Registration Dossier

Administrative data

Description of key information

Weight of Evidence: Skin Irritating, 2017

Read-Across - methyl-2-[[(2,4-dimethyl-3-cyclohexen-1-yl)methylene]amino]benzoate: Skin Irritation: skin irritating, rabbit, eq. or similar to EU Method B.4, 1985

Read-Across - oxydipropanol: Skin Irritation: not irritating, rabbit, US EPA OPP 81-1, 1995

 

Eye irritation: not eye irritating, in vitro EPIOCULAR RHE, OECD TG 492, 2016

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study follows protocols to similar to a recognised guideline under GLP.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in attachment to IUCLID section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is based on the hypothesis that the source and target substances have similar physico-chemical, toxicological properties and because of common breakdown products and therefore potential mechanisms of action. The read-across is assessed as part of a weight of evidence approach. Further information is included in attachment to IUCLID section 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source and target chemicals have comparable composition. Further information is included in attachment to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
The source substance is a common degradant of the target substance and with common degradants to the target under physiological conditions. The read-across in weight of evidence indicates with a common potential mode-of-action between source and target, adverse effects are seen in several studies. When the information is taken together by expert judgement the weight of evidence indicates that there is an indication of potential for adverse effects to be associated with the target.

4. DATA MATRIX
Further information is included in attachment to IUCLID section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: not reported
- Weight at study initiation: 2.4 - 3.0 Kg
- Housing: individually housed in grid-bottomed metal cages.
- Diet (e.g. ad libitum): certified pelleted diet ad libitum
- Water (e.g. ad libitum): mains water ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 51 - 77
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
0.5ml test material applied to clipped backs of test animals and over areas of approximately 6sqcm
Duration of treatment / exposure:
4 hours
Observation period:
skin reaction to the test article was assessed after one, 24, 48, 72, 168 and 336 hours. Additional assessments were carried out 7 and 14 days after dosing.
Number of animals:
4 females
Details on study design:
TEST SITE
- Area of exposure: doral surface of the trunk
- % coverage: not reported. 6 sq. centimetres is described as the treatment site using a 2.5 x 2.5 cm lint pad held in place with an adhesive bandage.
- Type of wrap if used: Elastoplast elastic adhesive bandage 10cm wide.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes - test substance application site cleansed using cotton wool soaked in warm water (37°C)
- Time after start of exposure: 4 hours

SCORING SYSTEM: Consistent with Draize et al. under European Communities No. L257, 16th September 1983 was used to classify the material. (Commission Directive of 29th July 1983 adapting technical progress for the fifth time, Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances) criteria.
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 1 hour
Score:
1.5
Max. score:
4
Reversibility:
not fully reversible within: 14d
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 24 hour
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 14d
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 48 hour
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 14d
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 72 hour
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 14d
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 7 days
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 14d
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 14 days
Score:
1.25
Max. score:
4
Reversibility:
not fully reversible within: 14d
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 1 hour
Score:
0.75
Max. score:
4
Reversibility:
not fully reversible within: 14d
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 24 hour
Score:
1.75
Max. score:
4
Reversibility:
not fully reversible within: 14d
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 48 hour
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 14d
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 72 hour
Score:
2.25
Max. score:
4
Reversibility:
not fully reversible within: 14d
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 7 days
Score:
3
Max. score:
4
Reversibility:
not fully reversible within: 14d
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 14 days
Score:
1
Max. score:
4
Reversibility:
not fully reversible within: 14d
Irritant / corrosive response data:
Individual scores are presented in the full study report and presented in table 1.

Table 1. individual scores

 

Time

Erythema

Oedema

Comments

 

 

 

 

 

1

1 hour

2

1

 

2

 

2

0

 

3

 

1

1

 

4

 

1

1

 

1

24 hours

2

2

 

2

 

2

1

 

3

 

2

2

 

4

 

2

2

 

1

48 hours

2

2

 

2

 

2

2

 

3

 

2

2

 

4

 

2

2

 

1

72 hours

2

3

 

2

 

2

2

 

3

 

2

2

 

4

 

2

2

Treated skin thickened

1

7 days

2

3

Treated skin thickened

2

 

2

3

Treated skin thickened

3

 

2

3

Treated skin thickened

4

 

2

3

Treated skin thickened

1

14 days

1

0

Slight desquamation

2

 

2

2

Desquamation

3

 

1

1

Slight desquamation

4

 

