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Description of key information


There are 4 studies for this endpoint:
-1 GLP reliable LLNA study performed with heptanol showing that heptanol is a week sensitizer
-2 GLP reliable magnusson and kligman studies perfomed with two different batches of heptanol showing negative results.
-1 Human patch study perfomed on 210 volunteers with a relaibility of 4 showing negative results.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 21 February 2011 to 16 March 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guideline; asaqsuate coherence between data, comments and conclusions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: on the first day of the treatment period, the animals of the preliminary and main tests were approximately 9 weeks old
- Weight at study initiation: a mean body weight ¿ standard deviation of 19.9 +/- 0.9 g
- Housing: individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm). Each cage contained (except for the 5 hours
following the 3H-TdR injections) autoclaved sawdust (SICSA, Alfortville, France).
- Diet (e.g. ad libitum): SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): tap water (filtered using a 0.22 micron filter) contained in bottles.
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): : 12 h/12 h (7:00 - 19:00).

IN-LIFE DATES: From: 23 February 2011 To: 16 March 2011.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 10, 25, 50 and 100%.
No. of animals per dose:
4 animals per dose.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The test item was prepared at the chosen concentrations in AOO by successive dilutions. The dosage form preparation
were homogenized by vortex.
The reference item was dissolved in AOO at the concentration of 25% (v/v).
Fresh dosage form preparations were made extemporaneously of the day of each administration and the dosage form preparations were stored at room temperature prior to use.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay (LLNA) based on the design adopted by ICCVAM (Interagency Coordination Committee on
the Validation of Alternative Methods, ICCVAM 1999) and ECETOC (Monograph No. 78 Skin sensitization Testing for the Purpose of Hazard
Identification and Risk Assessment, September 2000), with the addition of the evaluation of local irritation
- Criteria used to consider a positive response: stimulation index of 3.

TREATMENT PREPARATION AND ADMINISTRATION:
The maximum concentration was selected according to the criteria specified in the International Guidelines and on the basis of the data obtained in
the preliminary test:
- the concentrations were selected from the concentration series 100 (for liquids), 50, 25, 10, 5, 2.5, 1, 0.5%, etc.
- the maximum concentration was the highest achievable concentration, whilst avoiding overt systemic toxicity and excessive local irritation.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The threshold positive value of 3 for the SI was reached in the positive control group.
Parameter:
SI
Remarks on result:
other: DPM per node: Group 1: Vehicle: 104.13 Group 2: Test item at 5%: 149.13 Group 3: Test item at 10%: 119.88 Group 4: Test item at 25%: 176.50 Group 5: Test item at 50%: 442.63 Group 6: Test item at 100%: 372.50
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM per group: Group 1: Vehicle: 833 Group 2: Test item at 5%: 1193 Group 3: Test item at 10%: 959.00 Group 4: Test item at 25%: 1412 Group 5: Test item at 50%: 3541 Group 6: Test item at 100%: 2980
Interpretation of results:
other: weak sensitizer
Conclusions:
The test item, N-HEPTANOL, induced delayed contact hypersensitivity in the murine Local Lymph Node Assay.
According to the EC3 value obtained, the test item should be considered as a weak sensitizer.
Executive summary:
The objective of this study was to evaluate the potential of the test item, N-HEPTANOL, to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA). Evaluation of local irritation was also carried out in parallel. This study was conducted in compliance with the principles of Good Laboratory Practice. MethodsA preliminary test was first performed in order to define the concentrations of test item to be used in the main test. In the main test, 28 female CBA/J mice were allocated to seven groups: ·          five treated groups of four animals receiving the test item at the concentration of 5%, 10%, 25%, 50% and 100%in a mixture Acetone/olive oil (4/1; v/v) (vehicle), ·          one negative control group of four animals receiving the vehicle, ·          one positive control group of four animals receiving the reference item, a-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% in a mixture acetone/olive oil (4/1; v/v). During the induction phase, the test item, vehicle or reference item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6. ResultsFollowing the solubility assay, acetone/olive oil (4/1, v/v) was chosen as vehicle A solution was obtained at the maximum tested concentration of 50%. Consequently, the concentrations selected for the preliminary test were 10%, 25%, 50% and 100%. Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (100%). Clinical signs and mortalityNo treatment-related mortality and no clinical signs were observed during the observation period.Local irritation As all acceptance criteria were met, this experiment was therefore considered valid.  
The EC3value is equal to 38%.
ConclusionThe test item, N-HEPTANOL, induced delayed contact hypersensitivity in the murine Local Lymph Node Assay. According to the EC3 value obtained, the test item should be considered as a weak sensitizer.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 May 2011 - 22 July 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate coherence between data, comments and conclusions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
two animals were evaluated for skin reactions at 44.25h after removal of dressing instead of 48h
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
idem above
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A magnusson and Kligman was performed in order to verify the result obtained in the LLNA study as it is a maximization test..
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: at the beginning of the treatment period, the animals of the main test were 1-2 months old
- Weight at study initiation: 307 g ± 10 g for the males and 304 g ± 10 g for the females
- Housing: individually housed in polycarbonate cages with stainless steel lid
- Diet (e.g. ad libitum): free access to 106 pelleted diet
- Water (e.g. ad libitum): tap water (filtered with a 0.22 µm filter)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00).

