Registration Dossier

Administrative data

Description of key information

These endpoints were fulfilled using read across from Citronellol (3,7-dimethyl-6-Octen-1-ol, CAS 106 -22 -9).

Skin irritation/corrosion:

The skin irritating potential of the test substance was assessed in two in vitro human skin model studies.

The first study tested the substance in an in vitro topical test and in vitro patch test using the Skin Ethic reconstituted epidermal tissue. This study reported positive indication of irritation in both the direct topical application and patch test and levels of IL-1α were increased after treatment with the test item.

The second in vitro study was performed similar to OECD test guideline 431 and used the skin models EpiDerm, EPISKIN and PREDISKIN. This study also reported an outcome of irritating. 

 

Eye irritation:

Eye irritation was assessed in an in vivo study equivalent or similar to OECD Guideline 405 and according to Federal Register 38, No. 187, Para. 1500.42, 5. 27029; 27.09.73. Six rabbits were treated with the test item. All six rabbits had a corneal opacity score of 1, when calculated as the mean grading at 24/48/72 hours. The substance was therefore classified as Eye irritation, Category 2.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
In vitro direct topical test and In vitro patch test
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
In vitro direct topical test and In vitro patch test
GLP compliance:
not specified
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
other: not specified
Cell source:
other: not specified
Source strain:
not specified
Control samples:
yes, concurrent positive control
Species:
other: human tissue; SkinEthic (Nice, France)
Strain:
not specified
Type of coverage:
open
Preparation of test site:
not specified
Vehicle:
unchanged (no vehicle)
Controls:
yes
Duration of treatment / exposure:
4 h, 37°C
Observation period:
4 h
Details on study design:
In vitro direct topical test protocol:
Three reconstituted epidermal tissues of 0.63cm² on 0.3 ml defined maintenance medium in a 24-well plate were used per control or tested compound. Hundred microliters or 100 mg of test compounds were homogeneously displayed on the total surface of the reconstructed epidermis. Negative controls and positive controls were run in parallel for each experiment. Cultures were incubated for 4h at 37 °C, 5% CO2. The three cultures were then transferred into new wells of the same 24-well plate containing 0.3 ml of maintenance medium. Tissues were washed three times with 0.5 ml saline solution A. With solids (powders or crystals), the insert was turned upside down before washing, and— maintained in this position with forceps-knocked 2- to 3- fold on the inner wall of a beaker to mechanically remove most of the applied compound. Histology, MTT reduction, and IL-1α release endpoints were measured. Untreated tissues and H2O treated tissues were used as negative controls while SDS 20% (Basketter et al., 1997; Fentem et al., 2001) and nonanoic acid (Wahlberg and Maibach, 1980) treated tissues were used as positive controls. Negative controls were considered satisfactory if three criteria were met: a high cell viability measured by MTT reduction (≥85% of untreated epidermis), a normal histology (score≥75) (see histology scoring below) and no release of large amounts of IL-1α (<30pg/mL). Positive controls were considered satisfactory when a low cell viability was measured by MTT reduction (<50%), and when a necrosed histology (score<75) and an increase of the amount of secreted IL-1 α (≥30 pg/mL) were observed.

In vitro patch test protocol:
Three reconstituted epidermal tissues of 4 cm², placed on 1 ml defined maintenance medium in a 6-well plate, were used per control or test compound. Seventy-five microliters of the compound was homogeneously displayed on a 0.95 cm2 polypropylene chamber (Cincinnati, OH, USA), which was immediately applied, carefully, to the center of a 4 cm² culture. In case of solid compounds, 75 mg of the powder or crystals was spread on 0.95 cm² (same surface as for liquids) on the center of the culture, and covered immediately by the above chamber. A 5 mm large brush was used to improve the contact between the compound/patch and the epidermal tissue. The patches were homogeneously applied with delicacy; strong pressure was avoided. Negative controls and positive controls were performed in parallel for each experiment. The chamber was removed after a 4 h incubation at 37 °C, 5% CO2. No washing step was included in this protocol because most liquid compounds were absorbed by the patch. With solids, the culture was turned upside down, and—maintained in this position with forceps-knocked 2- to 3-fold on the inner wall of a beaker to mechanically remove most of the applied compound. Histology, MTT reduction, and IL-1α release endpoints were performed. Untreated tissues and H2O treated tissues were used as negative controls while SDS 20% (Basketter et al., 1997; Fentem et al., 2001) and nonanoic acid (Wahlberg and Maibach, 1980) treated tissues were used as positive controls. Negative controls were considered satisfactory if three criteria were met: a high cell viability measured by MTT reduction (≥85% of untreated epidermis), a normal histology (≥75) and no release of large amounts of IL-1α (<105 pg/mL). Positive controls were considered satisfactory when a low cell viability was measured by MTT reduction (<50%), and when a necrosed histology (<75) and an increase of the amount of secreted IL-1 α (≥105 pg/mL) were observed.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
> 50
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Other effects / acceptance of results:
Direct topical application: irritant: low cell viability, necrosed, amount of IL-1α increase
patch test: irritant: high cell viability, necrosed, amount of IL-1α increase
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
other: OECD 431
Principles of method if other than guideline:
Method: EpiDerm
GLP compliance:
yes
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
water
Details on test system:
EpiDerm (MatTek, Ashland, MA, USA), EPISKIN (Episkin-SNC, Chaponost, France) and PREDISKIN (BIOPREDIC, Rennes, France)
Type of coverage:
other: in medium
Preparation of test site:
not specified
Vehicle:
water
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml
- Concentration (if solution): 100%