1

1

Slight desquamation

 

 

 

 

 

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the study the test substance is considered to be irritating to skin.
Executive summary:

The study was performed in a method equivalent or similar to EU Method B.4 under GLP to assess the irritancy potential of the test substance to the skin of the New Zealand White rabbit. The test substance was applied over an area of approximately 2.5cm square to the clipped backs of 4 albino rabbits. The test substance was held in contact with the skin under a semi-occlusive patch for a 4 hour period. After this time the patches were removed and the skin reaction to the test substance was assessed after one, 24, 48 and 72 hours. Additional assessments were carried out 7 and 14 days after dosing to determine the degree of reversibility of the skin reaction. The response increased within twenty four hours of dosing when well defined erythema of the skin was noted in all four animals. Slight oedema was apparent and very slight oedema of the skin was observed. Well defined erythema and slight oedema were observed at the treated sites at the forty eight hour observation. Seventy two hours after dosing slight increase in oedematous reaction had occurred in one animal and thickening of the treated skin was observed in a second animal. Well defined erythema, moderate oedema and thickening of the treated skin was observed in all organisms seven days after dosing. This reaction declined and fourteen days after dosing well defined erythema and slight oedema remained in one organism, and very slight irritation was observed at the treated sites of the remaining organisms. Desquamation from the skin surface was noted in all four rabbits of the group. Applicant recalculation of the results indicates that under the conditions of this study, that the mean erythema scores between 24 and 72 hours were > 2.0 in at least 3 of 4 organisms and the response had not fully reversed within a 14 day period. The test item should be considered as irritating to the skin.

Endpoint:
skin irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1995
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria are met.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in attachment to IUCLID section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is based on the hypothesis that the source and target substances have similar physico-chemical, toxicological properties and because of common breakdown products and therefore potential mechanisms of action. Specifically, this source substance is a constituent of the source and target. With no indications of adverse effects. The read-across is assessed as part of a weight of evidence approach. Further information is included in attachment to IUCLID section 13.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source and target chemicals have comparable composition. Further information is included in attachment to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
The source substance is source substance is a constituent of the source and target. The read-across in weight of evidence indicates despite a common potential mode-of-action between source and target, no adverse effects are seen in several studies. When the information is taken together by expert judgement the weight of evidence indicates that there is an indication of potential for adverse effects to be associated with the target. However, these effects are not associated with this source substance.

4. DATA MATRIX
Further information is included in attachment to IUCLID section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
EPA OPP 81-5 (Acute Dermal Irritation)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: approximately 16 weeks
- Weight at study initiation: 2.0 to 2.3 Kg
- Housing: housed individually
- Diet (e.g. ad libitum): certified diet ad libitum
- Water (e.g. ad libitum): potable municipal water ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23 ºC
- Humidity (%): 35-65%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours dark on 12 hours light
Type of coverage:
occlusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 ml
- Concentration (if solution): Not applicable.
Duration of treatment / exposure:
4 hours
Observation period:
0.5 hour, 1 hour, cleaning at 4 hour and 24, 48 and 72 hours after patch removal
Number of animals:
6 (3 male: 3 female)
Details on study design:
TEST SITE
- Area of exposure: ca. 6 cm2
- % coverage: Not applicable.
- Type of wrap if used: Occlusive. Gauze pad secured by hypo-allergenic adhesive tape wrapped with non-irritating perforated plastic sheeting fastened at each end with tape.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes. 4 hours.
- Time after start of exposure: Skin was wiped with clean moistened paper towel to remove any test substance still remaining.
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: No effects observed.
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Reversibility:
other: No effects observed
Irritant / corrosive response data:
45 minutes after patch removal there was no irritation except for one site (1/6) which showed very slight erythema (score = 1). At 24 hours all test areas appeared normal and remained normal throughout during the remainder of the study.
Other effects:
No significant effects reported.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the study, the test substance is considered to be not irritating to skin.
Executive summary:

