6,8-dimethylnon-7-enal

Endpoint summary

Description of key information

Skin sensitiser (modified OECD 429, GLP,WoE, rel.2)

Skin sensitiser (OECD 406, GLP, WoE, rel.2)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

30 January to 04 February 2008.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted in compliance with OECD Guideline No. 429 without any deviation. The substance is adequately identified, but details on composition are missing. Therefore validation applies with restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

24 April 2002.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Directive 2004/73/EC

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

11 January 2007.

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier (F-53941 Le Genest Saint Isle)
- Age at study initiation: 8 weeks
- Weight at study initiation: 18-21 g
- Housing: Animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: foodstuff (M20, SDS), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21-24 °C
- Humidity: 33-50 %
- Air changes: Approximately 15 changes/h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: from 30 January to 04 February 2008.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Main test: 25%, 50% and 100% in the vehicle Acetone/olive oil (4/1, v/v)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Undiluted test item was used (100 %)
- Irritation: a preliminary screening test was not performed. Based on the disposal information (results of skin irritation according OECD n°404 & acute toxicity by oral route according OECD n° 423) the dose levels selected for the main test were 25% and 50%(v/v) in Acetone/Olive oil (4:1) and 100%.
Therefore, the concentration of 100% was chosen as the highest concentration for the main study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.4 compared to control values. Other relevant criteria such as dose-response and irritation level were also taken into account for the interpretation of the results. Any test item failing to produce a SI < 1.4 will be classified as a "non-sensitiser".


TREATMENT PREPARATION AND ADMINISTRATION:
25 μL of control or test substance was applied topically on the dorsal surface of both ears using a micropipette daily for three consecutive days (Days 1-3). On day 6 (end of the test), the animals were anaesthetised with sodium pentobarbital and administration continued to fatal levels. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. A single cell suspension of the lymph node cells of 4 mice of each group was prepared by gentle mechanical tissue disaggregation through a 200-mesh cell strainers in 4 mL of PBS (Ca2+ / Mg2+ - free) containing 0.5% BSA into a well of a multi-well 6. 10 μL of this cell suspension was diluted in 10 mL of physiological saline solution (NaCl 0.9%). The lymphocyte cells were counting using a cell counter (Beckman Coulter Z2). For the run, the lower size selected was 5 μm and the upper size selected was 15 μm (the average size of a lymphocyte is 8 μm).
The proliferation response of lymph node cells was expressed as the number of lymphocytes per lymph node and as the ratio of lymphocytes into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

None.

Positive control results:

Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of α- Hexylcinnamaldehyde, as a solution in acetone/olive oil (4:1, v/v) at concentrations of 5%,10% and 25% (v/v). With EC1.4 = 4.86%, α-Hexylcinnamaldehyde is considered to be a sensitiser under the conditions of the test.

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

25% (v/v) in Acetone/Olive oil (4:1)

Key result

Parameter:

SI

Value:

1.71

Test group / Remarks:

50% (v/v) in Acetone/Olive oil (4:1)

Key result

Parameter:

SI

Value:

1.79

Test group / Remarks:

100% (v/v) in Acetone/Olive oil (4:1)

Key result

Parameter:

other: EC1.4

Remarks:

%

Value:

25

Test group / Remarks:

Test item

Cellular proliferation data / Observations:

- A stimulation index of more than 1.4 was recorded for two concentration of the test item (50% and 100% (v/v) in acetone/olive oil).
The Stimulation Index (SI) calculated by pooled approach was respectively 1.40, 1.71 and 1.79 for the treated group at 25%, 50% and 100%. The EC1.4 value determined by linear interpolation of points on the dose-response curve was 25%.

LOCAL IRRITATION
- No cutaneous reaction was noted in the treated animals. Only, a slight dryness was registered at D6, in the treated group at 100%.
- A slight increase in ear thickness (+18.9%) and in ear weight (+8.3%) was recorded at the concentration of 100%. Therefore, the test item must be considered “slightly-irritant” at the concentration of 100% and “non-irritant” at the concentrations of 50% and 25%.
CLINICAL OBSERVATIONS
- No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test.

BODY WEIGHTS
- Bodyweight changes of the test animals between day 1 and day 6 were comparable to those observed in the corresponding control group animals over the same period.

