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Description of key information

Skin sensitisation: Skin sensitizer (Source substance, OECD 406, GLP, K, Rel.1)

Respiratory sensitisation: No data available.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
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 sensitisation profile. Although the protein binding potency was found to be different between the two isomers using SARs, this difference is not expected to have an impact on the skin sensitisation classification since the reaction-mass tested was composed of at least 60% of the target substance. Indeed, the reaction-mass should have been classified as Skin sensitizer 1A if the target substance was more potent than the other isomer which was not the case. Therefore, the read-across to the source substance was considered to be adequate.
The study design (OECD 406, 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 sensitisation study conducted 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, 8.7.2.

4. DATA MATRIX
See Iuclid section13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Positive control results:
alpha-Hexylcinnamaldehyde is a sensitizer under the conditions of this study.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
75% w/w in mineral oil
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
75% w/w in mineral oil
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
mineral oil
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
mineral oil
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation

Topical induction phase:

- Test animals (test substance, 100%): moderate to severe erythema (2 -3) was noted at all test sites one hour after removal of the topical induciton patch.

- Sham animals (mineral oil, 100%): very faint to severe erythema (0.5 -3) was noted for all sham control sites one hour after removal oh the topical induction patch.

- Historical positive control animals (HCA technical, applied as received): very faint to moderate erythema (0.5 -2) was noted at all test sites following the topical induction phase.

- Historical positive Vehicle control animals (mineral oil, 100%): very faint erythema (0.5) was noted at two of five vehicle control sites following the topical induction phase.

Challenge phase:

- Test animals (75% w/w mixture of the test substance in mineral oil): Eight of ten test animals exhibited signes of a sensitization response (faint erythema [1]) 24 hours following challenge patch removal. Similar indications were observed at seven sites through 48 hours. Very faint erythema (0.5) was noted at the other test sites following challenge.

- Sham animals (75% w/w mixture of the test substance in mineral oil): Very faint erythema (0.5) was noted at two of five sham sites 24 hours following the challenge patch removal. Irritation persisted at the two sites through 48 hours.

- Historical positive control animals (HCA technical, applied as a 75% w/w mixture in mineral oil): Eight of ten test animals exhibited signs of a sensitization response (faint to severe erythema [1 -3]) 24 hours following the challenge patch removal. Similar indications persisted at seven sites through 48 hours. Very faint erythema (0.5) was noted for two sites at the 24 -hour and three sites at the 48 -hour interval.

- Historical positive Vehicle control animals (HCA technical, applied as a 75% w/w mixture in mineral oil): Very faint erythema (0.5) was noted at three of five vehicle control sites 24 hours after challenge patch removal. All animals were free of irritation by 48 hours.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Based on available data on the source substance, the target substance is classified in Category 1B according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.
Executive summary:

In a Magnusson & Kligman maximisation study (GPMT) performed according to OECD Guideline 406 and in compliance with GLP, 10 Hartley guinea pigs (male/female) were induced with three pairs of intradermal injections (0.1 mL each) of 50 % solution of Freund’s Complete Adjuvant solution; 5 % concentration of the test article in mineral oil; 5 % concentration of the test article in FCA solution, on Day 1, on three different sites on dorsal area between the shoulder. Control group of 5 aimals was intradermally induced with 0.1 mL of 50 % solution of Freund’s Complete Adjuvant; mineral oil; mineral oil in FCA solution, on Day 1.

On Day 8, the topical induction phase was conducted. Prior to the topical induction, sites were re-clipped free of fur and the shoulder area of each est group animal was pre-treated with SLS in order to enhance sensitization by provoking a mild inflammatory reaction. The injection sites was topically induced with undiluted test article by occluded 2-ply gauze patch for 48 h in test group, whereas control group was applied with vehicle (mineral oil) alone.

On Day 22, a challenge dose of the test article (75 % w/w mixture in mineral oil) was applied to a naive site on each test and sham animal. The test concentrations for the main study were determined from a sighting study.