The study was performed under US EPA OPP 81-5 under GLP to assess the irritancy potential of the test substance to the skin of the New Zealand White rabbit. The test substance was applied over an area of approximately 6cm2 square to the clipped backs of 3 male and female albino rabbits. The test substance was held in contact with the skin under an occlusive patch for a 4 hour period. After this time the patches were removed and the skin reaction to the test substance was assessed after one, 24, 48 and 72 hours. No significant effects were observed at 24, 48 and 72 hours. Applicant recalculation of the results indicates that under the conditions of this study, that the mean erythema and oedema scores between 24 and 72 hours were < 2.0 in all organisms. There was no indications of irritation during the study The test item should be considered as not irritating to the skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Remarks:
Applicant assessment of the data indicates that since no irritation response interaction was observed (i.e. including enhanced sensitivity mean viability limit criteria: 65% suggested in OECD TG 492) with the test item in any of the test tissues that the acceptability of the assay was not impacted.
Qualifier:
according to
Guideline:
other: OECD Guideline 492: Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage (Adopted 28 July 2015).
Deviations:
yes
Remarks:
Minor methodology deviations deemed acceptable by expert judgement. Conclusion would acceptable by expert judgement.
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: September 2015; signature: November 2015
Species:
other: human-derived epidermal keratinocyte tissues
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: MatTek Corporation (USA)
- Housing: EpiOcular™ (OCL-200-EIT; lot 19595 kit G) tissues were shipped on medium-supplemented agarose gels in a 24-well plate. On the day of receipt the tissues were equilibrated (in its 24-well shipping container) to room temperature for about 15 minutes. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 hours at 37°C in 1.0 ml fresh prewarmed Assay medium. Assay medium was supplied by the manufacturer.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Prewarming: All incubations, with the exception of the last 25 minutes of test item incubation at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 56 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.2 - 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on the appropriate responses of reference controls these deviations are considered not to affect the study integrity.
Killed tissues (Lot: 19580 kit A, 19581 kit I and 19582 kit G): Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to (≤-15°C). The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them at room temperature. Further use of killed tissues was similar to living tissues.
Vehicle:
unchanged (no vehicle)
Controls:
other: TWO tissues were treated with 50 μl MilliQ water (negative control) and TWO tissues with 50 μl Methyl Acetate (positive control) respectively.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 uL
- Concentration (if solution): undiluted
Duration of treatment / exposure:
30 ±2 minutes
Observation period (in vivo):
After treatment the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed.
Number of animals or in vitro replicates:
The test was performed on a total of 2 tissues per test item together with a negative control and positive control.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): After pre-incubation of EpiOcular™ tissues and after completion of the treatment with 20 uL PBS, the negative and positive control and the test item was added into the insert atop the concerning EpiOcular duplicate tissues. The plates were placed into the incubator for 24 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2 and room temperature for 7 minutes (this deviation was not considered to impact the study integrity due to appropriate Positive Control and Negative Control responses). After the exposure period with the test item (30 ± 2 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS to remove residual test item. The test item was difficult to remove from the inserts, with the result that some test item precipitation stuck to the edges of the tissue and the inserts. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 ml of previously warmed Assay Medium (37°C) (the post-soak medium was brought to 37°C instead of room temperature; this deviation was considered to be acceptable since this is the normal culture conditions for the cells and post-soak procedure was performed at room temperature; additionally appropriate control responses were observed) in a pre-labeled 12-well plate for a 12 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 ml of warm Assay Medium and were incubated for 120 ± 15 minutes at 37°C.

After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2 ml isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 ml isopropanol refrigerated for 18 ± 2 hours in the dark. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the Plate Reader.

SCORING SYSTEM:
A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of formazan formed, as monitored by the absorbance. The results for the time-dependant cytotoxicity curves can be presented as the arithmetic mean ± standard deviation. By setting the absorption of the negative control to 100% cell survival the relative absorption calculated for the test item and the positive control can also be described as tissue viability. Appropriate freeze dried tissue samples and/or correction for colour interference is also conducted.

TOOL USED TO ASSESS SCORE: OD was read in a plate spectrophotometer at 570 nm without reference filter. Mean values were calculated from the 2 wells per tissue.
Irritation parameter:
other: Mean Relative Absorbance (% of negative control)
Value:
90
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: S.D. 0.045 or 28% (n=2); Corrected for colour interference and non-specific reduction of MTT by the test item.
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: NEGATIVE CONTROL: Mean Relative Absorbance (% of negative control): 100% with S.D. 0.045 (n=2)
- Acceptance criteria met for positive control: POSITIVE CONTROL (methyl acetate): Mean Relative Absorbance (% of negative control): 48% with S.D. 0.005 (n=2)
- Range of historical values if different from the ones specified in the test guideline: The positive control had a mean cell viability after 30 ± 2 minutes exposure of 48%. The absolute mean OD570 of the negative control tissues was within 0.8 and 2.6. The standard deviation value of the percentage viability of two tissues treated identically was less than 3% for the negative and positive controls, indicating that the test system functioned properly.
Other effects:
No pH effect of the test substance was observed on the rinsing medium.