None.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under these experimental conditions, the test material is classified as skin sensitiser in category 1B according to the annex I of the Regulation (EC) No. 1272/2008 and to the GHS.

Executive summary:

In a local lymph node assay performed according to OECD Guideline 429 and in compliance with GLP, three groups of CBA/J (CBA/J@Rj) strain mice (4 females/dose) were treated for three consecutive days (D1, D2, D3) with 50 μL (25 μL per ear) of the test item at concentrations of 25%, 50% and 100% (v/v) in Acetone/Olive oil (4:1). A further group of four animals was treated with Acetone/Olive oil (4:1). At D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting. The test concentrations for the main study were determined from the disposal information (results of skin irritation according OECD n°404 & acute toxicity by oral route according OECD n° 423). Based on the results of preliminary study, the dose levels selected for the main test were 25%, 50% and 100% (v/v) in Acetone/Olive oil (4:1).

 

No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test. Only, a slight dryness was registered at D6, in the treated group at 100%. A slight increase in ear thickness (+18.9%) and in ear weight (+8.3%) was recorded at the concentration of 100%. Therefore, the test item must be considered “slightly-irritant” at the concentration of 100% and “non-irritant” at the concentrations of 50% and 25%.

The Stimulation Index (SI) calculated by pooled approach was respectively 1.40, 1.71 and 1.79 for the treated group at 25%, 50% and 100%. The EC1.4 value determined by linear interpolation of points on the dose-response curve was 25%.

Under these experimental conditions, the test material is classified as skin sensitiser in category 1B (H317: May cause an allergic skin reaction) according to the annex I of the Regulation (EC) No. 1272 /2008 (CLP) and to the GHS.

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

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Between 18 February and 21 March 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted in compliance with OECD Guideline No. 406. The substance is adequately identified, but details on composition are missing. Therefore validation applies with restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

17 July 1992.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

Directive 96/54 E.E.C.

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

11 January 2007

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

According to REACH regulation, in vivo skin sensitisation studies that were carried out or initiated before May 2017 shall be considered appropriate to address this standard information requirement.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River (F-69592 L'Arbresle)
- Weight at study initiation: 294-346 g
- Housing: in groups of 2 or 3 in polycarbonate containers.
- Diet: ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-24 °C
- Humidity: 45-70 %
- Air changes: at least ten changes per hour.
- Photoperiod: 12 hours light/12 hours dark.

IN-LIFE DATES: 18 February to 21 March 2008.

Route:

intradermal

Vehicle:

olive oil

Concentration / amount:

6.25% for the 1st induction

Day(s)/duration:

Day 0 - 6.

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

100% for the 2nd induction

Day(s)/duration:

Day 7 - 10.

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

12.5% (MNIC)

Day(s)/duration:

Days 20-21

Adequacy of challenge:

highest non-irritant concentration

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

6.25% (1/2 MNIC)

Day(s)/duration:

Day 20 - 21

Adequacy of challenge:

other: 1/2 MNIC

No. of animals per dose:

MAIN STUDY:
GROUP 1 (negative control): 5 females
GROUP 2 (treated): 10 females

Details on study design:

PRELIMINARY STUDIES:
- Maximum Non Necrotizing Concentration (M.N.N.C.) determination by intradermal injection: 2 female guinea pigs were used. The test item was injected by intradermal route at the following concentrations: 100%, diluted at 50%, 25%, 12.5%, 6.25% and 3.125% in olive oil. No necrosis has been observed, since the concentration of 6.25%, the first induction has been carried out by intradermal injection at the same concentration.
- Pre-Maximum Non Irritant Concentration (Pre-M.N.I.C.) determination by topical application: 2 female guinea pigs were used. The test item was applied under an occlusive dressing during 24 hours, at the following concentrations 100%, diluted at 50%, 25% and 12.5% in paraffin oil. 24 hours after the removal of the occlusive dressings, no cutaneous reaction was recorded whatever the tested concentration. In view of these results, the concentration selected was 100% for the 2nd induction of the main study and the MNIC determination began at this concentration of 100%.
- Maximum Non Irritant Concentration (M.N.I.C.) determination by topical application: 3 female guinea pigs were used. After induction by intradermal injection with olive oil and by topical application with paraffin oil and a 17-day rest phase, the challenge phase under occlusive dressing for 24 hours consists in a single topical application of the test item at the following concentrations: 100% diluted at 50%, 25%, 12.5% in paraffin oil. 24 hours after removal of the occlusive dressings, a slight erythema was recorded in three animals on the treated area at 100% and 50% and a slight erythema was recorded in two animals on the treated area at 25%. In view of this result, the concentrations selected were 12.5% (MNIC) and 6.25% (1/2 MNIC) for the challenge phase.