 

Following challenge with test article at 75% w/w in mineral oil, eight of ten test animals responded positively at the 24h observation and seven of ten at the 48h observation, resulting in a worst-case response incidence of 80%. None of the sham animals responded positively to challenge with test material. A known sensitizer, HCA, which induced skin sensitization indicating the validity of the study.

 

Based on available data on the source substance, the target substance is classified in Category 1B according to 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 sensitisation endpoint.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 2 to October 3, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
At the time of study completion (2003), the LLNA OECD test method was not adopted.
Species:
guinea pig
Strain:
Hartley
Remarks:
albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elm Hill Breeding Laboratories, Chelmsford, MA
- Weight at study initiation: 324-377 g (test and sham groups: males)
- Housing: Animals were group housed in suspended stainless steel caging with mesh floors which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals DHEW (NIH). Litter paper was placed beneath the cage and was changed at least three times per week.
- Diet: Pelleted Purina Guinea Pig Chow #5025
- Water: Filtered tap water supplied by an automatic water dispensing system, ad libitum
- Acclimation period: 4 days (Test and Sham groups)

ENVIRONMENTAL CONDITIONS
- Temperature: 20-22 °C
- Humidity: 34-56 %
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: not reported
Route:
intradermal
Vehicle:
other: mineral oil
Concentration / amount:
5 % w/w
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 % w/w
Day(s)/duration:
Day 8 / 48 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: mineral oil
Concentration / amount:
75 % w/w
Day(s)/duration:
Day 22 / 24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
5 and 10 animals for sham and treatment group, respectively
Details on study design:
PRELIMINARY TEST:
- Intradermal injection ranging study: Two animals were used to determine the minimally irritating concentration (MIC) of test substance via itradermal injection. The fur was removed by clipping the dorsal area and flanks of each animal. This area was divided into six test sites on each animal. Each guinea pig received six ID injections; three concentrations of test material (0.1 mL each) at 1, 3 and 5 % w/w in mineral oil and the same three concentrations in an emulsion of CFA. 24 hours after the injections, each site was evaluated for local reactions (erythema). Based on these findings, the MIC selected for the ID injections of the main study was a 5% w/w mixture in mineral oil.
- Topical irritancy ranging study: Two animals were used to determine the minimally irritancy concentration (MIC). The previously clipped dorsal area of each guinea pig was divided into two sites. The test substance was applied neat and also diluted with a mineral oil to yield a w/w concentration of 75 %. Each concentration was applied to one of the to sites using a 2 cm x 4 cm, 2-ply gauze patch. The patch was covered with plastic wrap and secured in place with non-allergenic Durapore adhesive tape to avoid dislocation of the patch and to minimize loss of the test substance. After the 48-hour exposure period, the patches were removed and the test sites were gently cleansed of any residual test substance. One hour after removal, readings were made of local reactions (erythema).
Four animals were used to determine the Highest Non-irritating Concentration (HNIC). The fur was removed by clipping the dorsal area and flanks of each guinea pig. This area was divided into four test sites on each animal. The test substance was applied neat and also diluted with mineral oil to yield a w/w concentration of 75 %, 50% and 25%. Each concentration was applied to a test site using an occlusive 25 mm Hill Top Chamber. The sites were wrapped with non-allergenic Durapore adhesive tape. After 24-hours of exposure, the chambers were removed and the test sites were gently cleansed of any residual test subtance. Twenty-four hours after patch removal, each site was evaluated for local reactions (erythema).
Based on these findings, the MIC selected for topical application of the main study was 100% with a pretreatment of SLS. The HNIC established and used for challenge was a 75% w/w mixture in mineral oil.


MAIN STUDY
A. INDUCTION EXPOSURE: INTRADERMAL
- No. of exposures: One
- Test groups: Intradermally injected with 3 injections (0.1 mL each) of 50 % solution of Freund’s Complete Adjuvant and water; test article (5% w/w) in mineral oil; test article at a final concentration of 5% w/w in FCA/water (1:1), on Day 1.
- Control group: Intradermally injected with 3 injections (0.1 mL each) of 50 % solution of Freund’s Complete Adjuvant and water;undiluted vehicle (mineral oil); vehicle (mineral oil) in FCA/water (1:1), on Day 1.
- Exposure period: 6 days
- Site: dorsal area (anterior shoulder)
- Evaluation: 24 h after administration of the intradermal injection

Six days after administration of the intradermal injections, the injection sites were clipped free of fur. As the results of the topical range finding study indicated that undiluted test article was non-irritant, 10 % sodium lauryl sulphate in light liquid paraffin was applied to the test and control animals in an attempt to produce local irritation.