Table 1. Mean absorption in the EpiOcular™ test with the test item

 

A

(OD570)

B

(OD570)

Mean

(OD570)

 

SD

Negative control

1.674

1.611

1.643

±

0.045

Test item#1

1.809

1.163

1.486

±

0.457

Positive control

0.785

0.793

0.789

±

0.005

OD = optical density

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.0423). Isopropanol was used to measure the background absorption.

#1 Corrected for colour interference and non-specific reduction of MTT by the test item.

 

Table 2. Mean tissue viability in the EpiOcular™ test with the test item

 

Mean tissue viability (percentage of control)

Negative control

100

Test item

90

Positive control

48

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study the test substance is not a eye irritant or corrosive.
Executive summary:

The study was performed according to OECD TG 492 to assess the irritancy potential of the test material to the eye means of the Human Cornea Model Test using the EpiOcular™ model in accordance with GLP. The test item was tested through 50 μl topical application for 30 minutes of tissues of the human cornea model EpiOcular™. The methyl acetate positive control had a mean cell viability of 48% after 30 ± 2 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within 0.8 and 2.5. Thestandard deviation value of the percentage viability of two tissues treated identically was less than 3% for the negative and positive control, indicating that the test system functioned properly. The test item interacted with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) therefore in addition to the normal procedure, two freeze-killed tissues treated with test item and one freeze killed negative control treated tissues were used for the cytotoxicity evaluation with MTT. The nonspecific reduction of MTT by the test item was 0.9% of the negative control tissues. The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test item treated viable tissues. The test item showed colour interference in aqueous conditions. In addition to the normal procedure, two tissues were treated with test item. Instead of MTT solution these tissues were incubated with assay medium. The non-specific colour of the test item was 0.2% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test item treated viable tissues with MTT assay. Since the test item both interacted with MTT and showed colour interference, in addition to the normal procedure, two freeze-killed tissues were treated with test substance. Instead of MTT solution these tissues were incubated with assay medium. The non-specific colour in killed tissues was 0.3% of the negative control tissues. The OD of the freeze-killed treated tissues without MTT assay was added to the ODs of the test item treated viable tissues with MTT assay to avoid double correction. Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test item compared to the negative control tissues was 90%. The standard deviation of two tissues treated with the test item was 28% which is above the acceptance criterion of 20%. Since both individual viabilities were clearly negative (110 and 71%), this was judged to not affect the study outcome. Applicant assessment of the data indicates that since no irritation response interaction was observed (i.e. including enhanced sensitivity mean viability limit criteria: 65% suggested in OECD TG 492) with the test item in any of the test tissues that the acceptability of the assay was not impacted. Under the conditions of this study, since mean relative tissue viability for the test item was above 60% after 30 ± 2 minutes treatment the test item is considered to be non-irritant.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Read-Across - methyl-2-[[(2,4-dimethyl-3-cyclohexen-1-yl)methylene]amino]benzoate: In vivo, eq. or similar to EU Method B.4, 1985: The study was performed in a method equivalent or similar to EU Method B.4 under GLP to assess the irritancy potential of the test substance to the skin of the New Zealand White rabbit. The test substance was applied over an area of approximately 2.5cm square to the clipped backs of 4 albino rabbits. The test substance was held in contact with the skin under a semi-occlusive patch for a 4 hour period. After this time the patches were removed and the skin reaction to the test substance was assessed after one, 24, 48 and 72 hours. Additional assessments were carried out 7 and 14 days after dosing to determine the degree of reversibility of the skin reaction. The response increased within twenty four hours of dosing when well defined erythema of the skin was noted in all four animals. Slight oedema was apparent and very slight oedema of the skin was observed. Well defined erythema and slight oedema were observed at the treated sites at the forty eight hour observation. Seventy two hours after dosing slight increase in oedematous reaction had occurred in one animal and thickening of the treated skin was observed in a second animal. Well defined erythema, moderate oedema and thickening of the treated skin was observed in all organisms seven days after dosing. This reaction declined and fourteen days after dosing well defined erythema and slight oedema remained in one organism, and very slight irritation was observed at the treated sites of the remaining organisms. Desquamation from the skin surface was noted in all four rabbits of the group. Applicant recalculation of the results indicates that under the conditions of this study, that the mean erythema scores between 24 and 72 hours were > 2.0 in at least 3 of 4 organisms and the response had not fully reversed within a 14 day period. The test item should be considered as irritating to the skin.