Duration of treatment / exposure:
30, 60, 240, 480, 960 min
Details on study design:
Human skin derived keratinocytes are grown on specially prepaed Millicell cell cultures inserts, forming a multi-layered,differentiated, model of the human epidermis in vitro. Endpoint: time of exposure required to decrease tissue viability by 50% (ET50).
Reference: 20% sodium lauryl sulphate. Mean Irritation Potential (MIP) = (mean ET50 for 20% SLS:mean ET50 for test chemical); if MIP >= 0.8, test chemical is predicted to be irritant (I). 3 participants, each conducted the test three times for evaluation of the result.
Irritation / corrosion parameter:
other: Mean Irritation Potential
Run / experiment:
1a - ET50 = 47.9 min
Value:
0.8
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
other: Mean Irritation Potential
Run / experiment:
2a - ET50 = 65.4 min
Value:
1
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
other: Mean Irritation Potential
Run / experiment:
3a - ET50 = 73.5 min
Value:
1
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
other: Mean Irritation Potential
Run / experiment:
1b - ET50 = 74.4 min
Value:
1.1
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
other: Mean Irritation Potential
Run / experiment:
2b - ET50 = 83.0 min
Value:
1
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
other: Mean Irritation Potential
Run / experiment:
3b - ET50 = 54.1 min
Value:
1.3
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
other: Mean Irritaion Potential
Run / experiment:
1c - ET50 = 70.0 min
Value:
0.9
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
other: Mean Irritaion Potential
Run / experiment:
2c - ET50 = 66.1 min
Value:
0.3
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: Mean Irritation Potential
Run / experiment:
3c - ET50 = 69.6 min
Value:
0.3
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Mean irritant potential: (mean ET50 of 20%SLS)/(mean ET50 of the test material)
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
In vitro direct topical test and In vitro patch test
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substances and target substance have similar physical-chemical properties and (eco)toxicological properties because they are either stereoisomers of the target substance, are hydrolysed to the same substance or their chemical structure differs only by an additional double bond. This prediction is supported by data on the substances themselves.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance, L-Citronellol, is a mono-constituent substance (EC No. 231-415-7, CAS no. 7540-51-4 consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and a hydroxyl group. The substance is optically active, comprising a single, pure enantiomeric laevo form.

The source substance, DL-Citronellol, is a mono-constituent substance (EC No. 203-375-0, CAS no. 106-22-9, consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and a hydroxyl group. The substance is an equimolar mixture of two optical isomers (enantiomers).

The source substance, citronellyl acetate, is a mono-constituent substance (EC No. 205-775-0, CAS no. 150-84-5) consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and an acetate group.

The source substance, geraniol and it’s isomer, consist of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. The only difference between the isomers is the position of the first double bond.

The source substance, geraniol and nerol, is a multi-constituent substance of E/Z isomers (EC No. 906-125-5). The constituents consist of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group.

The source substance, geraniol, is a mono-constituent substance (EC No. 203-377-1, CAS no. 106-24-1), consisting of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. Geraniol is a pure form of the E-isomer.

The source substance, nerol, is a mono-constituent substance (EC No. 203-378-7, CAS no. 106-25-2), consisting of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. Nerol is a pure form of the Z-isomer.
The source and target substances are both of high purity with a low concentration of impurities.

