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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
Study period:
2012-04-25 to 2012-07-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The registration substance is a surfactant. The LLNA is not an appropriate test method.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts Variante TEA/Na Salz
Batch No: 12-SK020
Expiry Date: 02.03.2014
Physical State at RT: liquid
Colour: brown
pH: 9.0
Storage Conditions: at room temperature
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
- Semi barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to autoclaved hay and to Altromin 3122 maintenance diet for guinea pigs (lot no. 0950), rich in crude fibre
- Free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups in Terluran - cages on Altromin saw fibre bedding (lot no. 261111)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days) under laboratory conditions

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: physiological saline 0.9% NaCl (AlleMan Pharma GmbH, lot no. 111209, expiry date: 11/2014)
Concentration / amount:
For the intradermal injection (induction - first stage), 0.1 g of the test item was dissolved in 0.9% NaCl
to gain a final volume of 10 mL of a 1% solution (w/v).
For the topical application (induction – second stage), 3 g of the test item were dissolved in 0.9% NaCl
to gain a final volume of 6 mL of a 50% solution (w/v).
For the topical application (challenge), 3.13 g of the test item were dissolved in 0.9% NaCl
to gain a final volume of 25 mL of a 12.5% solution (w/v).
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: physiological saline 0.9% NaCl (AlleMan Pharma GmbH, lot no. 111209, expiry date: 11/2014)
Concentration / amount:
For the intradermal injection (induction - first stage), 0.1 g of the test item was dissolved in 0.9% NaCl
to gain a final volume of 10 mL of a 1% solution (w/v).
For the topical application (induction – second stage), 3 g of the test item were dissolved in 0.9% NaCl
to gain a final volume of 6 mL of a 50% solution (w/v).
For the topical application (challenge), 3.13 g of the test item were dissolved in 0.9% NaCl
to gain a final volume of 25 mL of a 12.5% solution (w/v).
No. of animals per dose:
10 test animals, 5 control animals and 5 animals for preliminary study
Details on study design:
Induction: First Stage, Intradermal Injection
Three pairs of intradermal injections of 0.1 mL volume were given in the shoulder region which was cleared of hair by clipping so that one of each pair lies on each side of the midline.
Test Group: Day 0
Injection 1: a 1:1 mixture (v/v) FCA/physiological saline 0.9% NaCl
Injection 2: a 1% concentration of the test item in physiological saline 0.9% NaCl
Injection 3: a 1% concentration of the test item formulated in a 1:1 mixture (v/v) FCA/physiological saline 0.9% NaCl
Control Group: Day 0
Injection 1: a 1:1 mixture (v/v) FCA/physiological saline 0.9% NaCl
Injection 2: 100% physiological saline 0.9% NaCl
Injection 3: a 50% (v/v) formulation of 0.9% NaCl in a 1:1 (v/v) mixture FCA/physiological saline 0.9% NaCl
Injections 1 and 2 were given close to each other and nearest to the head, while injection 3 was given toward the caudal part of the test area.

Induction: Second Stage, Topical Application
Test Group and Control Group: Day 6
Approximately twenty-four hours before the topical application the test area was painted with 0.5 g of 10% sodium lauryl sulphate
in vaseline after close clipping in order to create a local irritation.
Test Group: Day 7
The test item was dissolved in physiological saline 0.9% NaCl at a concentration of 50%. A patch was fully loaded with 0.5 mL of the
prepared test item. Then it was applied to the test area and held in contact with the help of an occlusive dressing for 48 hours.
Control Group: Day 7
A patch was fully loaded with 0.5 mL of physiological saline 0.9% NaCl. Then it was applied to the test area and held in contact with
the help of an occlusive dressing for 48 hours.
Challenge controls:
The flanks of treated and control animals were cleared of hair by close-clipping.
Test Group and Control Group: Day 20
The test item was dissolved in physiological saline 0.9% NaCl at a concentration of 12.5%. A patch, loaded with 0.5 mL
of the prepared test item was applied to the left flank of the animals and a patch loaded with 0.5 mL of the vehicle to the right flank
(intraspecific control). The patches were held in contact with the help of an occlusive dressing for 24 hours.
The application area was not rinsed.

