Registration Dossier

Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 16-May and 16-Jul-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well performed GLP and OECD Guideline study. The stability of the test item formulations under the test conditions is unknown. The formulation trial was performed before the study initiation date. Therefore, they are excluded from the GLP Statement of Compliance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA OPPTS 8702600 (Skin Sensitization), EPA 712-C-03-197
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
Buehler test
Justification for non-LLNA method:
Alkenes, C11-14, hydroformylation products, distn. residues, reaction products with maleic anhydride and sodium bisulfite, sodium salts has been classified as non-sensitiser in spite of a positive result of the Local Lymph Node Assay (LLNA). The LLNA is the most accepted in vivo method for the assessment of skin sensitization potential of chemicals however LLNA revealed some unexpected positive findings and discordant results for certain classes of chemicals as surfactants (cationic, anionic, non-ionic, sugar lipids), unsaturated fatty acids, fatty alcohols and silicone materials. Since Alkenes, C11-14, hydroformylation products, distn. residues, reaction products with maleic anhydride and sodium bisulfite, sodium salts fits in the definition of difficult substances to be tested with LLNA, because it is a surfactant with a long alkyl chain which easy contains double bonds, a positive results at the LLNA is questionable and confirmation with the Buehler Test Method, according to OECD TG 406 is beneficial. Before controlling the positive result of the LLNA by mean of a Buehler Test Method, it was advisable to first try an in vitro method. A so called hCLAT in-vitro method is available reporting a negative result, this result, in the light of REACH Annex XI provisions, must be either confirmed in vivo or inserted in a weight of evidence strategy.

Test material

Constituent 1
Reference substance name:
Alkenes, C11-14, hydroformylation products, distn. residues, reaction products with maleic anhydride and sodium bisulfite, sodium salts
EC Number:
938-654-2
Molecular formula:
not applicable
IUPAC Name:
Alkenes, C11-14, hydroformylation products, distn. residues, reaction products with maleic anhydride and sodium bisulfite, sodium salts

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS

- Animals: Albino Dunkin Hartley Guinea Pig, HsdDhl: DH, SPF
- Rationale: Recognized by international guidelines as a recommended test system. No valid non-animal model (in-vitro) is available at present for the test of contact sensitization.
- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 33 females (nulliparous and non-pregnant)
- Age at Delivery / Acclimatization Start: 4 to 6 weeks
- Body Weight at Delivery / Acclimatization Start: 333 - 404.8 g
- Identification: By individual ear tattoo
- Randomization: Randomly selected by hand at time of delivery. No computer randomization.
- Acclimatization: Under laboratory conditions after health examination. Thirteen days for control group and the test group. Five days for the animals used in the pretest. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. The animal room was air-conditioned with 10 - 15 air changes per hour. The air was continuously monitored for temperature and relative humidity. The ranges for room temperature and relative humidity were 20 ± 3 °C and 30 - 70%, respectively; although on 13-May, 17-Jun and 09/10-Jul-2013 the upper range for humidity was exceeded during room cleaning. In addition, on 08-May, 06-Jul and 07-Jul-2013, the upper ranges for temperature were exceeded for technical reasons. The animals were provided with an automatically controlled light cycle of 12 hours light and 12 hours darkness. Music was played during the daytime light period.
- Accommodation: In groups of up to ten in stainless steel cages with standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & Co KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland).
- Diet: Teklad Global Guinea pig diet 2040C (batch no. 68/12; Provimi Kliba AG, 4303 Kaiseraugst / Switzerland), ad libitum. A haystick 4646 (batch no. 58/12; Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was provided for environmental enrichment. Results of analyses for contaminants are archived at Harlan Laboratories Ltd.
Water: Community tap-water from Itingen ad libitum in water bottles. Results of bacteriological, chemical and contaminant analyses are archived at Harlan Laboratories Ltd.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Purified water
Concentration / amount:
Pretest: 75%, 50%, 25% and 10% (weight/weight) in purified water
Induction: three inductions at 75% (weight/weight) in purified water
Challenge: two challenges at 25% (weight/weight) in purified water
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Purified water
Concentration / amount:
Pretest: 75%, 50%, 25% and 10% (weight/weight) in purified water
Induction: three inductions at 75% (weight/weight) in purified water
Challenge: two challenges at 25% (weight/weight) in purified water
No. of animals per dose:
Pretest: 3 animals (Animal N° 1 to 3)
Control group (main study): 10 animals (Animal N° 4 to 13)
Test group (main study): 20 animals (Animal N° 14 to 33)
Details on study design:
APPLICATION OF THE TEST ITEM

The animal's fur was shaved with a fine clipper blade just prior to treatment. Closed patches were applied to the animals, containing 0.5 mL of a freshly prepared test item formulation in a 25 mm Hill Top Chamber (Hill Top Research Inc., Miamiville OH 45147 / USA). The test item formulation was delivered to the Hill Top Chamber using a 1 mL tuberculin syringe with the needle removed.