IN-LIFE DATES: From: 07 June 2011 To: 22 July 2011
Route:
intradermal and epicutaneous
Vehicle:
other: corn oil (intradermal); ethanol/drinking water (topical induction); acetone (topical challenge)
Concentration / amount:
1% intradermal, 100% topical induction, 10% topical challenge
Route:
epicutaneous, occlusive
Vehicle:
other: corn oil (intradermal); ethanol/drinking water (topical induction); acetone (topical challenge)
Concentration / amount:
1% intradermal, 100% topical induction, 10% topical challenge
No. of animals per dose:
in treated groups of main test:
10 males and 10 females
Details on study design:
RANGE FINDING TESTS:
Performed to determine maximum concentrations tested in the main test

MAIN STUDY
See executive summary
Challenge controls:
left flank: vehicle only
Positive control substance(s):
not required
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10% (challenge)
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
discrete erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10% (challenge). No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: discrete erythema.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10% (challenge)
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
discrete erythema; dryness of skin
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10% (challenge). No with. + reactions: 4.0. Total no. in groups: 20.0. Clinical observations: discrete erythema; dryness of skin.
Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item did not induce delayed contact hypersensitivity in guinea pigs.
Executive summary:

The objective of this study was to evaluate the potential of the test item, n-HEPTANOL (batch No. 1103016), to induce delayed contact hypersensitivity in guinea pigs.

This study was conducted in compliance with the principles of Good Laboratory Practice.

 

Methods

A preliminary test was first performed in order to determine the test item concentrations to be used in the main test.

In the main test, one group of 10 males and 10 females received the test item:

.          on day 1 by intradermal injections in the interscapular region at the concentration of 1%,

.          on day 8 by topical application to the clipped interscapular region at 100%,

.          on day 22 by topical application to the posterior right flank at 10%. The posterior left flank of the animals received the vehicle.

Another control group of five males and five females received the vehicles:

.             corn oil on day 1 in the interscapular region,

.            drinking water treated by reverse osmosis on day 8 in the interscapular region,

.            acetone on day 22 to the posterior left flank. The posterior right flank of animals received the test item at 10%.

 

On day 1, three pairs of intradermal injections were performed in the interscapular region of animals:

.          Freund's complete adjuvant (FCA) diluted to 50% (v/v) with 0.9% NaCl,

.          test item in vehicle or vehicle alone,

.          test item in FCA/0.9% NaCl (50/50, w/w) or vehicle at 50% (w/v) in FCA/0.9% NaCl (50/50, v/v).

 

On day 8, a filter paper (approximately 8 cm2) was fully-loaded with the dosage forms and then applied to the clipped interscapular region, over the intradermal injection sites. The filter paper was held in place by means of an occlusive dressing for 48 hours. The presence of local irritation was checked (but not scored).

The induction phase was followed by a 14-day rest period.

 

On day 22, a Finn Chamber filter paper was fully-loaded with the dosage forms. The chamber was held in contact with the skin by an occlusive dressing for 24 hours. Cutaneous reactions were evaluated before treatment and 24 and 48 hours after removal of the dressing.

 

Each animal was observed at least once a day for mortality and clinical signs during the treatment and observation periods. Body weight was recorded on day 1 and at the end of each observation period.

On completion of the observation period, the animals were sacrificed then discarded without macroscopic post-mortem examination. No skin samples were preserved.

 

Results

After the challenge application, in the control group, at the 24-hour reading, a discrete erythema was observed at right flank treated with test item of 1/10 animals. At the 48-hour reading, a discrete erythema was observed at left flank treated with vehicle of 1/10 animals.

 

In the test item-treated group, at the 24-hour reading, a discrete erythema was noted at right flank treated with test item of 1/20 animals.

