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Description of key information

CETONAL is irritant in the in vitro skin irritation test under the experimental conditions described in this report.

In the in vivo study, Cetonal was only observed at 24 hours after a single application and for up to 21 days following repeated application (days 1 to 21) at 3%, 10%, 30% and 100%.

When the in vitro experimental data is considered along side the in vivo study data, there is no evidence of corrosivity.

Therefore an in vitro corrosivity test is not required and Cetonal will be classified as a skin irritant.

The combination of the in vitro and in vivo tests indicate no eye irritation and therefore no classification is required.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
February 17th, 2017 to March 27th, 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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test material name (as stated in the report): Cetonal
Batch No.: SC00019822
Expiry date: 01 March 2019
Test system:
human skin model
Remarks:
EPISKIN Small ModelTM
Source species:
human
Cell type:
other: EPISKIN-SMTM, 0.38 cm2
Cell source:
other: SkinEthic Laboratories, Lyon, France.
Details on animal used as source of test system:
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Remarks:
The liquid test item was applied undiluted (25 µl) directly on top of the tissue
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Duration of treatment / exposure:
CETONAL was applied undiluted (25 µl) directly on top of the skin tissue for 15 ± 0.5 minutes.
Duration of post-treatment incubation (if applicable):
After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
second test after treatment with CETONAL
Value:
33
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

The positive control had a mean cell viability of 12% after 15 ± 0.5 minutes exposure. 

The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically with the control items was less than 7%, indicating that the test system functioned properly.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with CETONAL compared to the negative control tissues was 57% (individual values 38%, 100% and 33%). However, the standard deviation value between tissue replicates of CETONAL was 37% after 15 minutes exposure which is not within the acceptability criteria of the assay (≤18%). Therefore the test was repeated.

The relative mean tissue viability obtained in the second test after 15 ± 0.5 minutes treatment with CETONAL compared to the negative control tissues was 43% (individual values 59%, 44% and 26%).

The relative mean tissue viability obtained in the second test after treatment with CETONAL compared to the second set of negative control tissues with a standard deviation of 11% was 33% (individual values 46%, 34% and 20%).

The positive control had a mean cell viability of 7.3% after 15 ± 0.5 minutes exposure. 

Since the mean relative tissue viability for CETONAL was below 50% after 15 ± 0.5 minutes treatment in 4 out of 5 tissues, CETONAL is considered to be irritant.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
In conclusion, CETONAL is irritant in the in vitro skin irritation test under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate CETONAL for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)). The possible skin irritation potential of CETONAL was tested through topical application for 15 minutes.  The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch SC00019822 of CETONAL was a pale yellow liquid with a purity of 98.02%. CETONAL was applied undiluted (25 µl) directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure. 

The positive control had a mean cell viability of 12% after 15 ± 0.5 minutes exposure. 

The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. 

The standard deviation value of the percentage viability of three tissues treated identically with the control items was less than 7%, indicating that the test system functioned properly.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with CETONAL compared to the negative control tissues was 57% (individual values 38%, 100% and 33%). However, the standard deviation value between tissue replicates of CETONAL was 37% after 15 minutes exposure which is not within the acceptability criteria of the assay (≤18%). Therefore the test was repeated.

The relative mean tissue viability obtained in the second test after 15 ± 0.5 minutes treatment with CETONAL compared to the negative control tissues was 43% (individual values 59%, 44% and 26%).

The relative mean tissue viability obtained in the second test after treatment with CETONAL compared to the second set of negative control tissues with a standard deviation of 11% was 33% (individual values 46%, 34% and 20%).

 The positive control had a mean cell viability of 7.3% after 15 ± 0.5 minutes exposure. 

Since the mean relative tissue viability for CETONAL was below 50% after 15 ± 0.5 minutes treatment in 4 out of 5 tissues, CETONAL is considered to be irritant.

In conclusion, CETONAL is irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
September 16th, 1981 to October 23rd, 1981
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
other: Skin irritation and capacity of allergenic sensitization determined by Open Epicutaneous test on Guinea Pigs
Version / remarks:
Pre-Guidance but according to the practice devised by Klecak at the time.
Principles of method if other than guideline:
Single application to assess irritantcy prior to induction procedure and also after multiple application of the test substance up to 21 days.
GLP compliance:
no
Remarks:
Pre-GLP
Specific details on test material used for the study:
Test material name (as stated in the report): Cetonal Giv 2-1630
Species:
guinea pig
Strain:
not specified
Details on test animals or test system and environmental conditions:
One to six experimental groups and one control group of 6 to 8 guinea pigs were used.
Type of coverage:
open
Preparation of test site:
clipped
Vehicle:
other: either ethanol, acetone,m H2), vasoline or PEG.
Controls:
yes, concurrent no treatment
Amount / concentration applied:
Single application: 0.025 ml of undiluted test material and at 100%, 30%, 10%, and 3%.
Multiple application: 0.1 ml of test material at 100%, 30%, 10%, 3% for 21 days.
Duration of treatment / exposure:
Single application
Multiple application for 21 days
Observation period:
Single day and up to 21 days.
Number of animals:
6 to 8 guinea pigs were used per group.
Details on study design:
One day before starting the induction procedure the threshold toxic concentration of Cetonal was estimated.
A single application of 0.025mL of each concentration is simultaneously performed on one of the areas measuring 2 cm2 of the flank skin previously clipped and marked with a circular stamp.
The skin reactions were read 24 hours after the application of the test material.
The minimal irritant and the non-irritant concentrations were determined by an all or none criteria.