B. INDUCTION EXPOSURE: TOPICAL
- No. of exposures: One, on Day 8
- Exposure period: 48 h
- Test groups: 2-ply gauze patch (4 cm x 2 cm) with fivee-tenths of a milliliter of undiluted test material applied topically via occlusive dressing.
- Control group: 2-ply gauze patch (4 cm x 2 cm) with 0.5mL of vehicle (mineral oil) applied topically via occlusive dressing.
- Site: on the dorsal midline, between the injection sites
- Frequency of applications: Single application
- Evaluation: 24 h after removal of the topical induction patches in both test and control groups

C. CHALLENGE EXPOSURE: TOPICAL
- No. of exposures: One
- Day of challenge: Day 22
- Exposure period: 24 h
- test material at concentration of 75% w/w (HNIC) in mineral oil was applied to the right flank of all test and control animals using an occlusive 25 mm Hill Top Chamber.
- Evaluation (h after removal of challenge patch): 24 and 48 h

OTHER:
- Body weight of each animal was recorded at the start (Day 1) and end of the study (Day 25).
Challenge controls:
None
Positive control substance(s):
yes
Remarks:
alpha-Hexylcinnamaldehyde, technical (HCA)
Positive control results:
alpha-Hexylcinnamaldehyde is a sensitizer under the conditions of this study.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
75% w/w in mineral oil
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
75% w/w in mineral oil
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
mineral oil
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
mineral oil
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation

Topical induction phase:

- Test animals (test substance, 100%): moderate to severe erythema (2 -3) was noted at all test sites one hour after removal of the topical induciton patch.

- Sham animals (mineral oil, 100%): very faint to severe erythema (0.5 -3) was noted for all sham control sites one hour after removal oh the topical induction patch.

- Historical positive control animals (HCA technical, applied as received): very faint to moderate erythema (0.5 -2) was noted at all test sites following the topical induction phase.

- Historical positive Vehicle control animals (mineral oil, 100%): very faint erythema (0.5) was noted at two of five vehicle control sites following the topical induction phase.

Challenge phase:

- Test animals (75% w/w mixture of the test substance in mineral oil): Eight of ten test animals exhibited signes of a sensitization response (faint erythema [1]) 24 hours following challenge patch removal. Similar indications were observed at seven sites through 48 hours. Very faint erythema (0.5) was noted at the other test sites following challenge.

- Sham animals (75% w/w mixture of the test substance in mineral oil): Very faint erythema (0.5) was noted at two of five sham sites 24 hours following the challenge patch removal. Irritation persisted at the two sites through 48 hours.

- Historical positive control animals (HCA technical, applied as a 75% w/w mixture in mineral oil): Eight of ten test animals exhibited signs of a sensitization response (faint to severe erythema [1 -3]) 24 hours following the challenge patch removal. Similar indications persisted at seven sites through 48 hours. Very faint erythema (0.5) was noted for two sites at the 24-hour and three sites at the 48-hour interval.

- Historical positive Vehicle control animals (HCA technical, applied as a 75% w/w mixture in mineral oil): Very faint erythema (0.5) was noted at three of five vehicle control sites 24 hours after challenge patch removal. All animals were free of irritation by 48 hours.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the test conditions, the test material is classified in Category 1B according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.
Executive summary:

In a Magnusson & Kligman maximisation study (GPMT) performed according to OECD Guideline 406 and in compliance with GLP, 10 Hartley guinea pigs (male/female) were induced with three pairs of intradermal injections (0.1 mL each) of 50 % solution of Freund’s Complete Adjuvant solution; 5 % concentration of the test article in mineral oil; 5 % concentration of the test article in FCA solution, on Day 1, on three different sites on dorsal area between the shoulder. Control group of 5 aimals was intradermally induced with 0.1 mL of 50 % solution of Freund’s Complete Adjuvant; mineral oil; mineral oil in FCA solution, on Day 1.