 

Read-Across - oxydipropanol: In vivo, US EPA OPP 81-5, 1995: The study was performed under US EPA OPP 81-5 under GLP to assess the irritancy potential of the test substance to the skin of the New Zealand White rabbit. The test substance was applied over an area of approximately 6cm2 square to the clipped backs of 3 male and female albino rabbits. The test substance was held in contact with the skin under an occlusive patch for a 4 hour period. After this time the patches were removed and the skin reaction to the test substance was assessed after one, 24, 48 and 72 hours. No significant effects were observed at 24, 48 and 72 hours. Applicant recalculation of the results indicates that under the conditions of this study, that the mean erythema and oedema scores between 24 and 72 hours were < 2.0 in all organisms. There was no indications of irritation during the study The test item should be considered as not irritating to the skin.

 

Weight of evidence: The applicant assesses this study by expert judgement and indicates that the weight of evidence by read-across to relevant data on both a common breakdown product (expected in physiological conditions) and/or main constituent that there is evidence of transient skin irritation that is sufficient to meet the skin irritation criteria in accordance with Regulation (EC) 1272/2008.

 

Eye Irritation:

In vitro, OECD TG 492, 2016: The study was performed according to OECD TG 492 to assess the irritancy potential of the test material to the eye means of the Human Cornea Model Test using the EpiOcular™ model in accordance with GLP. The test item was tested through 50 μl topical application for 30 minutes of tissues of the human cornea model EpiOcular™. The methyl acetate positive control had a mean cell viability of 48% after 30 ± 2 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within 0.8 and 2.5. Thestandard deviation value of the percentage viability of two tissues treated identically was less than 3% for the negative and positive control, indicating that the test system functioned properly. The test item interacted with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) therefore in addition to the normal procedure, two freeze-killed tissues treated with test item and one freeze killed negative control treated tissues were used for the cytotoxicity evaluation with MTT. The nonspecific reduction of MTT by the test item was 0.9% of the negative control tissues. The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test item treated viable tissues. The test item showed colour interference in aqueous conditions. In addition to the normal procedure, two tissues were treated with test item. Instead of MTT solution these tissues were incubated with assay medium. The non-specific colour of the test item was 0.2% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test item treated viable tissues with MTT assay. Since the test item both interacted with MTT and showed colour interference, in addition to the normal procedure, two freeze-killed tissues were treated with test substance. Instead of MTT solution these tissues were incubated with assay medium. The non-specific colour in killed tissues was 0.3% of the negative control tissues. The OD of the freeze-killed treated tissues without MTT assay was added to the ODs of the test item treated viable tissues with MTT assay to avoid double correction. Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30 ± 2 minutes treatment with the test item compared to the negative control tissues was 90%. The standard deviation of two tissues treated with the test item was 28% which is above the acceptance criterion of 20%. Since both individual viabilities were clearly negative (110 and 71%), this was judged to not affect the study outcome. Applicant assessment of the data indicates that since no irritation response interaction was observed (i.e. including enhanced sensitivity mean viability limit criteria: 65% suggested in OECD TG 492) with the test item in any of the test tissues that the acceptability of the assay was not impacted. Under the conditions of this study, since mean relative tissue viability for the test item was above 60% after 30 ± 2 minutes treatment the test item is considered to be non-irritant.

Justification for selection of skin irritation / corrosion endpoint:

two in vivo GLP compliant Klimisch 4 read-across studies; selected study indicates potential for adverse effect and is used within a weight of evidence approach to the endpoint.

Justification for selection of eye irritation endpoint:

one in vitro GLP compliant Klimisch 1 study; selected study predicts no adverse effect for the substance

Justification for classification or non-classification

The substance meets the classification criteria under Regulation (EC) No 1272/2008 for skin irritation: category 2: H315

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for eye irritation.

For skin irritation the weight of evidence indicates by expert judgement by read-across to relevant data on both a common breakdown product (expected in physiological conditions) and/or main constituent that there is evidence of transient skin irritation that is sufficient to meet the skin irritation criteria in accordance with Regulation (EC) 1272/2008.