MAIN STUDY
Induction phase: The induction phase was performed by intradermal injection at D0 with the test item at 6.25% and by topical application at D7 with the test item at 100% during 48 hours, 24 hours after brushing with a solution of sodium lauryl sulfate.
A. INDUCTION EXPOSURE (INTRADERMAL - 1st induction: Day 0)
- No. of exposures: One
- 2 intradermal injections of the test item diluted at 6.25%; 2 intradermal injections of Freund’s Complete Adjuvant diluted at 50% in isotonic sodium chloride; 2 intradermal injections of a mixture with equal volumes - Freund’s Complete Adjuvant at 50% and the test item at 12.5%.
- Site: Each side of the mid-line on scapular zone
- Exposure period: Day 0-6
- Duration: 6 days
Day 6: The scapular zone of all the animals in each group, shorn beforehand, was brushed with a solution of sodium lauryl sulfate at 10% in thick vaseline, in order to create a local irritation.

B. INDUCTION EXPOSURE (TOPICAL - 2nd induction: Day 7)
- No. of exposures: One
- Topical application, on the same zone, with the test item at 100%, 24 hours after brushing with 0.5 mL of a solution of Sodium lauryl sulfate at 10%.
- Test groups: 0.5 mL of the test item at 100%
- Control group: 0.5 mL of paraffin oil
- Site: Same intradermally injected area of scapular zone
- Frequency of applications: Single application
Day 10: Occlusive dressing removal

REST PHASE
- The animals of both groups were left for 10 days.

C. CHALLENGE EXPOSURE - FIRST CHALLENGE
- No. of exposures: One
- Day(s) of challenge: Day 20
- Exposure period: 24 h
- Day 21: Occlusive dressing removal
- The experimental procedure of this phase was identical for both negative control and treated groups submitted to this experimentation: on the previously shorn dorso-lumbar zone, an application on either side of the spine, under occlusive dressing, was performed during 24 hours:
- 1 sample cup containing the test item at 12.5% (MNIC) and at 6.25% (1/2 MNIC).
- Evaluation (h after removal of challenge patch): 24, 48 and 72 h

Positive control substance(s):

yes

Remarks:

α-Hexylcinnamaldehyde

Positive control results:

α-Hexylcinnamaldehyde induced skin sensitisation indicating the validity of the study.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

12.5%

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

None

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

12.5%

No. with + reactions:

3

Total no. in group:

10

Clinical observations:

None

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

12.5%

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

12.5%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

12.5%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

negative control

Dose level:

12.5%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

6.25%

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

6.25%

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

6.25%

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

6.25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

6.25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

other: 3rd

Hours after challenge:

72

Group:

negative control

Dose level:

6.25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

25 and 50%

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

None

Remarks on result:

positive indication of skin sensitisation

- It was recorded a slight to moderate erythema, in respectively 50% (5/10), 30% (3/10) and 10% (1/10) of the animals from the treated group, 24, 48 and 72 hours after the challenge phase, on the treated area at 12.5%.

No cutaneous intolerance reaction was recorded in animals from the negative control group, on the treated area at 12.5%.

It was noted a slight to moderate erythema in 10% (1/10) of the animals from the treated group, 24, 48 and 72 hours after the challenge phase, on the treated area at 6.25%.

- No cutaneous intolerance reaction was recorded in animals from the negative control group, on the treated area at 6.25%

- Not any abnormality was recorded in the body weight gain of both groups.

- No mortality was registered during the main test.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under the test conditions, the test material is classified as skin sensitser in Category 1B according to the annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

In a Magnusson & Kligman maximisation study (GPMT) performed according to OECD Guideline 406 and in compliance with GLP, test item was assessed for skin sensitization in female Dunkin-Hartley Guinea pigs. After induction (intradermal injection at 6.25% and topical application at 100%) of 10 Guinea pigs of treated group with the test item and a 10-day rest phase, the challenge phase, under occlusive dressing for 24 hours, consisted of a single topical application of the test item at 6.25% and 12.5% and of a negative control (liquid paraffin). Control group of 5 females treated in similar manner. The test concentrations for the main study were determined from a preliminary study.