3. ANALOGUE APPROACH JUSTIFICATION
The read across hypothesis is based on structural similarity where the source substances only differ in the enantiomeric ratio or an additional double bond. Another source substance is expected to be hydrolysed to the same structure as the target substance.
In a non-chiral environment the target and source chemical DL-Citronellol will have identical properties, but in the chiral environment of living organisms the enantiomers may possess different carcinogenicity and teratogenicity (in a chiral environment, stereoisomers might experience selective absorption, protein binding, transport, enzyme interactions and metabolism, receptor interactions, and DNA binding). All endpoints read-across from DL-Citronellol are considered to be acceptable for this substance assuming that 50% of the target compound is available in the test material.
The source substance citronellyl acetate is read-across from as part of a weight of evidence approach in the repeated dose toxicity endpoint. As this substance is hydrolysed to Citronellol within 2 hours, this read-across endpoint is acceptable in the weight of evidence approach used.
The source substances geraniol, nerol and the reaction mass of geraniol/nerol differ from the target substance only by an additional double bond at C2. These structures are considered to represent a worst case scenario due to the additional potential reactive feature of the second double bond. The genotoxicity, repeated dose and reproductive toxicity endpoints read-across from these substances are therefore acceptable as a worst case assumption.

4. DATA MATRIX
Please refer to the data matrix included in the read-across justification document attached in Section 13.2.
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
no guideline followed
Principles of method if other than guideline:
In vitro direct topical test and In vitro patch test
GLP compliance:
not specified
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
other: not specified
Cell source:
other: not specified
Source strain:
not specified
Control samples:
yes, concurrent positive control
Species:
other: human tissue; SkinEthic (Nice, France)
Strain:
not specified
Type of coverage:
open
Preparation of test site:
not specified
Vehicle:
unchanged (no vehicle)
Controls:
yes
Duration of treatment / exposure:
4 h, 37°C
Observation period:
4 h
Details on study design:
In vitro direct topical test protocol:
Three reconstituted epidermal tissues of 0.63cm² on 0.3 ml defined maintenance medium in a 24-well plate were used per control or tested compound. Hundred microliters or 100 mg of test compounds were homogeneously displayed on the total surface of the reconstructed epidermis. Negative controls and positive controls were run in parallel for each experiment. Cultures were incubated for 4h at 37 °C, 5% CO2. The three cultures were then transferred into new wells of the same 24-well plate containing 0.3 ml of maintenance medium. Tissues were washed three times with 0.5 ml saline solution A. With solids (powders or crystals), the insert was turned upside down before washing, and— maintained in this position with forceps-knocked 2- to 3- fold on the inner wall of a beaker to mechanically remove most of the applied compound. Histology, MTT reduction, and IL-1α release endpoints were measured. Untreated tissues and H2O treated tissues were used as negative controls while SDS 20% (Basketter et al., 1997; Fentem et al., 2001) and nonanoic acid (Wahlberg and Maibach, 1980) treated tissues were used as positive controls. Negative controls were considered satisfactory if three criteria were met: a high cell viability measured by MTT reduction (≥85% of untreated epidermis), a normal histology (score≥75) (see histology scoring below) and no release of large amounts of IL-1α (<30pg/mL). Positive controls were considered satisfactory when a low cell viability was measured by MTT reduction (<50%), and when a necrosed histology (score<75) and an increase of the amount of secreted IL-1 α (≥30 pg/mL) were observed.

