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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Acceptable study performed following GPMT

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Name : 2-Ethylhexylal
Synonym : 2-Ethylhexylal pure grade
Both names correspond to the same test item.
CAS number : 22174-70-5
Batch number : 1204051500
Supplier : Lambiotte & Cie
Description : colourless liquid
Container : brown glass flask
Purity : 99.766%
Storage condition : at room temperature
Date of receipt : 14 May 2012
Expiry date : 05 April 2015

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Number:
. preliminary test: 17 females,
. main test: 20 females.
Females were nulliparous and non-pregnant.
- Strain and sanitary status: guinea pigs, Hartley Crl: (HA) BR, Caesarian obtained, Barrier sustained - Virus Antibody Free (COBS - VAF®).
- Reason for choice of species: the guinea pigs are accepted by Regulatory Authorities for this type of study. The strain used has been shown to produce satisfactory sensitization responses after application of known sensitizers (reference items).
- Breeder: Charles River Laboratories France, L’Arbresle, France.
- Age/weight: at the beginning of the treatment period, the animals were 1 to 2 months old and the females had a mean body weight of 347 g (range: 258 g to 486 g).
- Receipt: on arrival, the animals were given a clinical examination to ensure that they are in good condition.
- Acclimation: the animals were acclimated to the study conditions for a period of at least 5 days before treatment. A larger number of animals than necessary was acclimated to permit the selection and/or replacement of individuals.
- Allocation to study: during the acclimation period, the required number of animals were selected according to body weight and clinical condition, and allocated to the groups by a computerized randomization procedure. A new control group of five females was included in the study.
- Identification: the animals were individually identified by an ear tattoo (unique CiToxLAB France identity
number).

ENVIRONMENTAL CONDITIONS
From arrival at CiToxLAB France, the animals were housed in a barriered rodent unit.
The animal room conditions were set as follows:
. temperature : 22 ± 2°C,
. relative humidity : 50 ± 20%,
. light/dark cycle : 12 h/12 h,
. ventilation : approximately 12 cycles/hour of filtered, non-recycled air.
The corresponding instrumentation and equipment are checked and calibrated at regular intervals. The temperature and relative humidity are recorded continuously and the records checked daily and filed.

Housing
The animals were individually housed in polycarbonate cages with stainless steel lid (Tecniplast 2154, 940 cm²) containing autoclaved sawdust (SICSA, Alfortville, France).

Food and water
All animals had free access to 106 pelleted maintenance diet, batch No. 12010, (SAFE, Augy, France) and to tap water (filtered with a 0.22 μm filter).

Contaminant analyses
The batches of diet and sawdust were analyzed by the Suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants that could have interfered with or prejudiced the outcome of the study were found in the diet, drinking water or sawdust.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: 1st challenge: corn oil; 2nd challenge: acetone
Concentration / amount:
Please refer to Details on study design

Rationale for concentration selection
The maximum concentrations tested in the main test were selected according to the criteria specified in the OECD Guidelines and on the basis of the data that were obtained in the preliminary test:
. the concentration used for each induction exposure (intradermal injection or topical application) should be well-tolerated (i.e. no signs indicative of systemic toxicity should be observed) and should be the highest to cause mild-to-moderate skin irritation,
. the concentration used for the challenge exposure (topical application) should be the highest non-irritant concentration,
. the concentrations were selected from the amongst the following: 100% (for liquid or pasty test items), 75%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc.,
. the maximum tested concentration of the test item were the highest achievable concentration, whilst avoiding overt signs indicative of systemic toxicity and excessive local skin irritation.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: 1st challenge: corn oil; 2nd challenge: acetone
Concentration / amount:
Please refer to Details on study design