The Hill Top Chambers were firmly secured by an elastic plaster (Dermaplast® Isomed® 5 cm x 10 m or 10 cm x 10 m, provided by IVF, 8212 Neuhausen, Switzerland) wrapped around the trunk of the animal and secured with impervious adhesive tape (Dermaplast® Isopor® Vlies, 2.5 cm x 10 m, provided by IVF, 8212 Neuhausen / Switzerland). The occlusive dressing was left in place for 6 ± 0.25 hours.

The same treatment method was used for the pretest, the induction and the challenges. The control animals were treated in the same manner with the vehicle only during the induction.

PRETEST

The pretest was performed during the acclimatization of the main study animals. The test item concentrations used were selected during the preliminary solubility testing. The test item was formulated in the vehicle.

The positions of the different test item concentrations used for the pretest are shown in the section “any other information on material and methods incl. tables” below. Four different concentrations were used on each animal. The concentrations for the pretest were A = 75%, B = 50%, C = 25% and D = 10% (weight/weight) in purified water.

The allocation of the different test item concentrations A, B, C and D on the animals was alternated in order to minimize site-to-site variation of the skin reactions.

INDUCTION (MAIN STUDY)

The induction of the main test was performed within three weeks. A total of three inductions (one per week) was performed. The test item concentration used for the induction was determined based on the results of the pretest. A test item concentration of 75% (weight/weight) in purified water was well-tolerated systemically and caused mild-to-moderate skin irritation.

The repeated application was performed at the same site on the left shoulder of the animals. The positions of the application sites are shown in the section “any other information on material and methods incl. tables” below. After the last induction exposure the animals were left untreated for approximately 2 weeks before the challenge.
Challenge controls:
The first challenge was performed approximately two weeks after the last induction. The test item concentration used for the challenge was determined based on the results of the pretest. A test item concentration of 25% (weight/weight) in purified water was well-tolerated systemically and was the highest non-irritant concentration.

Due to the equivocal results during the first challenge, a second challenge was performed using the same test group. The animals were challenged with 25% (weight/weight) in purified water.

The positions of the application sites are shown in the section “any other information on material and methods incl. tables” below.
Positive control substance(s):
not required
Remarks:
The results from the most recent positive control study (Harlan Study Number D65986, performed from 25-Oct to 07-Dec-2012 confirm alpha-hexylcinnamaldehyde as skin sensitizer, as it produced allergic contact dermatitis in >15% of the test animals.

Results and discussion

Positive control results:
The sensitivity and reliability of the experimental method was assessed at least twice a year by use of positive control items such as alpha-hexylcinnamaldehyde or 2-mercapto-benzothiazole which are recommended by the Commission Regulation (EC) No 440/2008, B.6 and OECD 406 and are known to have moderate skin sensitization properties in the guinea pig. The results from the most recent positive control study (Harlan Study Number D65986, performed from 25-Oct-2012 to 07-Dec-2012 confirm alpha-hexylcinnamaldehyde as skin sensitizer, as it produced allergic contact dermatitis in >15% of the test animals.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Purified water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No findings noted
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Purified water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No findings noted.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
purified water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
purified water
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: purified water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: purified water.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25% test item in purified water
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
Discrete or patchy erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25% test item in purified water. No with. + reactions: 4.0. Total no. in groups: 20.0. Clinical observations: Discrete or patchy erythema.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25% test item in purified water
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
Discrete or patchy erythema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25% test item in purified water. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: Discrete or patchy erythema.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
25% test item in purified water
No. with + reactions:
8
Total no. in group:
20
Clinical observations:
Discrete or patchy erythema with or without scale as well as moderate and confluent erythema with scale
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 25% test item in purified water. No with. + reactions: 8.0. Total no. in groups: 20.0. Clinical observations: Discrete or patchy erythema with or without scale as well as moderate and confluent erythema with scale.
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
25% test item in purified water
No. with + reactions:
7
Total no. in group:
20
Clinical observations:
Discrete or patchy erythema with or without scale as well as moderate and confluent erythema with scale
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 25% test item in purified water. No with. + reactions: 7.0. Total no. in groups: 20.0. Clinical observations: Discrete or patchy erythema with or without scale as well as moderate and confluent erythema with scale.

Any other information on results incl. tables

VIABILITY / MORTALITY

 

All animals survived the scheduled observation period.

 

CLINICAL SIGNS

 

No clinical signs were recorded in any animal.

 

SKIN REACTIONS

 

- Induction: Discrete or patchy to moderate and confluent erythema were observed in all animals of the test group after treatment with 75% test item in purified water. One animal showed intense erythema and swelling at the 3rd week of induction. No skin reactions were observed in the control group after treatment with purified water.

 

- First Challenge: No skin reactions were observed in the control group (previously not exposed to the test item) after treatment with purified water. Discrete or patchy erythema was observed in 4 of 20 animals of the test group 24 hours after application of 25% test item in purified water, but 2 of these dermal reactions had resolved at the 48 hours reading.

 

- Second Challenge: Since the control group animals were already exposed to the test item during the first challenge, they were not exposed to the test item during the second challenge, because they were no longer negative controls.