It was recorded a slight to moderate erythema, in respectively 50% (5/10), 30% (3/10) and 10% (1/10) of the animals from the treated group, 24, 48 and 72 hours after the challenge phase, on the treated area at 12.5%. No cutaneous intolerance reaction was recorded in animals from the negative control group, on the treated area at 12.5%. It was noted a slight to moderate erythema in 10% (1/10) of the animals from the treated group, 24, 48 and 72 hours after the challenge phase, on the treated area at 6.25%. No cutaneous intolerance reaction was recorded in animals from the negative control group, on the treated area at 6.25%.

The sensitivity of the guinea-pig was checked with known sensitiser i.e., α-Hexylcinnamaldehyde indicating the validity of the study.

Under these experimental conditions, the test material is classified as skin sensitiser in category 1B (H317: May cause an allergic skin reaction) according to the annex I of the Regulation (EC) No. 1272 /2008 (CLP) and to the GHS.

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The two available studies and considered within a Weight of Evidence approach (modified OECD 429, GLP, rel.2) and (OECD 406, GLP, rel.2).

 

- In a Magnusson & Kligman maximisation study (GPMT) performed according to OECD Guideline 406 and in compliance with GLP (Phycher, 2008), test item was assessed for skin sensitization in female Dunkin-Hartley Guinea pigs. After induction (intradermal injection at 6.25% and topical application at 100%) of 10 Guinea pigs of treated group with the test item and a 10-day rest phase, the challenge phase, under occlusive dressing for 24 hours, consisted of a single topical application of the test item at 6.25% and 12.5% and of a negative control (liquid paraffin). Control group of 5 females treated in similar manner. The test concentrations for the main study were determined from a preliminary study.

It was recorded a slight to moderate erythema, in respectively 50% (5/10), 30% (3/10) and 10% (1/10) of the animals from the treated group, 24, 48 and 72 hours after the challenge phase, on the treated area at 12.5%. No cutaneous intolerance reaction was recorded in animals from the negative control group, on the treated area at 12.5%. It was noted a slight to moderate erythema in 10% (1/10) of the animals from the treated group, 24, 48 and 72 hours after the challenge phase, on the treated area at 6.25%. No cutaneous intolerance reaction was recorded in animals from the negative control group, on the treated area at 6.25%.

Under these experimental conditions, the test item is considered as a skin sensitizer.

 

- In a local lymph node assay performed according to OECD Guideline 429 and in compliance with GLP, three groups of CBA/J (CBA/J@Rj) strain mice (4 females/dose) were treated for three consecutive days (D1, D2, D3) with 50 μL (25 μL per ear) of the test item at concentrations of 25%, 50% and 100% (v/v) in Acetone/Olive oil (4:1). A further group of four animals was treated with Acetone/Olive oil (4:1). At D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting. The test concentrations for the main study were determined fromthe disposal information(results of skin irritation according OECD n°404 & acute toxicity by oral route according OECD n° 423). Based on the results of preliminary study, the dose levels selected for the main test were 25%, 50% and 100% (v/v) in Acetone/Olive oil (4:1). No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test.Only, a slight dryness was registered at D6, in the treated group at 100%. A slight increase in ear thickness (+18.9%) and in ear weight (+8.3%) was recorded at the concentration of 100%. Therefore, the test item must be considered “slightly-irritant” at the concentration of 100% and “non-irritant” at the concentrations of 50% and 25%.

The Stimulation Index (SI) calculated by pooled approach was respectively 1.40, 1.71 and 1.79 for the treated group at 25%, 50% and 100%. The EC1.4 value determined by linear interpolation of points on the dose-response curve was 25%.

Under these experimental conditions, the test item is considered as a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Harmonized classification:

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

 

Self classification:

Based on the available data, the substance is classified as skin sensitiser in Category 1B (H317: May cause an allergic skin reaction) according to the CLP and to the GHS.

 

No data was available for respiratory sensitisation.