In vitro patch test protocol:
Three reconstituted epidermal tissues of 4 cm², placed on 1 ml defined maintenance medium in a 6-well plate, were used per control or test compound. Seventy-five microliters of the compound was homogeneously displayed on a 0.95 cm2 polypropylene chamber (Cincinnati, OH, USA), which was immediately applied, carefully, to the center of a 4 cm² culture. In case of solid compounds, 75 mg of the powder or crystals was spread on 0.95 cm² (same surface as for liquids) on the center of the culture, and covered immediately by the above chamber. A 5 mm large brush was used to improve the contact between the compound/patch and the epidermal tissue. The patches were homogeneously applied with delicacy; strong pressure was avoided. Negative controls and positive controls were performed in parallel for each experiment. The chamber was removed after a 4 h incubation at 37 °C, 5% CO2. No washing step was included in this protocol because most liquid compounds were absorbed by the patch. With solids, the culture was turned upside down, and—maintained in this position with forceps-knocked 2- to 3-fold on the inner wall of a beaker to mechanically remove most of the applied compound. Histology, MTT reduction, and IL-1α release endpoints were performed. Untreated tissues and H2O treated tissues were used as negative controls while SDS 20% (Basketter et al., 1997; Fentem et al., 2001) and nonanoic acid (Wahlberg and Maibach, 1980) treated tissues were used as positive controls. Negative controls were considered satisfactory if three criteria were met: a high cell viability measured by MTT reduction (≥85% of untreated epidermis), a normal histology (≥75) and no release of large amounts of IL-1α (<105 pg/mL). Positive controls were considered satisfactory when a low cell viability was measured by MTT reduction (<50%), and when a necrosed histology (<75) and an increase of the amount of secreted IL-1 α (≥105 pg/mL) were observed.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
> 50
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Other effects / acceptance of results:
Direct topical application: irritant: low cell viability, necrosed, amount of IL-1α increase
patch test: irritant: high cell viability, necrosed, amount of IL-1α increase
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substances and target substance have similar physical-chemical properties and (eco)toxicological properties because they are either stereoisomers of the target substance, are hydrolysed to the same substance or their chemical structure differs only by an additional double bond. This prediction is supported by data on the substances themselves.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance, L-Citronellol, is a mono-constituent substance (EC No. 231-415-7, CAS no. 7540-51-4 consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and a hydroxyl group. The substance is optically active, comprising a single, pure enantiomeric laevo form.

The source substance, DL-Citronellol, is a mono-constituent substance (EC No. 203-375-0, CAS no. 106-22-9, consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and a hydroxyl group. The substance is an equimolar mixture of two optical isomers (enantiomers).

The source substance, citronellyl acetate, is a mono-constituent substance (EC No. 205-775-0, CAS no. 150-84-5) consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and an acetate group.

The source substance, geraniol and it’s isomer, consist of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. The only difference between the isomers is the position of the first double bond.

The source substance, geraniol and nerol, is a multi-constituent substance of E/Z isomers (EC No. 906-125-5). The constituents consist of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group.

The source substance, geraniol, is a mono-constituent substance (EC No. 203-377-1, CAS no. 106-24-1), consisting of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. Geraniol is a pure form of the E-isomer.

The source substance, nerol, is a mono-constituent substance (EC No. 203-378-7, CAS no. 106-25-2), consisting of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. Nerol is a pure form of the Z-isomer.
The source and target substances are both of high purity with a low concentration of impurities.

3. ANALOGUE APPROACH JUSTIFICATION
The read across hypothesis is based on structural similarity where the source substances only differ in the enantiomeric ratio or an additional double bond. Another source substance is expected to be hydrolysed to the same structure as the target substance.
In a non-chiral environment the target and source chemical DL-Citronellol will have identical properties, but in the chiral environment of living organisms the enantiomers may possess different carcinogenicity and teratogenicity (in a chiral environment, stereoisomers might experience selective absorption, protein binding, transport, enzyme interactions and metabolism, receptor interactions, and DNA binding). All endpoints read-across from DL-Citronellol are considered to be acceptable for this substance assuming that 50% of the target compound is available in the test material.
The source substance citronellyl acetate is read-across from as part of a weight of evidence approach in the repeated dose toxicity endpoint. As this substance is hydrolysed to Citronellol within 2 hours, this read-across endpoint is acceptable in the weight of evidence approach used.
The source substances geraniol, nerol and the reaction mass of geraniol/nerol differ from the target substance only by an additional double bond at C2. These structures are considered to represent a worst case scenario due to the additional potential reactive feature of the second double bond. The genotoxicity, repeated dose and reproductive toxicity endpoints read-across from these substances are therefore acceptable as a worst case assumption.

4. DATA MATRIX
Please refer to the data matrix included in the read-across justification document attached in Section 13.2.
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
other: OECD 431
Principles of method if other than guideline:
Method: EpiDerm
GLP compliance:
yes
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
water
Details on test system:
EpiDerm (MatTek, Ashland, MA, USA), EPISKIN (Episkin-SNC, Chaponost, France) and PREDISKIN (BIOPREDIC, Rennes, France)
Type of coverage:
other: in medium
Preparation of test site:
not specified
Vehicle:
water
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml
- Concentration (if solution): 100%