Induction - Application of 0.1 ml for the OET procedure.

Repeated application of Cetonal on days 1 to 21 were also performed with reactions noted on days 7, 14 and 21.
Irritation parameter:
overall irritation score
Basis:
mean
Remarks:
100%
Time point:
7 d
Score:
4
Max. score:
4
Reversibility:
not reversible
Remarks on result:
probability of severe irritation
Remarks:
Applied days 1 to 7
Irritation parameter:
overall irritation score
Basis:
mean
Remarks:
100%
Time point:
24 h
Score:
4
Max. score:
4
Reversibility:
other: only 24h conducted
Remarks on result:
positive indication of irritation
Irritation parameter:
overall irritation score
Basis:
mean
Remarks:
100%
Time point:
14 d
Score:
4
Max. score:
4
Reversibility:
not reversible
Remarks on result:
probability of severe irritation
Remarks:
Applied days 1 to 14
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
A 30% alcoholic solution caused moderate to strong irritation, a 10% solution very slight to sligh irritation and a 3% solution was well tolerated if applied repeatedly for up to 21 days.
Executive summary:

The Cetonal was tested in this study by Open Epicutaneous test on Guinea Pigs Pre-Guidance but according to the practice devised by Klecak at the time.

One day before starting the induction procedure the threshold toxic concentration of Cetonal was estimated.

A single application of 0.025mL of each concentration is simultaneously performed on one of the areas measuring 2 cm2 of the flank skin previously clipped and marked with a circular stamp.

The skin reactions were read 24 hours after the application of the test material.

The minimal irritant and the non-irritant concentrations were determined by an all or none criteria.

Induction - Application of 0.1 ml for the OET procedure.

Repeated application of Cetonal on days 1 to 21 were also performed with reactions noted on days 7, 14 and 21.

A 30% alcoholic solution caused moderate to strong irritation, a 10% solution very slight to sligh irritation and a 3% solution was well tolerated if applied repeatedly for up to 21 days.

According to this test, Cetronal is clasified Category 2 (irritant) based on GHS criteria.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1966
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
according to guideline
Guideline:
other:
Version / remarks:
The method of procedure is that suggested by Dr. Draize and described in "Appraisal of the safety of chemicals in foods, drugs amd cosmetics" published by the Association of Food and Drug Officials of the United States.
GLP compliance:
no
Remarks:
Test conducted prior to the GLP guidelines
Specific details on test material used for the study:
Test material name (as stated in the report): Cetonal GIV 2 -1613
Sample received on October 14, 1966
Species:
rabbit
Strain:
other: albino rabbits
Details on test animals or tissues and environmental conditions:
Three normal, healthy albino rabbit were used in this experiment
Vehicle:
other: 80% aqueous propylene glycol
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.1mL of 2.0% of test material in 80% aqueous propylene glycol
Duration of treatment / exposure:
instillation
Observation period (in vivo):
7 days after instillation
Number of animals or in vitro replicates:
3 rabbits
Details on study design:
Ecah animal had 0.1 mL of test sample instilled into the right eye with no further treatment.
Both the treated and control eyes were examined every twenty four hours for 4 days and then again on th seventh day.
Irritation parameter:
cornea opacity score
Basis:
animal: animal #1 ; animal #2 and animal 3#
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: Na
Irritation parameter:
iris score
Basis:
animal: animal #1 ; animal #2 and animal 3#
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: Na
Irritation parameter:
conjunctivae score
Basis:
animal: animal #1 ; animal #2 and animal 3#
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: Na
Irritation parameter:
chemosis score
Basis:
animal: animal #1 ; animal #2 and animal 3#
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: Na

The scorings recorded were made according to the Draize scale for scoring ocular lesions.

Interpretation of results:
GHS criteria not met
Conclusions:
Instillation of the test material as described did not produce any irritation in any of the treated eyes. Hence, Cetonal does not meet the criteria to be as Eye irritant Category 2 according to the CLP Regulation (EC) No. 1272/2008.
Executive summary:

The Cetonal was tested in this study to determine his eye irritant potential according to the procedure is that suggested by Dr. Draize and described in "Appraisal of the safety of chemicals in foods, drugs amd cosmetics" published by the Association of Food and Drug Officials of the United States.

