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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:
2012-02-13 to 2012-05-03
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 test material has detergent properties. Substances of this type regularly show false positive results in the LLNA in mice. Therefore another method was selected.

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-(octylimino)bisethanol
EC Number:
239-555-0
EC Name:
2,2'-(octylimino)bisethanol
Cas Number:
15520-05-5
Molecular formula:
C12H27NO2
IUPAC Name:
2-[(2-hydroxyethyl)(octyl)amino]ethan-1-ol

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
(preliminary test and main study: lot no. 0930), 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
(preliminary test: lot no. 110811, main study: lot no. 061211)
- 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)

Induction
Route:
intradermal and epicutaneous
Vehicle:
other: physiological saline 0.9% NaCl ; vaseline
Concentration / amount:
For the intradermal injection (induction - first stage), 0.05 g of the test item was dissolved in physiological saline 0.9% NaCl
to gain a final volume of 10 mL of a 0.5% solution (w/v).
For the topical application (induction – second stage), 2.5 g of the test item were dissolved in vaseline
to gain a final weight of 10 g of a 25% solution (w/w).
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: vaseline
Concentration / amount:
For the topical application (challenge), 0.2 g of the test item were dissolved in vaseline
to gain a final weight of 20 g of a 1% solution (w/w).
Day(s)/duration:
1
Adequacy of challenge:
not specified
No. of animals per dose:
10 test animals and 5 control animals
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 0.5% concentration of the test item in physiological saline 0.9% NaCl
Injection 3: a 0.5% concentration of the test item formulated in a 1:1 mixture (v/v) FCA/physiological saline 0.9% NaCl

Negative-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 physiological saline 0.9% NaCl in a 1:1 (v/v) mixture FCA/physiological saline 0.9% NaCl

Positive-Control Group: Day 0
Injection 1: a 1:1 mixture (v/v) FCA/physiological saline 0.9% NaCl
Injection 2: 2% mercaptobenzothiazole in cottonseed oil
Injection 3: a 50% (v/v) formulation of 2% mercaptobenzothiazole 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 close clipped.

Test Group: Day 7
The test item was dissolved in vaseline at a concentration of 25%. A patch was fully loaded with 0.5 g 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.

Negative-Control Group: Day 7
A patch was fully loaded with 0.5 g of vaseline. Then it was applied to the test area and held in contact with
the help of an occlusive dressing for 48 hours.

Positive-Control Group: Day 7
A patch was fully loaded with the positive-control substance at a concentration of 25%, applied to the test
area and held in contact by an occlusive dressing for 48 hours.

Challenge: Topical Application
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 vaseline at a concentration of 1%. A patch, loaded with 0.5 g of the prepared
test item was applied to the left flank of the animals and a patch loaded with 0.5 g 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.

Positive control group: day 20-22
A patch loaded with the positive-control substance at a concentration of 15% was applied to the left flank of the animals
and, a patch loaded with the vehicle to the right flank (intraspecific control), respectively. The patches were held in contact
by an occlusive dressing for 24 hours.
The application area was not rinsed.
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 vaseline at a concentration of 1%. A patch, loaded with 0.5 g of the prepared test item was applied to the left flank of the animals and a patch loaded with 0.5 g 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.
Positive control group: day 20-22
A patch loaded with the positive-control substance at a concentration of 15% was applied to the left flank
of the animals and, a patch loaded with the vehicle to the right flank (intraspecific control), respectively.
The patches were held in contact by an occlusive dressing for 24 hours.
The application area was not rinsed.
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole

Results and discussion

Positive control results:
There was only evidence of sensitisation at the positive-control animals and the percentage of sensitised animals was 100%.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0,5 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0,5 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
15 %
No. with + reactions:
5
Total no. in group:
5
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:
0,5 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
15 %
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
no
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Preliminary Test

1 animal was treated intradermally with concentrations of 5% and 2.5% of the test item (dissolved inphysiological saline 0.9% NaCl, AlleMan Pharma, lot no. 111036, expiry date: 09/2014).

1 animal was treated intradermally with concentrations of 1% and 0.5% of the test item (dissolved inphysiological saline 0.9% NaCl, AlleMan Pharma, lot no. 111036, expiry date: 09/2014).

1 animal was treated topically with concentrations of 100% and 50% of the test item (dissolved in vaseline, Euro OTC Pharma, lot no. 2912017-01, expiry date: 30/04/2013) for 24 hours.

1 animal was treated topically with concentrations of 100% and 50% of the test item (dissolved in vaseline, Euro OTC Pharma, lot no. 2912017-01, expiry date: 30/04/2013) for 48 hours.