On Day 8, the topical induction phase was conducted. Prior to the topical induction, sites were re-clipped free of fur and the shoulder area of each est group animal was pre-treated with SLS in order to enhance sensitization by provoking a mild inflammatory reaction. The injection sites was topically induced with undiluted test article by occluded 2-ply gauze patch for 48 h in test group, whereas control group was applied with vehicle (mineral oil) alone.

On Day 22, a challenge dose of the test article (75 % w/w mixture in mineral oil) was applied to a naive site on each test and sham animal. The test concentrations for the main study were determined from a sighting study.

 

Following challenge with test article at 75% w/w in mineral oil, eight of ten test animals responded positively at the 24h observation and seven of ten at the 48h observation, resulting in a worst-case response incidence of 80%. None of the sham animals responded positively to challenge with test material. A known sensitizer, HCA, which induced skin sensitization indicating the validity of the study.

 

Under the test conditions, the test material is classified in Category 1B according to 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 sensitisation endpoint.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

No study was located on the target substance, therefore the study performed on the source substance was used as the key study to cover this endpoint (see Iuclid section 13 for read-across justification.

In the guinea-pig maximisation test (PSL, 2003, Rel.1) performed on the source substance according to OECD Guideline 406 and in compliance with GLP, guinea pigs (male/female) were induced with three pairs of intradermal injections (0.1 mL each) of 50 % solution of Freund’s Complete Adjuvant solution; 5 % concentration of the source substance in mineral oil; 5 % concentration of the source substance in FCA solution, on Day 1, on three different sites on dorsal area between the shoulder. Control group of 5 aimals was intradermally induced with 0.1 mL of 50 % solution of Freund’s Complete Adjuvant; mineral oil; mineral oil in FCA solution, on Day 1. On Day 8, the topical induction phase was conducted. Prior to the topical induction, sites were re-clipped free of fur and the shoulder area of each est group animal was pre-treated with SLS in order to enhance sensitization by provoking a mild inflammatory reaction. The injection sites was topically induced with undiluted source substance by occluded 2-ply gauze patch for 48 h in test group, whereas control group was applied with vehicle (mineral oil) alone. On Day 22, a challenge dose of the source substance (75 % w/w mixture in mineral oil) was applied to a naive site on each test and sham animal. The test concentrations for the main study were determined from a sighting study.  

Following challenge with the source substance at 75% w/w in mineral oil, eight of ten test animals responded positively at the 24h observation and seven of ten at the 48h observation, resulting in a worst-case response incidence of 80%. None of the sham animals responded positively to challenge with the source substance. A known sensitizer, HCA, induced skin sensitization indicating the validity of the study.  

Under the test conditions, the source substance is a skin sensitizer.

In addition, the OASIS TIMES v. 2.27.19 software was used to predict the skin sensitisation of the target substance. Structural alert was identified for skin sensitisation (skin sensitiser). The target substance falls inside the applicability domain of the model.

Based on the available data, the target substance is considered to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Under the REACH regulation there is no legal standard information requirement in Annexes VII to X to perform any specific test for respiratory sensitisation. In addition, no validated or widely recognised in vitro or in vivo test methods specific to respiratory sensitisation are available yet. No human or animal data are available on the source or on the target substances to address respiratory sensitisation. No alert for respiratory sensitisation were found by the OECD QSAR Toolbox.

Justification for classification or non-classification

Harmonized classification:

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

 

Self-classification:

Based on the available data on the source substance, the target substance is classified as Skin Sens. 1B, H317 (May cause an allergic skin reaction) according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS, since > 60% (80%) of the animals were sensitised to an intradermal induction concentration > 1% (5%) in a GPMT.

 

No direct scientific data are available on the substance to address respiratory sensitisation