At the 48-hour reading, a discrete erythema was observed at left flank treated with vehicle of 1/20 animals and at right flank treated with test item of 4/20 animals. In addition, dryness of the skin was noted at right flank of 1/20 animals.


As the cutaneous reactions were similar at right flank treated with test item and at left flank treated with vehicle, they were considered not to be attributed to delayed contact hypersensitivity.

Conclusion

The test item did not induce delayed contact hypersensitivity in guinea pigs. 

Therefore, the test item should not be considered as sensitizing.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

 


 


For this endpoint, 4 studies are available.


 


1/Local Lymph Node Assay (LLNA) with heptanol (batch No.1103007)


The objective of this study was to evaluate the potential of the test item, N-HEPTANOL, to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA). Evaluation of local irritation was also carried out in parallel. This study was conducted in compliance with the principles of Good Laboratory Practice. Following the solubility assay, acetone/olive oil (4/1, v/v) was chosen as vehicle. A solution was obtained at the maximum tested concentration of 50%. Consequently, the concentrations selected for the preliminary test were 10%, 25%, 50% and 100%. Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (100%).No treatment-related mortality and no clinical signs were observed during the observation period.
The EC3value was equal to 38%.
Conclusion The test item, N-HEPTANOL, induced delayed contact hypersensitivity in the murine Local Lymph Node Assay. According to the EC3value obtained, the test item should be considered as a weak sensitizer.


 


2/Magnusson and Kligman study (OECD 406) with heptanol (batch No. 1103007)


The objective of this study was to evaluate the potential of the test item, n-HEPTANOL (batch No. 1103007), to induce delayed contact hypersensitivity in guinea pigs. This study was conducted in compliance with the principles of Good Laboratory Practice and OECD 406 guideline.


After the challenge application, no erythema was observed at left flank treated with vehicle and at right flank treated with test item of control animals. Only dryness of the skin was noted at right flank of 1/10 animals at the 48-hour reading.


 In the test item-treated group, at the 24-hour reading, a discrete or moderate erythema was noted at right flank treated with test item of 3/20 animals. At the 48-hour reading, a discrete erythema was observed at left flank treated with vehicle of 1/20 animals and at right flank treated with test item of 2/20 animals. In addition, dryness of the skin was noted at right flank of 3/20 animals.


As the cutaneous reactions were similar at right flank treated with test item and at left flank treated with vehicle, they were considered not to be attributed to delayed contact hypersensitivity.


Conclusion


The test item did not induce delayed contact hypersensitivity in guinea pigs. Therefore, the test item should not be considered as sensitizing.


 


3/Magnusson and Kligman (OECD 406) with heptanol (batch No.1103016)


 


The objective of this study was to evaluate the potential of the test item, n-HEPTANOL (batch No. 1103016), to induce delayed contact hypersensitivity in guinea pigs. This study was conducted in compliance with the principles of Good Laboratory Practice.


After the challenge application, in the control group, at the 24-hour reading, a discrete erythema was observed at right flank treated with test item of 1/10 animals. At the 48-hour reading, a discrete erythema was observed at left flank treated with vehicle of 1/10 animals.


In the test item-treated group, at the 24-hour reading, a discrete erythema was noted at right flank treated with test item of 1/20 animals.


At the 48-hour reading, a discrete erythema was observed at left flank treated with vehicle of 1/20 animals and at right flank treated with test item of 4/20 animals. In addition, dryness of the skin was noted at right flank of 1/20 animals.


As the cutaneous reactions were similar at right flank treated with test item and at left flank treated with vehicle, they were considered not to be attributed to delayed contact hypersensitivity.


Conclusion


The test item did not induce delayed contact hypersensitivity in guinea pigs.  Therefore, the test item should not be considered as sensitizing.


 


4/ Human patch test


A maximization test was carried out on 20 volunteers. The material was tested at a concentration of 1% in petrolatum and produced no sensitization reactions.


 


 



Respiratory sensitisation

Endpoint conclusion
Additional information:
Migrated from Short description of key information:
There is no information available on the potential for Heptanol to produce respiratory sensitisation in animals.

Justification for classification or non-classification

No classification is warranted based on the two negative Magnusson and Kligman studies performed with two different batches of Heptanol and the negative Human patch test performed on 20 volonteers.

The positive results obtained in the LLNA study was considered to be a false positive results : Indeed, the major issue of debate for the LLNA is the degree to which the assay is associated with false positive and false negative outcomes especially when the tested item is an irritant as it is the case with heptanol. In conclusion, considering the weight of evidence assessment from data generated from the two Magnusson ans Kligman studies and the Human patch test, no classification is warranted for the sensitization endpoint.