Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irritation: not irritating (Source substance, OECD 404, GLP, K, rel. 1)

Eye irritation: not irritating (Target substance, OECD 438, GLP, K, rel.1 & Source substance, OECD 405, GLP, K, rel. 1)

Respiratory irritation:  No data was available, however the substance not being classified for skin and eye irritation, no classification is expected for respiratory irritation.

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:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to the Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, (eco)toxicological and environmental fate properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances are structurally related, in that both are 1-[(x,x)-dimethyl-1-cyclohexen-1-yl]-pent-4-en-1-one, which can exist as alpha (1-[(5,5)-dimethyl...) or beta (1-[(3,3)-dimethyl...) forms, meaning the position of the double bond in the hexane cycle differs.
The target substance is the isomer alpha (1-(5,5-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one).
The source substance is a mixture of isomer alpha (1-(5,5-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one), present as the major constituent between 60 and 75% in the mixture, and corresponding to the target (mono-constituent) substance; and isomer beta (1-(3,3-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one), present between 25 and 35%.

3. ANALOGUE APPROACH JUSTIFICATION
Based on structural similarity and comparable physicochemical and toxicological properties, the source and the target substances are expected to have similar skin irritation profile.
In an in vitro study, the target substance was predicted as irritant to the skin. However, the result was suspected to be a false positive response (See Iuclid section 7.3 for further detail). Therefore, a read-across to the in vivo study performed on the source substance was used for the skin irritation endpoint. The study design (OECD 404, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the skin irritation study conducted in the rabbits with the source substance are likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, 8.6.1.

4. DATA MATRIX
See Iuclid section13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 h
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 48 h
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
- Application of the test article to healthy intact rabbit skin resulted in a primary irritation score of 0.22.
- Local signs (mean values from 24 to 72 hours) consisted of grade 0.11 erythema and grade 0.11 oedema. In one animal, very slight erythema and very slight swelling were observed until 24 hours after treatment. All signs of irritation were reversible after 48 hours.
- No irreversible alterations of the treated skin were observed nor were corrosive effects evident on the skin.
Other effects:
- No clinical signs of systemic toxicity were observed in the animals during the test and observation period, and no mortality occured.
- No staining by the test article of the treated skin was observed.
- The body weight of all rabbits were considered to be within the normal range of variability.

Table 7.3.1/1: Skin irritation scores - individual and mean values

Score at time point

Erythema (Animal no 1 / 2 / 3)

Max. score 4

Oedema (Animal no 1 / 2 / 3)

Max. score 4

1 h

1 / 0 / 0

1 / 0 / 0

24 h

1 / 0 / 0

1 / 0 / 0

48 h

0 / 0 / 0

0 / 0 / 0

72 h

0 / 0 / 0

0 / 0 / 0

Average 24, 48 and 72 h

0.33 / 0.0 / 0.0

0.33 / 0.0 / 0.0

Interpretation of results:
GHS criteria not met
Conclusions:
Based on available data on the source substance, the target substance is not classified as irritant to the skin according to the criteria of the Annex VI of Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.
Executive summary:

In a dermal irritation study performed according to the EU Method B.4 and according to the OECD Guideline No. 404, and in compliance with GLP, 0.5 mL of an analogue of the registered substance was applied on the clipped skin of three New Zealand White rabbits (1 male and 2 females). The test material was held in contact with the skin by means of a semi-occlusive dressing for 4 h. Skin reactions were assessed and scored according to the Draize scale at 1, 24, 48 and 72 h after removal of the dressing and test article.

Formation of erythema and oedema of score 1 in the male rabbit were observed 1 h and 24 h after patch removal. Observed oedematous and erythematous reactions had both completely disappeared within 48 h. No cutaneous reaction was observed in the female rabbits at any time.

The individual scores for each animal within 3 scoring times (24, 48 and 72 h) were 0.33 / 0.0 / 0.0 for erythema and 0.33 / 0.0 / 0.0 for oedema.

Based on available data on the source substance, the target substance is not classified as irritant to the skin according to the criteria of the Annex VI of Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 14 to 17, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Elevage Scientifique des Dombes, 01400 Chatillon sur Chalaronne, France
- Age at study initiation: 13 weeks
- Weight at study initiation: Animal n°1: 3034g ; Animal n°2: 2861g ; Animal n°3: 2447g
- Housing: Animals were housed individually in stainless steel cages equipped with feed hoppers, drinking water bowls, with wood and haysticks for gnawing.
- Diet: Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad libitum (batch no. 64/99) from Provimi Kliba AG, 4303 Kaiseraugst, Switzerland
- Water: Community tap water from Itingen, ad libitum, in water bowls.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 20±3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: June 08, 1999 To: June 17, 1999.

