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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:
17 August 2006 to 15 September 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
17 July 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Directive 96/54
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
EPA 712-C-03-197
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
At the time of testing (2006) the OECD 406 was still an accepted OECD test method.

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
466-080-8
EC Name:
-
Molecular formula:
C23H31N3O4
IUPAC Name:
466-080-8
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Name: LFC 2098
Product/common name: LFC 2098
Purpose: Industrial chemical
Colour: white
Physical state: solid, powder
Purity: 99. 69%
Storage: at room temperature, protected from light
Molecular formula: C23H31O4N3
Molecular weight: 413
Safety precautions: 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:
10/5 (test/control) Hsd Poc : DH — guinea pigs (Full-Barrier) , Sex: female, nulliparous, nonpregnant, body weight at the commencement of the study 300 — 500 g.
5 (range finding) Hsd Poe : DH — guinea pigs (Full-Barrier) , Sex: female, nulliparous, nonpregnant, body weight at the commencement of the study 300 - 500 g.

A health inspection was performed to ensure the good state of health of theanimals:
The animals were derived from a controlled full barrier maintained breeding system (SPF).
Source: Harlan Winkelmann GmbH, D-33178 Borchen.
According to Art. 9.2, No.7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

Animal Husbandry

The animals were barrier maintained (semi—barrier) in an air conditioned room
— Temperature: 22 ± 3 °C
— Rel. humidity: 55 ± 10%
— Artificial light, sequence being 12 hours light, 12 hours dark
— Air change: at least 10 x / hour
— Feeding ad libitum, ssniff Ms-H, 4mm V2233-000 complete diet for guinea pigs, rich in crude fiber, totally pathogen-free (TPF)
— Free access to tap water (drinking water, municipal residue control, microbiol. controlled periodically)
— The animals were kept in groups in Terluran - cages on Lignocel bedding
— Certificates of food, water and bedding are filed at BSL Bioservice
— Adequate acclimatization period

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: 1:1 FCA/Saline
Concentration / amount:
0.1 ml
Day(s)/duration:
Day 1 / single injection
Adequacy of induction:
not specified
Route:
intradermal
Vehicle:
cotton seed oil
Concentration / amount:
2.5% / 0.1 ml
Day(s)/duration:
Day 1 / single injection
Adequacy of induction:
not specified
Route:
intradermal
Vehicle:
other: 1:1 FCA/Saline
Concentration / amount:
2.5% test material / 0.1ml
Day(s)/duration:
Day 1 / single injection
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
not specified
Concentration / amount:
0.5g test material
Day(s)/duration:
Day 7 / 48 hours
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
cotton seed oil
Concentration / amount:
0.5 ml
Day(s)/duration:
Day 7 / 48 hours
Adequacy of induction:
not specified
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.5g test material
Day(s)/duration:
Day 20 / 24 hours
Adequacy of challenge:
highest non-irritant concentration
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
cotton seed oil
Concentration / amount:
0.5 ml
Day(s)/duration:
Day 20 / 24 hours
Adequacy of challenge:
not specified
No. of animals per dose:
10 test
5 control
5 range-finder
Details on study design:
Induction: First Stage, Intruder-moi Injection
Three pairs of intraderrnal 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 rnidline.

Test group: Day 0
- Injection 1: a 1:1 mixture (v/v) FCA/physiological saline
- Injection 2: a 2.5% concentration of the test substance in Cotton Seed Oil
- lnj ection 3: a 2.5% concentration of the test substance formulated in a 1:1 mixture (v/v) FCA/physiological saline

Control group: Day 0
- Injection 1: a 1:1 mixture (v/v) FCA/physiological saline
- Injection 2: Cotton Seed Oil at a concentration of 100%
- Injection 3: a 50% (v/v) formulation of Cotton Seed Oil in a 1:1 (v/v) mixture FCA/physiological saline
- Injections l and 2 were given close to each other and nearest to the head, while 3 is given toward the caudal part of the test area.

Induction: Second Stage, Topical Application
- Test and Control Group: Day 6
Approximately twenty-four hours before the topical induction application the test area was close-clipped.
- Test Group: Day 7
A patch was fully loaded with 0.5 g of the prepared test item, applied to the test area and held in contact by an occlusive dressing for 48 hours.
- Control Group: Day 7
A patch was fully loaded with 0.5 mL of Cotton Seed Oil and 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 and Control Group : Day 20
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 mL Cotton Seed Oil to the right flank (intraspecific control), respectively. The patches were held in contact by an occlusive dressing for 24 hours.

