0



0



0



0



0



mean



0.0



0.0



0.0



0.0



0.0



0.0



ICE class



 



I



Fluorescein retention



4



0.5



1



-



-



-



-



5



0.5



3



-



-



-



-



6



0.5



0.5



-



-



-



-



Mean



0.5



1.5



-



-



-



-



ICE class



 



II



-



-



-



-



Corneal thickness



4



0.58



0.58



0.58



0.58



0.58



0.58



5



0.57



0.57



0.57



0.57



0.57



0.57



6



0.56



0.56



0.56



0.56



0.56



0.56



Corneal swelling



4



-



0



0



0



0



0



5



-



0



0



0



0



0



6



-



0



0



0



0



0



Mean



-



0



0



0



0



0



ICE class



I



Combination of the 3 Endpoints



2 x I, 1 x II



CLASSIFICATION



No category



TEST ITEM:


1-butyl-5-[4-chlorophenyl]azo]-1,2-dihidro-6-hidroxy-4-methyl-2-oxonicotinonitrile


 


Application: 30 mg of pure test item


Application date: 10 September 2018


Table 9


 


Note:


No morphological effects were noted, whatever the examination time.


 


 


 


 


 

Interpretation of results:
GHS criteria not met
Conclusions:
In ccordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitriledoes not require classification for eye irritation and serious eye damage as defined by the UN GHS (No Category).
No signal word and hazard statement are required.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification

OECD 431: Taking into account the results obtained in the current study, it is concluded that, under the assayed experimental conditions and according to the OECD guideline for the Testing of Chemicals Nº. 431, the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile can be categorised as Non-Corrosive according to the United Nations Globally Harmonized System of Classification and Labelling of Chemicals.


 


OECD 438: the combination of the three endpoints was 2 x I, 1 x II, in accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No category).


 


OECD 439: Taking into account the results obtained in the current study, it is concluded that, under the assayed experimental conditions and according to the OECD guideline for the Testing of Chemicals Nº. 439, the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile was ranked as nonirritant to skin (No Category; no-label) in accordance with the United Nations Globally Harmonized System of Classification and Labelling of Chemicals.