Type of coverage:
semiocclusive
Preparation of test site:
clipped
Remarks:
the dorsal fur was clipped with an electric clipper approximately 3 days before treatment
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL

Duration of treatment / exposure:
4 h
Observation period:
1, 24, 48 and 72 h after patch removal.
Number of animals:
1 male and 2 females
Details on study design:
TEST SITE
- Area of exposure: Dorsal area of the trunk (left side only)
- Type of wrap if used: Test material was applied to approximately 6 cm² of the intact skin of the clipped area. It was covered with a 2.5 x 2.5 cm patch of surgical gauze and the gauze was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with tape.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The dressing was removed and the skin was flushed with lukewarm tap water to clean the application site so that any reactions were clearly visible at that time.
- Time after start of exposure: 4 h

OBSERVATION TIME POINTS:
- viability: daily
- skin reaction: at approximately 1, 24, 48 and 72 hours after the removal of the dressing, gauze patch and test article.
- clinical signs: daily during the observation period
- body weight: at start of acclimatization, on the first day of application and at termination of observation

SCORING SYSTEM: The skin reaction was assessed according to the numerical scoring system listed in the EEC Commission Directive 92/69/EEC, July 31, 1992.
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 h
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 48 h
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
- Application of the test article to healthy intact rabbit skin resulted in a primary irritation score of 0.22.
- Local signs (mean values from 24 to 72 hours) consisted of grade 0.11 erythema and grade 0.11 oedema. In one animal, very slight erythema and very slight swelling were observed until 24 hours after treatment. All signs of irritation were reversible after 48 hours.
- No irreversible alterations of the treated skin were observed nor were corrosive effects evident on the skin.
Other effects:
- No clinical signs of systemic toxicity were observed in the animals during the test and observation period, and no mortality occured.
- No staining by the test article of the treated skin was observed.
- The body weight of all rabbits were considered to be within the normal range of variability.

Table 7.3.1/1: Skin irritation scores - individual and mean values

Score at time point

Erythema (Animal no 1 / 2 / 3)

Max. score 4

Oedema (Animal no 1 / 2 / 3)

Max. score 4

1 h

1 / 0 / 0

1 / 0 / 0

24 h

1 / 0 / 0

1 / 0 / 0

48 h

0 / 0 / 0

0 / 0 / 0

72 h

0 / 0 / 0

0 / 0 / 0

Average 24, 48 and 72 h

0.33 / 0.0 / 0.0

0.33 / 0.0 / 0.0

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, test material is not classified as irritant to the skin according to the criteria of the Annex VI of Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.
Executive summary:

In a dermal irritation study performed according to the EU Method B.4 and according to the OECD Guideline No. 404, and in compliance with GLP, 0.5 mL of the test material was applied on the clipped skin of three New Zealand White rabbits (1 male and 2 females). The test material was held in contact with the skin by means of a semi-occlusive dressing for 4 h. Skin reactions were assessed and scored according to the Draize scale at 1, 24, 48 and 72 h after removal of the dressing and test article.

Formation of erythema and oedema of score 1 in the male rabbit were observed 1 h and 24 h after patch removal. Observed oedematous and erythematous reactions had both completely disappeared within 48 h. No cutaneous reaction was observed in the female rabbits at any time.

The individual scores for each animal within 3 scoring times (24, 48 and 72 h) were 0.33 / 0.0 / 0.0 for erythema and 0.33 / 0.0 / 0.0 for oedema.

Under the test conditions, test material is not classified as irritant to the skin according to the criteria of the Annex VI of Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to the Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, (eco)toxicological and environmental fate properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances are structurally related, in that both are 1-[(x,x)-dimethyl-1-cyclohexen-1-yl]-pent-4-en-1-one, which can exist as alpha (1-[(5,5)-dimethyl...) or beta (1-[(3,3)-dimethyl...) forms, meaning the position of the double bond in the hexane cycle differs.
The target substance is the isomer alpha (1-(5,5-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one).
The source substance is a mixture of isomer alpha (1-(5,5-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one), present as the major constituent between 60 and 75% in the mixture, and corresponding to the target (mono-constituent) substance; and isomer beta (1-(3,3-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one), present between 25 and 35%.