Duration of treatment / exposure:
30, 60, 240, 480, 960 min
Details on study design:
Human skin derived keratinocytes are grown on specially prepaed Millicell cell cultures inserts, forming a multi-layered,differentiated, model of the human epidermis in vitro. Endpoint: time of exposure required to decrease tissue viability by 50% (ET50).
Reference: 20% sodium lauryl sulphate. Mean Irritation Potential (MIP) = (mean ET50 for 20% SLS:mean ET50 for test chemical); if MIP >= 0.8, test chemical is predicted to be irritant (I). 3 participants, each conducted the test three times for evaluation of the result.
Irritation / corrosion parameter:
other: Mean Irritation Potential
Run / experiment:
1a - ET50 = 47.9 min
Value:
0.8
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
other: Mean Irritation Potential
Run / experiment:
2a - ET50 = 65.4 min
Value:
1
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
other: Mean Irritation Potential
Run / experiment:
3a - ET50 = 73.5 min
Value:
1
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
other: Mean Irritation Potential
Run / experiment:
1b - ET50 = 74.4 min
Value:
1.1
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
other: Mean Irritation Potential
Run / experiment:
2b - ET50 = 83.0 min
Value:
1
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
other: Mean Irritation Potential
Run / experiment:
3b - ET50 = 54.1 min
Value:
1.3
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
other: Mean Irritaion Potential
Run / experiment:
1c - ET50 = 70.0 min
Value:
0.9
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
other: Mean Irritaion Potential
Run / experiment:
2c - ET50 = 66.1 min
Value:
0.3
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: Mean Irritation Potential
Run / experiment:
3c - ET50 = 69.6 min
Value:
0.3
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Mean irritant potential: (mean ET50 of 20%SLS)/(mean ET50 of the test material)
Interpretation of results:
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 vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro eye irritation study does not need to be conducted because adequate data from an in vivo eye irritation study are available
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report. Test procedure according to national standards
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Qualifier:
according to
Guideline:
other: FEDERAL REGISTER 38, NO. 187, PARA. 1500.42, 5. 27029; 27.09.73
Principles of method if other than guideline:
Method: Draize Test
GLP compliance:
not specified
Test material information:
Composition 1
Species:
rabbit
Strain:
Vienna White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Sex: female
- Weight at study initiation: mean 2.57 kg (2.33 - 3.15 kg)
- Diet: SSNIFF sold by Intermast
Vehicle:
unchanged (no vehicle)
Controls:
other: left eye untreated, serves as control
Amount / concentration applied:
0.1 ml
Duration of treatment / exposure:
application into the conjunctival sac of the lower right eyelid
Observation period (in vivo):
8 days
Number of animals or in vitro replicates:
6
Details on study design:
SCORING SYSTEM:

turgor/swelling of conjunctiva or opacity of cornea:
0 = nonexistant
1 = slight
2 = distinct
3 = intense
4 = severe

affected area of cornea:
1 = 0 < A < 1/4
2 = 1/4 <= A < 1/2
3 = 1/2 <= A < 3/4
4 = 3/4 <= A

redness of conjunctiva:
0 = regular
1 = slightly increased
2 = distinct
3 = intense

secretion:
0 = regular
1 = slightly increased
2 = distinctly increased
3 = highly increased

iris:
1 = ciliary injection
2 = iritia
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
24/48/72 h
Score:
30.5
Max. score:
39
Reversibility:
not fully reversible within: 8 days
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
not fully reversible within: 8 days
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0.28
Max. score:
2
Reversibility:
fully reversible within: 8 days
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
1.72
Max. score:
3
Reversibility:
not fully reversible within: 8 days
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
1.44
Max. score:
4
Reversibility:
fully reversible within: 8 days
Other effects:
- scars in all animals
- suppuration in one animal
- pupil contraction in one animal

Results:

 cornea      iris conjunctiva     calculated irritation scores    
time animal opacity area redness turgor/swelling secretion other cornea iris conjunctiva total mean
24 h 1 1 4 0 2 2 2 scar 20 0 12 32 33,67
2 1 4 0 2 2 2 scar 20 0 12 32
3 1 4 1 2 2 2 scar 20 5 12 37
4 1 4 0 2 2 2 scar 20 0 12 32
5 1 4 1 2 2 2 scar 20 5 12 37
6 1 4 0 2 2 2 scar 20 0 12 32
48 h 1 1 4 0 1 1 1 scar 20 0 6 26 29,00
2 1 4 0 2 2 2 scar 20 0 12 32
3 1 4 1 2 2 3 scar/suppuration 20 5 14 39
4 1 4 0 2 1 1 scar 20 0 8 28
5 1 3 0 1 1 1 scar 15 0 6 21
6 1 4 0 2 1 1 scar/pupil contraction 20 0 8 28
72 h 1 1 4 0 1 1 1 scar 20 0 6 26 28,83
2 1 4 1 2 1 2 scar 20 5 10 35
3 1 4 1 2 2 3 scar/suppuration 20 5 14 39
4 1 4 0 1 0 1 scar 20 0 4 24
5 1 3 0 1 1 1 scar 15 0 6 21
6 1 4 0 2 1 1 scar/pupil contraction 20 0 8 28
8 d 1 0 0 0 0 0 0 scar 0 0 0 0 5,17
2 1 1 0 2 0 1 scar 5 0 6 11
3 0 0 0 1 0 1 scar 0 0 4 4
4 1 1 0 1 0 0 scar 5 0 2 7
5 1 1 0 1 0 0 scar 5 0 2 7
6 0 0 0 1 0 0 scar 0 0 2 2
Mean 24-72 h 1 1.0 4.0 0.0 1.3 1.3 1.3 20.0 0.0 8.0 28.0
2 1.0 4.0 0.3 2.0 1.7 2.0 20.0 1.7 11.3 33.0
3 1.0 4.0 1.0 2.0 2.0 2.7 20.0 5.0 13.3 38.3
4 1.0 4.0 0.0 1.7 1.0 1.3 20.0 0.0 8.0 28.0
5 1.0 3.3 0.3 1.3 1.3 1.3 16.7 1.7 8.0 26.3
6 1.0 4.0 0.0 2.0 1.3 1.3 20.0 0.0 9.3 29.3
Mean 24-72 h 1 3.89 0.28 1.72 1.44 1.67 19.4 1.4 9.7 30.5

24h after application of 0,1 ml of test substance:
slight opacity of complete cornea and in 2/6 cases ciliary injections; 
all animals: distinct redness and turgor/swelling of conjunctiva, increased secretion  of tears and scarred retractions at the eyelid

72h: 
redness and turgor of conjunctiva and secretion in regression

8d: 
3/6 animals show very light opacity of small areas of cornea, 
5/6  show slight redness of conjunctiva, 
2/6 show slightly increased secretion of tears, 
1/6 shows temporary suppuration, 
1/6 shows temporary  contraction of the pupil; 
all animals: irreversible scarred alterations of the eyelid

primary irritation score: 31 -> moderately irritating

Interpretation of results:
Category 2A (irritating to eyes) based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
 The test material induced mean corneal opacity scores (24/48/72 hr) of 1 in 6/6 rabbits. Therefore, according the CLP criteria, the substance is classified as Eye irritation, Category 2.
Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report. Test procedure according to national standards
Justification for type of information:
ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source substances and target substance have similar physical-chemical properties and (eco)toxicological properties because they are either stereoisomers of the target substance, are hydrolysed to the same substance or their chemical structure differs only by an additional double bond. This prediction is supported by data on the substances themselves.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance, L-Citronellol, is a mono-constituent substance (EC No. 231-415-7, CAS no. 7540-51-4 consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and a hydroxyl group. The substance is optically active, comprising a single, pure enantiomeric laevo form.

The source substance, DL-Citronellol, is a mono-constituent substance (EC No. 203-375-0, CAS no. 106-22-9, consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and a hydroxyl group. The substance is an equimolar mixture of two optical isomers (enantiomers).

The source substance, citronellyl acetate, is a mono-constituent substance (EC No. 205-775-0, CAS no. 150-84-5) consisting of a C8 carbon backbone, methyl substituents at C3 and C7, one double bond and an acetate group.

The source substance, geraniol and it’s isomer, consist of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. The only difference between the isomers is the position of the first double bond.

The source substance, geraniol and nerol, is a multi-constituent substance of E/Z isomers (EC No. 906-125-5). The constituents consist of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group.

The source substance, geraniol, is a mono-constituent substance (EC No. 203-377-1, CAS no. 106-24-1), consisting of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. Geraniol is a pure form of the E-isomer.

The source substance, nerol, is a mono-constituent substance (EC No. 203-378-7, CAS no. 106-25-2), consisting of a C8 carbon backbone, methyl substituents at C3 and C7, two double bonds and a hydroxyl group. Nerol is a pure form of the Z-isomer.
The source and target substances are both of high purity with a low concentration of impurities.