 

At 24-hour reading discrete or patchy erythema was observed in 4 of 20 animals, discrete or patchy erythema with scaling was observed in 3 of 20 animals and moderate and confluent erythema with scaling was observed in one animal of the test group.

 

At 48-hour reading discrete or patchy erythema was observed in 5 of 20 animals, discrete or patchy erythema with scaling was observed in 1 of 20 animals and confluent erythema with scaling was observed in one animal of test group. In addition scaling was observed in 7 of 20 animals of the test group.

 

BODY WEIGHTS

 

The body weight of the animals was within the range commonly recorded for animals of this strain and age.

 

NECROPSY

 

No unscheduled deaths occurred, hence no necropsy was performed. 

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the above mentioned findings in a non-adjuvant sensitization test in guinea pigs and in accordance with Regulation (EC) No 1272/2008, Alkenes, C11-14, hydroformylation products, distn. residues, reaction products with maleic anhydride and sodium disulfite, sodium salts is as a skin sensitizer.
Executive summary:

GENERAL

 

The purpose of this study was to assess the skin sensitization potential ofAlkenes, C11-14, hydroformylation products, distn. residues, reaction products with maleic anhydride and sodium disulfite, sodium saltsaccording to OECD guideline 406 and Commission Regulation (EC) No. 440/2008 using the Buehler test.

 

Twenty female Albino Dunkin Hartley guinea pigs of the test group were treated topically with 75% (weight/weight) test item in purified water once a week for a 3-week induction phase. Two weeks after the last induction, the animals were challenged with 25% (weight/weight) test item in purified water. Two weeks after the first challenge, the test group was rechallenged with 25% (weight/weight) test item in purified water.

 

The ten animals of the control group were treated with purified water during the induction. The animals were challenged with 25% (weight/weight) test item in purified water during the first challenge.

 

RESULTS

 

- Induction:

 

Discrete or patchy to moderate and confluent erythema were observed in all animals of the test group after treatment with 75% test item in purified water. One animal showed intense erythema and swelling at the 3rdweek of induction. No skin reactions were observed in the control group after treatment with purified water.

 

- First Challenge:

 

No skin reactions were observed in the test control group (previously not exposed to the test item) after treatment with purified water.

 

Discrete or patchy erythema was observed in 4 of 20 animals of the test group 24 hours after application of 25% test item in purified water, but 2 of these dermal reactions resolved by 48 hours reading.

 

Incidence and Severity of Skin Reactions after Topical Challenge with 25% Test Item in Purified Water:

 

Erythema Score

Test Group

20 Animals

Control Group

10 Animals

 

24 Hours

48 Hours

24 Hours

48Hours

0

16

18

10

10

1

4

2

0

0

2

0

0

0

0

3

0

0

0

0

No. with grades ≥ 1

4

2

0

0

No. tested

20

20

10

10

Incidence*

4/20

0/10

Severity**

0.15

0

 

*Number of animals showing a response of grade 1 or greater at either 24- or 48-hour reading out of the total animals.

** Total sum of 24- and 48-hour response readings divided by the number of readings (maximum of 3).

 

- Second Challenge

 

Since the control group animals were already exposed to the test item during the first challenge, they were not exposed to the test item during the second challenge, because they were no longer negative controls.

 

At 24-hour reading discrete or patchy erythema was observed in 4 of 20 animals, discrete or patchy erythema with scaling was observed in 3 of 20 animals and moderate and confluent erythema with scale was observed in one animal of test group.

 

At 48-hour reading discrete or patchy erythema was observed in 5 of 20 animals, discrete or patchy erythema with scale was observed in 1 of 20 animals and confluent erythema with scale was observed in one animal of test group. In addition scales were observed in 7 of 20 animals of the test group.

 

Incidence and Severity of Skin Reactions after Topical Challenge with 25% Test Item in Purified Water:

 

Erythema Score

 

Test Group

20 animals

 

24 Hours

48 Hours

0

12

13

1

7

6

2

1

1

3

0

0

No. with grades ≥ 1

8

7

No. tested

20

20

Incidence

12/20

Severity

0.425

 

* Number of animals showing a response of grade 1 or greater at either 24- or 48-hour reading out of the total animals.

** Total sum of 24- and 48-hour response readings divided by the number of readings (maximum of 3).

 

CONCLUSION

 

In this non-adjuvant sensitization test, the overall incidence in the first challenge was 20%, which is higher than 15 % threshold for sensitizers as defined by the OECD and EC guidelines.

 

Therefore, a second challenge was performed, for confirmation of the results of the first challenge, using the same concentration of 25% test item in purified water. In this second challenge, an overall incidence of 60% was observed, which was clearly higher than 20% observed in the first challenge and above the 15 % threshold. Therefore the test item is considered to be a sensitizer.

 

Based on the above mentioned findings in a non-adjuvant sensitization test in guinea pigs and in accordance with Regulation (EC) No 1272/2008, Alkenes, C11-14, hydroformylation products, distn. residues, reaction products with maleic anhydride and sodium disulfite, sodium salts is as a skin sensitizer.

 

Categories Display