3. ANALOGUE APPROACH JUSTIFICATION
Based on structural similarity and comparable physicochemical and toxicological properties, the source and the target substances are expected to have similar eye irritation profile.
In an in vitro study, the target substance was predicted as non-irritant to eyes. The in vivo study performed on the source substance was used to confirm the relevance of the read-across approach. The study design (OECD 405, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the eye irritation study conducted in the rabbits with the source substance are likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, 8.6.2.

4. DATA MATRIX
See Iuclid section13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Irritation parameter:
cornea opacity score
Basis:
animal: #1, #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal: #1, #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal: #1, #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
fully reversible within: 24 h
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 24 h
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal: #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Application of the test article to healthy rabbit conjunctivae resulted in a primary irritation score of 0.00. In all animals, slight reddening of the conjuctivae as well as moderate watery discharge was observed and in one animal slight conjunctival swelling was noted, one hour after treatment. In one animal, slight watery discharge persisted until 24 hours. All signs of irritation were reversible after 48 hours.
Other effects:
- No clinical signs of systemic toxicity were observed in the animals during the test and observation period, and no mortality occured.
- No staining by the test article was observed.
- The body weight of all rabbits were considered to be within the normal range of variability.

Table 7.3.2/1: Eye irritation response data for each animal at each observation time

Score at time point Cornea Iris Conjunctivae
Opacity (/4) (/2) Redness (/3) Chemosis (/4)
1 h (Day 1) 0 / 0 / 0 0 / 0 / 0 1 / 1 / 1 1 / 0 / 0
24 h (Day 1) 0 / 0 / 0 0 / 0 / 0 0 / 0 / 0 0 / 0 / 0
48 h (Day 2) 0 / 0 / 0 0 / 0 / 0 0 / 0 / 0 0 / 0 / 0
72 h (Day 3) 0 / 0 / 0 0 / 0 / 0 0 / 0 / 0 0 / 0 / 0
Average 24, 48 and 72 h 0 / 0 / 0 0 / 0 / 0 0 / 0 / 0 0 / 0 / 0
Interpretation of results:
GHS criteria not met
Conclusions:
Based on available data on the source substance, the target substance is not classified as irritating to eyes according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according the GHS.
Executive summary:

In an eye irritation study performed according to the EU Method B.5 and OECD Guideline No. 405, and in compliance with GLP, 0.1 mL of undiluted test material was instilled into the left eye of 3 New Zealand White rabbits (1 male and 2 females). The lids are then gently held together for about one second to prevent loss of the article. The right eye remained untreated and served as the reference control. Animals were observed at 1, 24, 48 and 72 h after test material instillation. The reactions in the conjunctiva (redness, chemosis and discharge), the iris and the cornea (opacity) were scored according to the Draize scale.

In all animals, slight reddening of the conjuctivae as well as moderate watery discharge was observed and in one animal slight conjunctival swelling was noted, one hour after treatment. In one animal, slight watery discharge persisted until 24 hours. All signs of irritation were reversible after 48 hours.

Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 0.0 / 0.0 / 0.0 for cornea score, iris score, conjunctivae score and chemosis score.

Based on available data on the source substance, the target substance is not classified as irritating to eyes according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 21 to 24, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
1987
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Elevage Scientifique des Dombes, 01400 Chatillon sur Chalaronne, France
- Age at study initiation: 13 weeks
- Weight at study initiation: Animal n°1: 2613g ; Animal n°2: 2726g ; Animal n°3: 2581g
- Housing: Animals were housed individually in stainless steel cages equipped with feed hoppers, drinking water bowls, with wood and haysticks for gnawing.
- Diet: Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad libitum (batch no. 64/99) from Provimi Kliba AG, 4303 Kaiseraugst, Switzerland
- Water: Community tap water from Itingen, ad libitum, in water bowls.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 20±3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: June 15, 1999 To: June 24, 1999.
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated eye served as control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
Duration of treatment / exposure:
No washing was done.
Observation period (in vivo):
1, 24, 48 and 72 h after instillation
Number of animals or in vitro replicates:
1 male and 2 females
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: Not done

SCORING SYSTEM: The ocular reaction was assessed according to the numerical scoring system listed in the EEC Commission Directive 92/69/EEC, July 31, 1992.