3. ANALOGUE APPROACH JUSTIFICATION
The read across hypothesis is based on structural similarity where the source substances only differ in the enantiomeric ratio or an additional double bond. Another source substance is expected to be hydrolysed to the same structure as the target substance.
In a non-chiral environment the target and source chemical DL-Citronellol will have identical properties, but in the chiral environment of living organisms the enantiomers may possess different carcinogenicity and teratogenicity (in a chiral environment, stereoisomers might experience selective absorption, protein binding, transport, enzyme interactions and metabolism, receptor interactions, and DNA binding). All endpoints read-across from DL-Citronellol are considered to be acceptable for this substance assuming that 50% of the target compound is available in the test material.
The source substance citronellyl acetate is read-across from as part of a weight of evidence approach in the repeated dose toxicity endpoint. As this substance is hydrolysed to Citronellol within 2 hours, this read-across endpoint is acceptable in the weight of evidence approach used.
The source substances geraniol, nerol and the reaction mass of geraniol/nerol differ from the target substance only by an additional double bond at C2. These structures are considered to represent a worst case scenario due to the additional potential reactive feature of the second double bond. The genotoxicity, repeated dose and reproductive toxicity endpoints read-across from these substances are therefore acceptable as a worst case assumption.

4. DATA MATRIX
Please refer to the data matrix included in the read-across justification document attached in Section 13.2.
Reason / purpose:
read-across source
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Qualifier:
according to
Guideline:
other: FEDERAL REGISTER 38, NO. 187, PARA. 1500.42, 5. 27029; 27.09.73
Principles of method if other than guideline:
Method: Draize Test
GLP compliance:
not specified
Test material information:
Composition 1
Species:
rabbit
Strain:
Vienna White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Sex: female
- Weight at study initiation: mean 2.57 kg (2.33 - 3.15 kg)
- Diet: SSNIFF sold by Intermast
Vehicle:
unchanged (no vehicle)
Controls:
other: left eye untreated, serves as control
Amount / concentration applied:
0.1 ml
Duration of treatment / exposure:
application into the conjunctival sac of the lower right eyelid
Observation period (in vivo):
8 days
Number of animals or in vitro replicates:
6
Details on study design:
SCORING SYSTEM:

turgor/swelling of conjunctiva or opacity of cornea:
0 = nonexistant
1 = slight
2 = distinct
3 = intense
4 = severe

affected area of cornea:
1 = 0 < A < 1/4
2 = 1/4 <= A < 1/2
3 = 1/2 <= A < 3/4
4 = 3/4 <= A

redness of conjunctiva:
0 = regular
1 = slightly increased
2 = distinct
3 = intense

secretion:
0 = regular
1 = slightly increased
2 = distinctly increased
3 = highly increased

iris:
1 = ciliary injection
2 = iritia
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
24/48/72 h
Score:
30.5
Max. score:
39
Reversibility:
not fully reversible within: 8 days
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
not fully reversible within: 8 days
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0.28
Max. score:
2
Reversibility:
fully reversible within: 8 days
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
1.72
Max. score:
3
Reversibility:
not fully reversible within: 8 days
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
1.44
Max. score:
4
Reversibility:
fully reversible within: 8 days
Other effects:
- scars in all animals
- suppuration in one animal
- pupil contraction in one animal

Results:

 cornea      iris conjunctiva     calculated irritation scores    
time animal opacity area redness turgor/swelling secretion other cornea iris conjunctiva total mean
24 h 1 1 4 0 2 2 2 scar 20 0 12 32 33,67
2 1 4 0 2 2 2 scar 20 0 12 32
3 1 4 1 2 2 2 scar 20 5 12 37
4 1 4 0 2 2 2 scar 20 0 12 32
5 1 4 1 2 2 2 scar 20 5 12 37
6 1 4 0 2 2 2 scar 20 0 12 32
48 h 1 1 4 0 1 1 1 scar 20 0 6 26 29,00
2 1 4 0 2 2 2 scar 20 0 12 32
3 1 4 1 2 2 3 scar/suppuration 20 5 14 39
4 1 4 0 2 1 1 scar 20 0 8 28
5 1 3 0 1 1 1 scar 15 0 6 21
6 1 4 0 2 1 1 scar/pupil contraction 20 0 8 28
72 h 1 1 4 0 1 1 1 scar 20 0 6 26 28,83
2 1 4 1 2 1 2 scar 20 5 10 35
3 1 4 1 2 2 3 scar/suppuration 20 5 14 39
4 1 4 0 1 0 1 scar 20 0 4 24
5 1 3 0 1 1 1 scar 15 0 6 21
6 1 4 0 2 1 1 scar/pupil contraction 20 0 8 28
8 d 1 0 0 0 0 0 0 scar 0 0 0 0 5,17
2 1 1 0 2 0 1 scar 5 0 6 11
3 0 0 0 1 0 1 scar 0 0 4 4
4 1 1 0 1 0 0 scar 5 0 2 7
5 1 1 0 1 0 0 scar 5 0 2 7
6 0 0 0 1 0 0 scar 0 0 2 2
Mean 24-72 h 1 1.0 4.0 0.0 1.3 1.3 1.3 20.0 0.0 8.0 28.0
2 1.0 4.0 0.3 2.0 1.7 2.0 20.0 1.7 11.3 33.0
3 1.0 4.0 1.0 2.0 2.0 2.7 20.0 5.0 13.3 38.3
4 1.0 4.0 0.0 1.7 1.0 1.3 20.0 0.0 8.0 28.0
5 1.0 3.3 0.3 1.3 1.3 1.3 16.7 1.7 8.0 26.3
6 1.0 4.0 0.0 2.0 1.3 1.3 20.0 0.0 9.3 29.3
Mean 24-72 h 1 3.89 0.28 1.72 1.44 1.67 19.4 1.4 9.7 30.5