Rationale for concentration selection
The maximum concentrations tested in the main test were selected according to the criteria specified in the OECD Guidelines and on the basis of the data that were obtained in the preliminary test:
. the concentration used for each induction exposure (intradermal injection or topical application) should be well-tolerated (i.e. no signs indicative of systemic toxicity should be observed) and should be the highest to cause mild-to-moderate skin irritation,
. the concentration used for the challenge exposure (topical application) should be the highest non-irritant concentration,
. the concentrations were selected from the amongst the following: 100% (for liquid or pasty test items), 75%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc.,
. the maximum tested concentration of the test item were the highest achievable concentration, whilst avoiding overt signs indicative of systemic toxicity and excessive local skin irritation.
No. of animals per dose:
Main test: 10 females in test item group; 5 females in each control group
Details on study design:
VEHICLES
The vehicle used in this study was selected from the results of solubility assays performed by the CiToxLAB France Pharmacy and on tests to check the easy passage of dosage form through a needle (for intradermal injections), prior to the preliminary test. For each route of administration, the highest concentration with adequate solubility and homogeneity was called the maximum practicable concentration
(all concentrations and proportions: w/v).
For intradermal injection, in the absence of recommendation from the Sponsor, in order to select the most appropriate vehicle, the solubility assay first started at the concentration of 25% and the first choice vehicle was 0.9% NaCl. As unsatisfactory solubility of the test item was obtained in 0.9% NaCl, corn oil (vegetable oil) was used.
A solution was obtained at the concentration of 25% in corn oil.
. Name : corn oil
. Batch number : MKBH4894V
For topical application, in the absence of recommendation from the Sponsor, in order to select the most appropriate vehicle, the solubility assay first started at the concentration of 75% and the first choice vehicle was drinking water treated by reverse osmosis. As unsatisfactory solubility of the test item was obtained in drinking water treated by reverse osmosis, ethanol/ drinking water treated by reverse osmosis (80/20) for induction and acetone for challenge, and corn oil were tested.
A solution was obtained at the concentration of 75% in corn oil.
. Name : corn oil
. Batch number : MKBH4894V

As the test item is a liquid, the maximum achievable concentration for topical applications will be 100%.
As local reactions were observed on the left flank (treated with vehicle) at the challenge phase, additional solubility assays were performed using acetone.
A solution was obtained at the concentration of 75% in acetone. Acetone was used as vehicle for the second challenge phase only.
. Name : acetone
. Batch number : I605612

PREPARATION OF THE ANIMALS
For each test, the animals were clipped and/or shaved as follows:
. the interscapular region was clipped (approximately 4 cm x 3 cm) the day before intradermal injection,
. the interscapular region was clipped the day before topical application of the dosage from for induction phase of preliminary test and of main test,
. each posterior flank was clipped and shaved (approximately 3 cm x 3 cm) the day before topical application (challenge phase of preliminary test and first challenge phase of main test),
. each median flank was clipped and/or shaved the day before topical application (second challenge phase of main test),
. the application sites were shaved again after dressing removal (induction phase of preliminary test) and after the 24-hour scoring of cutaneous reactions (challenge phase of preliminary test and firs and second challenge phase of main test).

Details on range finding and main tests are shown in "Any other information on M&M"

Clinical examinations
- Morbidity and mortality: Each animal was checked for mortality and morbidity at least once a day during the treatment and observation periods, including weekends and public holidays.
- Clinical signs: Each animal was observed at least once a day, at approximately the same time, for the recording of clinical signs.
- Cutaneous reactions:
Cutaneous reactions were scored in each animal according to the following scale:
. no visible change ..............................................................................................................................0
. discrete or patchy erythema ...........................................................................................................1
. moderate and confluent erythema..................................................................................................2
. intense erythema ...............................................................................................................................3
Any observations of edema or other lesions were recorded.

Body weight: The body weight of each animal was recorded the day of group allocation, then on the first day of treatment, on day 25 and at sacrifice.

PATHOLOGY
Sacrifice: On completion of the observation period, all animals were sacrificed by an intraperitoneal injection of pentobarbital sodium (see § Study plan adherence).
Macroscopic examination: No macroscopic post-mortem examination was performed in any animals.
Preservation of tissues: No skin samples were preserved.






Challenge controls:
Please refer to "Any other information on M&M"
Positive control substance(s):
yes
Remarks:
Benzothiazole-2-thiol (CAS 149-30-4) was used to check sensitivity and reliability of the experimental technique used is assessed every 6 months. The reference item was not assayed in this study and is referred as historical data.