TOOL USED TO ASSESS SCORE: fluorescein
Irritation parameter:
cornea opacity score
Basis:
animal: #1, #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal: #1, #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal: #1, #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Reversibility:
fully reversible within: 24 h
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 24 h
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal: #2 and #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Application of the test article to healthy rabbit conjunctivae resulted in a primary irritation score of 0.00. In all animals, slight reddening of the conjuctivae as well as moderate watery discharge was observed and in one animal slight conjunctival swelling was noted, one hour after treatment. In one animal, slight watery discharge persisted until 24 hours. All signs of irritation were reversible after 48 hours.
Other effects:
- No clinical signs of systemic toxicity were observed in the animals during the test and observation period, and no mortality occured.
- No staining by the test article was observed.
- The body weight of all rabbits were considered to be within the normal range of variability.

Table 7.3.2/1: Eye irritation response data for each animal at each observation time

Score at time point Cornea Iris Conjunctivae
Opacity (/4) (/2) Redness (/3) Chemosis (/4)
1 h (Day 1) 0 / 0 / 0 0 / 0 / 0 1 / 1 / 1 1 / 0 / 0
24 h (Day 1) 0 / 0 / 0 0 / 0 / 0 0 / 0 / 0 0 / 0 / 0
48 h (Day 2) 0 / 0 / 0 0 / 0 / 0 0 / 0 / 0 0 / 0 / 0
72 h (Day 3) 0 / 0 / 0 0 / 0 / 0 0 / 0 / 0 0 / 0 / 0
Average 24, 48 and 72 h 0 / 0 / 0 0 / 0 / 0 0 / 0 / 0 0 / 0 / 0
Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, the test material is not classified as irritating to eyes according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according the GHS.
Executive summary:

In an eye irritation study performed according to the EU Method B.5 and OECD Guideline No. 405, and in compliance with GLP, 0.1 mL of undiluted test material was instilled into the left eye of 3 New Zealand White rabbits (1 male and 2 females). The lids are then gently held together for about one second to prevent loss of the article. The right eye remained untreated and served as the reference control. Animals were observed at 1, 24, 48 and 72 h after test material instillation. The reactions in the conjunctiva (redness, chemosis and discharge), the iris and the cornea (opacity) were scored according to the Draize scale.

In all animals, slight reddening of the conjuctivae as well as moderate watery discharge was observed and in one animal slight conjunctival swelling was noted, one hour after treatment. In one animal, slight watery discharge persisted until 24 hours. All signs of irritation were reversible after 48 hours.

Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 0.0 / 0.0 / 0.0 for cornea score, iris score, conjunctivae score and chemosis score.

Under the test conditions, the test material is not classified as irritating to eyes according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.

This study is considered as acceptable and satisfies the requirement for eye irritation endpoint.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 438 without any deviation.
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:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected on 05 July 2016 / signed on 28 October 2016
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Multiple chicken heads, were supplied by Baileys Turkeys Ltd., Cheshire, UK where they were killed for human consumption were used for this assay.
- Characteristics of donor animals (e.g. age, sex, weight): Spring chickens (Gallus Gallus e.g. Ross 308 Broiler) weighed 3kg and were approximately 56 days old.
- Storage, temperature and transport conditions of ocular tissue: Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline. The temperature of the chambers was at 32 ±1.5 °C.
Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
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 µL of the test item was applied, as supplied, to the cornea
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
The test item was applied for 10 seconds and then rinsed from the eye using 20mL of isotonic saline
Number of animals or in vitro replicates:
Test item: 3 eyes
Positive control: 3 eyes
Negative control: 2 eyes
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
- Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are =< 0.5.
- Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner.
- Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ±1.5 °C.
- Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit lamp microscope at the center of each cornea.
- Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.
- After the approval process the eyes were incubated for 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES: 2, 3 and 3 eyes for negative & positive control and test item, respectively.

NEGATIVE CONTROL USED: sodium chloride 0.9% w/v

POSITIVE CONTROL USED: 5% Benzalkonium chloride

APPLICATION DOSE AND EXPOSURE TIME
- Immediately following the zero reference measurements, each test eye (including clamp) was removed from the superfusion apparatus and placed horizontally (cornea facing upwards) into a petri dish. 0.03 mL of test item was applied to the cornea. The entire surface of the cornea was evenly covered. The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline. The treated eye (including clamp) was subsequently returned to the superfusion apparatus in the original upright position.

OBSERVATION PERIOD
- Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the eyes had been decontaminated with the isotonic saline.