0.1mL of 2.0% of test material in 80% aqueous propylene glycol was instilled into the right eye of three normal, healthy albino rabbit, with no further treatment. The untreated left eye of ecah animal served as it own control. Both the treated and control eyes were examined every twenty four hours for 4 days  and then again  on th seventh day.

Instillation of the test material as described did not produce any irritation in any of the treated eyes. Hence, Cetonal does not meet the criteria to be as Eye irritant Category 2  according to the CLP Regulation (EC) No. 1272/2008.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 10th, 2014
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 Ocular Corrosives and Severe Irritants)
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Test material name (as stated in the report): Cetonal
Batch No.: SC00011028
Expiry date: 23 June, 2016
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
The eyes collected from Chickens obtained from a slaughterhouse where they are killed for human consumption have been used for this assay. The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse. (approximately 7 weeks old 1.5 - 2.5 Kg).
Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding . Because eyes were dissected in the laboratory , the intact heads were transported from the slaughterhouse at ambient temperature in polystyrene boxes humidified with towels moistened with physiological saline.

The eyelids are 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 is pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles are cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye is placed on an absorbent pad and the nictitating membrane and other connective tissue are cut away.
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
30 microL of test item was applied as supplied to the cornea such taht the entire surface of the cornea is evenly covered with the test item.
Duration of treatment / exposure:
Test item was applied for 10 seconds and then rinsed from the eye with 20 mL of physiological saline at ambient temperature. As residual test item was observed on the cornea, four rinses of 10 mL of physiological saline were added.
Duration of post- treatment incubation (in vitro):
Treated corneas are evaluated pretreatment and starting at 30, 75, 120, 180 and 240 minutes after the post treatment rinse.
Number of animals or in vitro replicates:
3 enucleated chicken eyes
Details on study design:
Damages by the test substance were assessed by determination of corneal swilling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.
Irritation parameter:
cornea opacity score
Run / experiment:
maximal mean score
Value:
1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: corresponding to ICE class II
Irritation parameter:
fluorescein retention score
Run / experiment:
mean score
Value:
0.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: corresponding to ICE class II
Irritation parameter:
percent corneal swelling
Run / experiment:
maximal mean score
Value:
32
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: at 240 post dose, corresponding to ICE class III
Other effects / acceptance of results:
The combination of the three endpoints for the positive control, 5% Benzalconium chloride was 3 x IV. Therefore, the positive controle is classified as "Corrosive/Severe irritant" as expected.
The combination of the three endpoints for the negative control, physiological saline was 3 x I. Therefore, the positive controle is classified as "No category" as expected.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 1.0 corresponding to ICE class II

- mean score of fluorescein retention: 0.7 corresponding to ICE class II

- maximal mean corneal swelling : + 32 at 240 minutes post dose corresponding to ICE class III

Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained, under this experimental conditions enable to conclude that the test item Cetonal does not have to be classified in category 1 "irreversible effects on eye".
Executive summary:

This experience on Cetonal has been performed according to the OECD guideline No 438 adopted 26 July 2013.

The eyes collected from Chickens obtained from a slaughterhouse where they are killed for human consumption have been used for this assay. The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse. (approximately 7 weeks old 1.5 - 2.5 Kg). Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding . Because eyes were dissected in the laboratory , the intact heads were transported from the slaughterhouse at ambient temperature in polystyrene boxes humidified with towels moistened with physiological saline. The eyelids are 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 is pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles are cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye is placed on an absorbent pad and the nictitating membrane and other connective tissue are cut away. 30 microL of test item was applied as supplied to the cornea such taht the entire surface of the cornea is evenly covered with the test item. Test item was applied for 10 seconds and then rinsed from the eye with 20 mL of physiological saline at ambient temperature. As residual test item was observed on the cornea, four rinses of 10 mL of physiological saline were added. Damages by the test substance were assessed by determination of corneal swilling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose. The ocular reactions observed in eyes treated with the test item were: - maximal mean score of corneal opacity: 1.0 corresponding to ICE class II - mean score of fluorescein retention: 0.7 corresponding to ICE class II - maximal mean corneal swelling : + 32 at 240 minutes post dose corresponding to ICE class III The combination of the three endpoints for the positive control, 5% Benzalconium chloride was 3 x IV. Therefore, the positive controle is classified as "Corrosive/Severe irritant" as expected. The combination of the three endpoints for the negative control, physiological saline was 3 x I. Therefore, the positive controle is classified as "No category" as expected. The results obtained, under this experimental conditions enable to conclude that the test item Cetonal does not have to be classified in category 1 "irreversible effects on eye" according to the CLP Regulation (EC) No. 1272/2008.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification

When the in vitro experimental data is considered alongside the in vivo study data, there is no evidence of corrosivity.

Therefore an in vitro corrosivity test is not required and Cetonal will be classified as a skin irritant.

The combination of the in vitro and in vivo tests indicate no eye irritation and therefore no classification is required.