24h after application of 0,1 ml of test substance:
slight opacity of complete cornea and in 2/6 cases ciliary injections; 
all animals: distinct redness and turgor/swelling of conjunctiva, increased secretion  of tears and scarred retractions at the eyelid

72h: 
redness and turgor of conjunctiva and secretion in regression

8d: 
3/6 animals show very light opacity of small areas of cornea, 
5/6  show slight redness of conjunctiva, 
2/6 show slightly increased secretion of tears, 
1/6 shows temporary suppuration, 
1/6 shows temporary  contraction of the pupil; 
all animals: irreversible scarred alterations of the eyelid

primary irritation score: 31 -> moderately irritating

Interpretation of results:
Category 2A (irritating to eyes) based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
 The test material induced mean corneal opacity scores (24/48/72 hr) of 1 in 6/6 rabbits. Therefore, according the CLP criteria, the substance is classified as Eye irritation, Category 2.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Skin corrosion / irritation

Skin corrosion is defined as the production of irreversible damage to the skin following application of the test substance. Skin irritation is the production of reversible damage to the skin following application of the test substance. 

In the in vitro skin topical application test and in vitro patch test, the criteria for the positive control was a low cell viability was measured by MTT reduction (<50%), and when a necrosed histology (score<75) and an increase of the amount of secreted IL-1 α (≥30 pg/mL in the topical test and ≥105 pg/mL in the patch test) were observed. In the topical test a result of low cell viability, necrosed, and increase in IL-1α was reported. In the patch test a result of high cell viability, necrosed, and increase in IL-1α was reported. Therefore the substance was considered as irritating under the conditions of this test.

In a second in vitro skin irritation study, carried out using methods equivalent or similar to OECD 431, the exposure time required to decrease tissue viability by 50% was measured. Under the conditions of this test, the test item was predicted to be an irritant.

Considering the positive result in both in vitro tests, it was concluded that the test substance should be classified as a skin irritant, Category 2 according to CLP.

 

Eye irritation

Serious eye damage is defined as the production of tissue damage in the eye, or serious physical decay or vision following application of the test substance to the anterior surface of the eye which is not fully reversible. Eye irritation means the production of changes in the eye following application of the test substance which is fully reversible. 

Eye irritation was assessed using an in-vivo test method equivalent or similar to OECD Guideline 405.

During in vivo testing, substances are classified as having irreversible effects in the eye if at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or have not fully reversed within the observation period; or at least in 4 of 6 tested animals, a positive response of corneal opacity ≥ 3 and/or iritis > 1,5 calculated as the mean scores following grading at 24, 48 and 72 hours. A substance is classified as having reversible effects on the eye if when applied to the eye of an animal, a substance produces: at least in 4 of 6 tested animals, a positive response of: corneal opacity ≥ 1 and/or iritis ≥ 1, and/or conjunctival redness ≥ 2 and/or conjunctival oedema (chemosis) ≥ 2 calculated as the mean scores following grading at 24, 48 and 72 hours.

 The test material induced mean corneal opacity scores (24/48/72 hr) of 1 in 6/6 rabbits. Therefore, according the CLP criteria, the substance is classified as Eye irritation, Category 2.