METHODS FOR MEASURED ENDPOINTS:
- All observations of the cornea and measurement of corneal thickness were performed using a HaagStreit BP900 slit-lamp microscope with depth-measuring device no. 1.
- Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test substance exposure) were determined at each of the above time points.

SCORING SYSTEM:
- Mean corneal swelling (%): Percentage corneal swelling was assessed from corneal thickness measurements. Mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point.
Corneal swelling (%) = ((corneal thickness at time t - corneal thickness at time = 0) / (corneal thickness at time = 0)) x 100
- Mean maximum opacity score: Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.
0: No opacity
0.5: Very faint opacity
1: Scattered or diffuse areas; details of the iris clearly visible
2: Easily discernible translucent area; details of the iris are slightly obscured
3: Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4: Complete corneal opacity; iris invisible
- Mean fluorescein retention score at 30 minutes post-treatment
0: No fluorescein retention
0.5: Very minor single cell staining
1: Single cell staining scattered throughout the treated area of the cornea
2: Focal or confluent dense single cell staining
3: Confluent large areas of the cornea retaining fluorescein
- Pitting, loosening (sloughing), roughening of the corneal surface, and adhering of test item are all morphological effects that can be noted on the cornea. The classification of these findings was subject to interpretation.

DECISION CRITERIA:
Results from corneal opacity, swelling, and fluorescein retention should be evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint are then combined to generate an Irritancy Classification for each test item.
Irritation parameter:
cornea opacity score
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Value:
0.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Value:
6.34
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
The ocular reactions observed in eyes treated with the test item were:
- maximal mean score of corneal opacity: 0.5, corresponding to the ICE class I;
- mean score of fluorescein retention: 0.3, corresponding to the ICE class I;
- maximal mean corneal swelling: 6.34% corresponding to the ICE class II.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The combination of the three endpoints for the negative control, physiological saline solution, was 2 x I, 1 x II. 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, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

HISTORICAL CONTROL DATA:
In order to confirm the acceptability of the test, a comparison was made with historical control data for negative and positive controls obtained by the laboratory. The test was considered acceptable as the concurrent negative and positive controls were identified as GHS Non-Classified and GHS Category 1 respectively.

Table 7.3.2/5: Individual and average values for evaluation of corneal lesions after treatment with the test item

 

End Point

Eye Number

Time
(after decontamination)

0
minutes

30 minutes

75 minutes

120 minutes

180 minutes

240 minutes

Corneal Opacity

3A

0

0

0

0.5

0.5

0.5

6A

0

0.5

0.5

0.5

0.5

0.5

8A

0.5

0.5

0.5

0.5

0.5

0.5

Mean

0.2

0.3

0.3

0.5

0.5

0.5

ICE Class

I

Fluorescein Retention

3A

 

0.5

 

 

 

 

6A

 

0

 

 

 

 

8A

 

0.5

 

 

 

 

Mean

 

0.3

 

 

 

 

ICE Class

I

Corneal Thickness

3A

0.67

0.73

0.76

0.69

0.74

0.72

6A

0.68

0.68

0.69

0.72

0.70

0.75

8A

0.70

0.68

0.68

0.68

0.74

0.70

Mean

0.68

0.70

0.81

0.70

0.73

0.72

Mean Corneal Swelling (%)

 

1.95

3.90

1.95

6.34

5.85

ICE Class

II

ICE Classes Combined:

2 x I, 1 x II

Classification:

No category

- Corneal opacity scores: Very faint opacity was noted in the test item and one of the negative control treated eyes. (Complete corneal opacity; iris invisible) was noted in all positive control treated eyes. Sloughing was noted in two of the positive control treated eyes.

- Fluorescein retention scores: Very minor single cell staining was noted in two of the test item treated eyes. Confluent large areas of the cornea retaining fluorescein were noted in all positive control treated eyes. No fluorescein retention was noted in both negative control treated eyes.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, test substance is not classified according to the Annex VI of Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

An in vitro eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

 

The test item was applied, as supplied, at the dose of 30 µL, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed using 20 mL of isotonic saline. Three eyes were treated in the same manner with a positive control and two eyes with a negative control. Damages by the test substance were assessed by determination of corneal swelling, 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: 0.5, corresponding to the ICE class I;

- mean score of fluorescein retention: 0.3, corresponding to the ICE class I;

- maximal mean corneal swelling: 6.34% corresponding to the ICE class II.