Observation
Test Group and Control Group
Approximately 20 hours after removing the patch, the challenge area was cleared of hair by the use of a depilatory cream.
Approximately 24 and 48 hours after removing the patch the skin reaction was observed and recorded according to the grades shown below.
Additionally all animals were observed for signs of toxicity at least once daily during the test period.
Positive control substance(s):
not required
Remarks:
performed periodically every 6 months

Results and discussion

Positive control results:
The recent reliability check was performed in March/April 2012. The raw data of this study are kept in the BSL archives (BSL Project ID 120793).
The reliability checks are audited by the QA-unit periodically.
Positive-control substance: mercaptobenzothiazole, purity > 98%,
Fluka Chemica, Lot No. 41107195, expiry date: 20/01/2014
Concentrations: 2% induction I phase (in cottonseed oil)
25% induction II phase (in vaseline)
15% challenge (in Vaseline)
The sensitisation rate after application of the positive-control substance mercaptobenzothiazole (15% in vaseline)
was 100%, confirming the reliability of the test system.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Induction: 1% intradermal, 50% topical; Challenge: 12.5% topical
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Induction: 1% intradermal, 50% topical; Challenge: 12.5% topical
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Challenge: 12.5% topical
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Challenge: 12.5% topical
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Preliminary Test

1 animal was treated intradermally with concentrations of 1%, 1.5%, 2.5% and 5% of the test item (dissolved in physiological saline 0.9% NaCl).

1 animal was treated topically with concentrations of 50% (emulsified with vaseline)and 100% (undiluted) of the test itemfor 24 hours.

1 animal was treated topically with concentrations of 50% (emulsified with vaseline)and 100% (undiluted) of the test itemfor 48 hours.

1 animal was treated topically with concentrations of 12.5%, 25% (two application sites) and 50% of the test item (dissolved in physiological saline 0.9% NaCl)for 24 hours.

1 animal was treated topically with concentrations of 12.5%, 25% (two application sites) and 50% of the test item (dissolved in physiological saline 0.9% NaCl)for 48 hours.

Based on the results of this preliminary test, a concentration of 1% was chosen for the intradermal application of the main test and a concentration

of 50% was selected for the dermal induction. These concentrations caused slight signs of irritation, without leading to systemic effects.

A concentration of 12.5% was found to be the highest dose which did not cause any signs of irritation after a topical treatment over a period

of 24 hours and therefore was chosen for the challenge application in the main test.

Main Test

Signs of irritation during the induction:

Intradermal Induction I (24-hour reading):

Injection site 1:  erythema grade 1 in 5/5 control and 10/10 test animals, oedema grade 1 in 5/5 control and 10/10 test animals.

Injection site 2: erythema grade 1, oedema grade 1 and eschar in 10/10 test animals.

Injection site 3:erythema grade 1 in 5/5 control and 10/10 test animals, oedema grade 1 in 5/5 control and 10/10 test animals.

Intradermal Induction I (48-hour reading):

Injection site 1:erythema grade 1 in 5/5 control and 10/10 test animals, oedema grade 1 in 5/5 control and 10/10 test animals.

Injection site 2: erythema grade 1, oedema grade 1 and eschar in 10/10 test animals.

Injection site 3: erythema grade 1 in 5/5 control and 10/10 test animals, oedema grade 1 in 5/5 control and 10/10 test animals.

Dermal Induction II (48-hour exposure, occlusive):

Immediately after

removing the patch:     no signs of irritation in any of the test or control animals.

24 hours after

removing the patch:     no signs of irritation in any of the test or control animals.

Challenge Exposure (24-hour exposure, occlusive):

Neither erythema nor oedema was observed in any animal at any time of observation.

There was no evidence of sensitisation and the percentage of sensitised animals was 0%.

Table:  Challenge Exposure

Challenge Concentrations of Test Substance: 12.5%

 

Number of Animals Showing Skin Reactions after

24 hours

48 hours

Test Group

0

0

Negative-Control Group

0

0

Body Weight Development

The body weight development was within the biological range for all animals compared to historical data.

All animals of both groups survived throughout the test period.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The skin sensitization potential was investigated according to the OECD Guideline 406. No significant skin sensitization potential was found. No classification is warranted.
Executive summary:

The skin sensitization potential of Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts was investigated according to the OECD Guideline 406. No significant skin sensitization potential was found. No classification is warranted. The induction doses were 1 % and 50% for intradermal and epidermal respectively. The challenge was performed at the concentration of 12.5%. None of the treated 10 animals responded upon challenge. Based on the obtained result, Pentapropylensuccinic anhydride, reaction products with ethanolamine, sodium and triethanolamine salts is considered to be a non skin sensitizer.