1 animal was treated topically with concentrations of 25% and 12.5% of the test item (dissolved in vaseline, Euro OTC Pharma, lot no. 2912017-01, expiry date: 30/04/2013) for 24 hours.

1 animal was treated topically with concentrations of 25% and 12.5% of the test item (dissolved in vaseline, Euro OTC Pharma, lot no. 2912017-01, expiry date: 30/04/2013) for 48 hours.

1 animal was treated topically with concentrations of 6.25% and 3% of the test item (dissolved in vaseline, Euro OTC Pharma, lot no. 2912017-01, expiry date: 30/04/2013) for 48 hours.

1 animal was treated topically with concentrations of 6.25% and 3% of the test item (dissolved in vaseline, Euro OTC Pharma, lot no. 2912017-01, expiry date: 30/04/2013) for 24 hours.

1 animal was treated topically with concentrations of 2% and 1% of the test item (dissolved in vaseline, Euro OTC Pharma, lot no. 2912017-01, expiry date: 30/04/2013) for 24 hours.

Based on the results of this preliminary test, a concentration of 0.5% was chosen for the intradermal application of the main test and a concentration of 25% was selected for the dermal induction. These concentrations caused slight signs of irritation, without leading to systemic effects.

A concentration of 1% 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 negative- and positive-control and 10/10 test animals,
                                  oedema grade 1 in 5/5 negative- and positive-control and 10/10 test animals

Injection site 2:            erythema grade 1 in 5/5 positive-control animals,
                                  oedema grade 1 in 4/5 positive-control animals

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

Intradermal Induction I (48-hour reading):

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

Injection site 2:            erythema grade 1 in 5/5 positive-control animals,
                                  oedema grade 1 in 5/5 positive-control animals

Injection site 3:            erythema grade 1 in 5/5 negative- and positive-control and 10/10 test animals,
                                  oedema grade 1 in 3/5 negative-control, 5/5 positive-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 the negative-control and test animals at any observation time.

24 hours after removing the patch: Erythema grade 1 in 2 out of 5 positive-control animals and erythema grade 2 in 3 out of 5 positive-control animals at the test site.

48 hours after removing the patch: Erythema grade 1 in 2 out of 5 positive-control animals and erythema grade 2 in 3 out of 5 positive-control animals at the test site.

Eschar in 4 out 5 positive-control animals at the test site

Table3:  Challenge Exposure

Challenge Concentrations of Test Substance: 1%

 

Number of Animals Showing Skin Reactions after

24 hours

48 hours

Test Group

0

0

Negative-Control Group

0

0

Positive-Control Group
(15% concentration)

5

5

Body Weight Development

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

All animals of the test and both control groups survived throughout the test period.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
Under the conditions of the present study it can be stated that the test item Genamin 3920 caused no reactions identified as
sensitisation at the tested concentration.
According to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 2001/59/EC]) labelling is not necessary.
Executive summary:

On the basis of the test results given below and in conformity with the criteria given in Annex VI to Commission Directive 2001/59/EC the substance should be:

classified

 

  

 

unclassified

X

Daily clinical observations of the animals did not reveal any visible clinical symptoms. No mortality occurred.

Species/strain: Crl: HA - Guinea Pigs

Number of animals (test group): 10

Number of animals (negative-control group): 5

Number of animals (positive-control group): 5

The following concentrations were determined by the preliminary test:

For each induction the highest concentration to cause mild-to-moderate skin irritation, but well tolerated systemically, were used as follows:

Intradermal induction: 0.5% of the test item, vehicle:physiological saline 0.9% NaCl

Dermal induction: 25% of the test item, vehicle: vaseline

Challenge exposure (highest non-irritating dose): 1% of the test item, vehicle: vaseline

During the induction slight signs of irritation were observed. Those findings corresponded to the results of the preliminary test and confirmed the validity of the study.


Challenge Exposure:

Neither erythema nor oedema was observed in the negative-control and test animals at any observation time.

24 hours after removing the patch:

Erythema grade 1 in 2 out of 5 positive-control animals and erythema grade 2 in 3 out of 5 positive-control animals at the test site.

48 hours after removing the patch:

Erythema grade 1 in 2 out of 5 positive-control animals and erythema grade 2 in 3 out of 5 positive-control animals at the test site.

Eschar in 4 out 5 positive-control animals at the test site.

There was only evidence of sensitisation at the positive-control animals for the challenge and the percentage of sensitised animals was 100%.

Table1:  Challenge Exposure

Challenge Concentrations of Test Substance: 1%

 

Number of Animals Showing Skin Reactions after

24 hours

48 hours

Test Group

0

0

Negative-Control Group

0

0

Positive-Control Group
(15% concentration)

5

5

Method (type of test): OECD 406
EC 440/2008
OPPTS 870.2600