 

The combination of the three endpoints for the negative control, physiological saline solution, was 2 x I, 1 x II. Therefore, the negative control is classified as "No Category", as expected. The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

 

Under the test conditions, test substance is not classified according to the Annex VI of Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for in vitro eye irritation endpoint.

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

Respiratory irritation

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

Additional information

Skin irritation / corrosion:

A key in vivo study was identified on the source substance (RCC, 1999, rel.1) (see Iuclid section 13 for read-across justification). This a dermal irritation study was performed according to the OECD Guideline No. 404, and in compliance with GLP. Formation of erythema and oedema of score 1 in the male rabbit were observed 1 h and 24 h after patch removal. Observed oedematous and erythematous reactions had both completely disappeared within 48 h. No cutaneous reaction was observed in the female rabbits at any time. The individual scores for each animal within 3 scoring times (24, 48 and 72 h) were 0.33 / 0.0 / 0.0 for erythema and 0.33 / 0.0 / 0.0 for oedema.

Under the test conditions, the source substance is not a skin irritant.

A new in vitro test was performed on the target substance itself to assess the read-across approach (Envigo, 2017). This Episkin study was performed according to the OECD Guideline 439 and in compliance with GLP. The target substance with a mean tissue viability of 47.2 ± 12.4 %, was predicted as irritant to the skin. However, the result was suspected to be a false positive response based on the following information:

- in the in vivo study described above, the source substance was not a skin irritant. The source substance is a reaction-mass between 4-​Penten-​1-​one, 1-​(5,​5-​dimethyl-​1-​cyclohexen-​1-​yl)​- (= the target substance) present at 60 -75% w/w/ and 1-(3,3-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one present at 25 -35 % w/w. The difference between the two constituents, i.e. the position of the double bound within the cycle, is not expected to have an impact on the irritation potential.

- in the eye irritation studies, both the source substance (in vivo) and the target substance (in vitro) were not irritating to the eyes.

- in the acute dermal toxicity study, no sign of irritation was observed up to 1000 mg/kg bw in rabbits (maximum dose-level tested) with the target substance. Although the estimate of the amount of test substance per cm² is lower than the one employed in a standard OECD 404, this study brings additional arguments for the absence of irritation potential.

- in the GPMT, no sign of irritation was observed with the undiluted source substance. Although the skin of the guinea-pigs is known to be less sensitive than that of the rabbits, this study brings additional arguments for the absence of skin irritation potential.

Scientifically, the in vivo studies have prevalence over the in vitro studies, therefore it was concluded that the target and the source substances are not irritating to skin.

Eye irritation:

A key in vivo study was identified on the source substance (RCC, 1999, rel.1) (See Iuclid section 13 for read-across justification). This eye irritation study was performed according to the OECD Guideline No. 405, and in compliance with GLP. In all animals, slight reddening of the conjuctivae as well as moderate watery discharge was observed and in one animal slight conjunctival swelling was noted, one hour after treatment. In one animal, slight watery discharge persisted until 24 hours. All signs of irritation were reversible after 48 hours. Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 0.0 / 0.0 / 0.0 for cornea score, iris score, conjunctivae score and chemosis score.

Under the test conditions, the source substance is not an eye irritant.

A new in vitro study was performed on the target substance itself to assess the read-across approach (Envigo, 2017, rel.1). An ICE study was performed according to the OECD Guideline 438 and in compliance with GLP. This test method was selected based on its applicability domain (i.e. including ketones).

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

- maximal mean score of corneal opacity: 0.5, corresponding to the ICE class I;

- mean score of fluorescein retention: 0.3, corresponding to the ICE class I;

- maximal mean corneal swelling: 6.34% corresponding to the ICE class II.

The combination of the three endpoints for the negative control, physiological saline solution, was 2 x I, 1 x II. Therefore, the negative control is classified as "No Category", as expected. The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe irritant", as expected.

Under the test conditions, the target substance is not an eye irritant.

Based on these two studies, it can be concluded that both the source and the target substance are not irritating to eyes. The relevance of the read-across in terms of local effects is thus confirmed.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

 

Self-classification:

Skin irritation:

Based on the available information, no additional self-classification is proposed regarding skin irritation according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.

Eye irritation:

Based on the available information, no additional self-classification is proposed regarding eye irritation according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.

 

Respiratory irritation:

No data was available, however the substance not being classified for skin and eye irritation, no classification is expected for respiratory irritation.