Results and discussion

Positive control results:
Historical data with Mercaptobenzothiazole show that the reference item induced positive skin sensitization reactions in 80% (8/10) guinea pigs.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
other: First challenge - first reading
Hours after challenge:
24
Group:
other: Group 6 - Negative control (corn oil at 1st challenge - acetone at 2nd challenge)
Dose level:
Induction: 0%; 1st Challenge: 0% in left posterior flank and 50% in right posterior flank
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
Y40189 showed cutaneous reaction of score 1 on right flank (RF)
Remarks on result:
other: see Remark
Remarks:
Reading: other: First challenge - first reading. . Hours after challenge: 24.0. Group: other: Group 6 - Negative control (corn oil at 1st challenge - acetone at 2nd challenge). Dose level: Induction: 0%; 1st Challenge: 0% in left posterior flank and 50% in right posterior flank. No with. + reactions: 1.0. Total no. in groups: 5.0. Clinical observations: Y40189 showed cutaneous reaction of score 1 on right flank (RF).
Reading:
other: First challenge - second reading
Hours after challenge:
48
Group:
other: Group 6 - Negative control (corn oil at 1st challenge - acetone at 2nd challenge)
Dose level:
Induction: 0%; 1st Challenge: 0% in left posterior flank and 50% in right posterior flank
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
Y40188 and Y40192: score 2 on LF and score 1 with dryness of the skin on RF; Y40189: score 1 on LF and score 2 with dryness of the skin on RF; Y40190: score 1 on RF; Y40191: score 2 on LF
Remarks on result:
other: see Remark
Remarks:
Reading: other: First challenge - second reading. . Hours after challenge: 48.0. Group: other: Group 6 - Negative control (corn oil at 1st challenge - acetone at 2nd challenge). Dose level: Induction: 0%; 1st Challenge: 0% in left posterior flank and 50% in right posterior flank. No with. + reactions: 5.0. Total no. in groups: 5.0. Clinical observations: Y40188 and Y40192: score 2 on LF and score 1 with dryness of the skin on RF; Y40189: score 1 on LF and score 2 with dryness of the skin on RF; Y40190: score 1 on RF; Y40191: score 2 on LF.
Reading:
other: Second challenge - first reading
Hours after challenge:
24
Group:
other: Group 6 - Negative control (corn oil at 1st challenge - acetone at 2nd challenge)
Dose level:
Induction: 0%; 1st Challenge: 0% in left posterior flank and 50% in right posterior flank; 2nd challenge: 0.25% in left median flank and 0% in right median flank
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: see Remark
Remarks:
Reading: other: Second challenge - first reading. . Hours after challenge: 24.0. Group: other: Group 6 - Negative control (corn oil at 1st challenge - acetone at 2nd challenge). Dose level: Induction: 0%; 1st Challenge: 0% in left posterior flank and 50% in right posterior flank; 2nd challenge: 0.25% in left median flank and 0% in right median flank. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
other: Second challenge - second reading
Hours after challenge:
48
Group:
other: Group 6 - Negative control (corn oil at 1st challenge - acetone at 2nd challenge)
Dose level:
Induction: 0%; 1st Challenge: 0% in left posterior flank and 50% in right posterior flank; 2nd challenge: 0.25% in left median flank and 0% in right median flank
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: see Remark
Remarks:
Reading: other: Second challenge - second reading. . Hours after challenge: 48.0. Group: other: Group 6 - Negative control (corn oil at 1st challenge - acetone at 2nd challenge). Dose level: Induction: 0%; 1st Challenge: 0% in left posterior flank and 50% in right posterior flank; 2nd challenge: 0.25% in left median flank and 0% in right median flank. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
other: First challenge - first reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Induction: 5% for intradermal injection; 75% for cutaneous application; 1st Challenge: 0% in left posterior flank and 50% in right posterior flank
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
Y40193, Y40194, Y40196, Y40201 and Y40202: score 1 on RF; Y40198: score 2 on RF
Remarks on result:
other: see Remark
Remarks:
Reading: other: First challenge - first reading. . Hours after challenge: 24.0. Group: test group. Dose level: Induction: 5% for intradermal injection; 75% for cutaneous application; 1st Challenge: 0% in left posterior flank and 50% in right posterior flank. No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: Y40193, Y40194, Y40196, Y40201 and Y40202: score 1 on RF; Y40198: score 2 on RF.
Reading:
other: First challenge - second reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Induction: 5% for intradermal injection; 75% for cutaneous application; 1st Challenge: 0% in left posterior flank and 50% in right posterior flank
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
Y40194 and Y40196 with dryness of the skin on RF; Y40195, Y40197, Y40201 and Y40202: score 1 and dryness of the skin on RF; Y40193 and Y40201: score 2 and dryness of the skin on RF
Remarks on result:
other: see Remark
Remarks:
Reading: other: First challenge - second reading. . Hours after challenge: 48.0. Group: test group. Dose level: Induction: 5% for intradermal injection; 75% for cutaneous application; 1st Challenge: 0% in left posterior flank and 50% in right posterior flank. No with. + reactions: 8.0. Total no. in groups: 10.0. Clinical observations: Y40194 and Y40196 with dryness of the skin on RF; Y40195, Y40197, Y40201 and Y40202: score 1 and dryness of the skin on RF; Y40193 and Y40201: score 2 and dryness of the skin on RF.
Reading:
other: Second challenge - first reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Induction: 5% for intradermal injection; 75% for cutaneous application; 1st Challenge: 0% in left posterior flank and 50% in right posterior flank; 2nd challenge: 0.25% in left median flank and 0% in right median flank
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: see Remark
Remarks:
Reading: other: Second challenge - first reading. . Hours after challenge: 24.0. Group: test group. Dose level: Induction: 5% for intradermal injection; 75% for cutaneous application; 1st Challenge: 0% in left posterior flank and 50% in right posterior flank; 2nd challenge: 0.25% in left median flank and 0% in right median flank. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
other: Second challenge - second reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Induction: 5% for intradermal injection; 75% for cutaneous application; 1st Challenge: 0% in left posterior flank and 50% in right posterior flank; 2nd challenge: 0.25% in left median flank and 0% in right median flank
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: see Remark
Remarks:
Reading: other: Second challenge - second reading. . Hours after challenge: 48.0. Group: test group. Dose level: Induction: 5% for intradermal injection; 75% for cutaneous application; 1st Challenge: 0% in left posterior flank and 50% in right posterior flank; 2nd challenge: 0.25% in left median flank and 0% in right median flank. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
other: Second challenge - first reading
Hours after challenge:
24
Group:
other: Group 9 - Negative control (acetone at 2nd challenge)
Dose level:
2nd challenge: 0.25% in left median flank and 0% in right median flank
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: see Remark
Remarks:
Reading: other: Second challenge - first reading. . Hours after challenge: 24.0. Group: other: Group 9 - Negative control (acetone at 2nd challenge). Dose level: 2nd challenge: 0.25% in left median flank and 0% in right median flank. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
other: Second challenge - second reading
Hours after challenge:
48
Group:
other: Group 9 - Negative control (acetone at 2nd challenge)
Dose level:
2nd challenge: 0.25% in left median flank and 0% in right median flank
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: see Remark
Remarks:
Reading: other: Second challenge - second reading. . Hours after challenge: 48.0. Group: other: Group 9 - Negative control (acetone at 2nd challenge). Dose level: 2nd challenge: 0.25% in left median flank and 0% in right median flank. No with. + reactions: 0.0. Total no. in groups: 5.0.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the experimental conditions of this study, the test item, 2-Ethylhexylal, did not induce delayed contact hypersensitivity in guinea pigs.
Therefore, the test item should not be considered as skin sensitizing.
Executive summary:

The objective of this study was to evaluate the potential of the test item, 2-Ethylhexylal, to induce delayed contact hypersensitivity in guinea pigs.

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 ten females received the test item:

. on day 1 by intradermal injections in the interscapular region at the concentration of 5% in corn oil,

. on day 8 by topical application to the clipped interscapular region at 75% in corn oil,

. on day 22 by topical application to the posterior right flank at 50% in corn oil. The posterior left flank of the animals received the vehicle (corn oil).

Another control group of females received the vehicle, corn oil, on days 1 and 8 in the interscapular region and on day 22 to the posterior left flank; the posterior right flank of animals received the test item at 50% in corn oil.

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 (corn oil) or corn oil alone,

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

As local reactions were observed on the left flank (treated with vehicle, i.e. corn oil) at the first challenge phase, additional solubility and preliminary assays were performed in order to determine the test item concentrations in acetone to be used for the second challenge phase.

The second challenge application was performed on day 40 under the same experimental conditions than in the first challenge, except that the test item dose formulations were applied at the concentration of 0.25% in acetone to the shaved median left flank of animals and the vehicle, acetone, was applied to the shaved median right flank. A naïve control group of five females was included in the study for the second challenge phase.

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, on day 25 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

No unscheduled deaths occurred during the main test.

No clinical signs indicative of systemic toxicity were observed in any animals.

The body weight change of the test item-treated animals was similar to that of controls.

First challenge application (corn oil used as vehicle)

In the control group 6, at the 24-hour reading, a discrete erythema was observed at right flank (treated with

test item) of 1/5 females. At the 48-hour reading, a discrete or moderate erythema was observed at

left flank (treated with vehicle) of 4/5 animals and at right flank (treated with test item) of 4/5 females

associated with dryness of the skin in three of them.

In the test item-treated group 7, at the 24-hour reading, a discrete or moderate erythema was noted at

right flank (treated with test item) of 6/10 females. At the 48-hour reading, a discrete or moderate erythema

was noted at right flank (treated with test item) of 6/10 animals and dryness of the skin was observed

in 8/10 females.

As local reactions were observed on the vehicle treated flanks of the control group, a second challenge

application with another vehicle (acetone) was performed.

Second challenge application (acetone used as vehicle)

No cutaneous reaction was noted on the right and left flanks of any animals.

Conclusion

Under the experimental conditions of this study, the test item, 2-Ethylhexylal, did not induce delayed contact hypersensitivity in guinea pigs.

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