Observation
Test and Control Group
Approximately 21 hours after removing the patch the challenge area was cleaned with tap water and cleared of hair by the use of a depilation creme.
Approximately 24 hours after removing the patch the skin reaction was observed and recorded according to the grades shown below. Two more observations were recorded 48 and 72 hours after patch removal.
Additionally all animals have been observed for signs of toxicity at least once daily during the test period.

Evaluation of Results
The percentage of animals sensitized in comparison to the negative control group and to the intraspecific control was calculated for each time of observation. The highest percentage was used for the rating of the test item.
Individual data were recorded in tabular form.
The body weights of the test- and control group were determined at the commencement and at the end of the study.

Reliability Check
The recent reliability check was performed in July/August 2006. The raw data of this study are kept in the ESL archives (B SL Project ID 062189). The reliability checks were audited by the QA-unit periodically.
Positive control substance: Mercaptobenzothiazole, Purity > 98%, CAS No. 149-30-4, Lot 835857 306, Merck
Concentrations:
- 2% induction 1 phase
- 25% induction ll phase
- 15% challenge

The sensitization rate after application of the positive control substance Mercaptobenzothiazole (15% in Vaseline) was 90 %, confirming the reliability of the test system.
Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole; 15% in Vaseline

Results and discussion

Positive control results:
See Table 2 below.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0.5g test material
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.5g test material
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
0.5g test material
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0.5g test material
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.5g test material
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0.5g test material
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
Mercaptobenzothiazole; 15% in Vaseline
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
Mercaptobenzothiazole; 15% in Vaseline
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
positive control
Dose level:
Mercaptobenzothiazole; 15% in Vaseline
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Table 2. Frequency of Sensitization in Positive Control Animals

10 animals used for positive control group

  E0 E1 E2 E3 E4 O0 O1 O2 O3 O4 % animals diseased
P24 1 7 2 0 0 10 - - - - 90
P48 3 7 0 0 0 10 - - - - 70
P72 9 1 0 0 0 10 - - - - 10

E = Erytherna

O = Oedema

0-4 = Grade

P: Positive Control group (Mercaptobenzothiazole; 15% in Vaseline)

24, 48, 72 hours after end of challenge treatment;

Table 3. Grade of Reactions in Preliminary

Test Skin Reactions after Inrtrademial Injection

Animal No. Conc. % (left/right) 24 hours 48 hours 72 hours
Erythema Oedema Erythema Oedema Erythema Oedema
1 2.5 1 0 0 0 1 0
2.5 1 0 0 0 1 0

Skin Reactions after Topical Application

Animal No. Conc. % (left/right) 24 hours 48 hours 72 hours
Erythema Oedema Erythema Oedema Erythema Oedema
10 (24 hour exposure) 100 0 0 0 0 0 0
50 0 0 0 0 0 0
12 (48 hour exposure) 100 0 0 0 0 0 0
50 0 0 0 0 0 0
11 (24 hour exposure) 100 0 0 0 0 0 0
50 0 0 0 0 0 0
13 (48 hour exposure) 100 0 0 0 0 0 0
50 0 0 0 0 0 0

Main Test

Induction readings:

1. Induction i: After 24 hours, at injection site 2 erythema grade 1 in 6 of 10, at injection site 3 erytherna grade l in 4 of 10 animals. After 48 hours, at injection site 2 erytherna grade 1 in 8 of 10, at injection site 3 erytherna grade 1 in 6 of 10 animal . No other signs of irritations were observed in any of the animals (Induction first stage).

2. No signs of irritation were observed in any of the animals after the topical application (Induction second stage).

Challenge readings:

The results of the experimental animals at the challenge phase were compared with the results of the control animals.

No signs of irritation were observed after the challenge.

The maximum percentage of animals sensitized was 0%.

For single data. see tables 4a and 4b.

Animals of both groups survived throughout the test period. Animals of the test group showed no reduced weight gain compared to historical data (table 5).

Table 4a. Frequency of Sensitization in Comparison to Control Animals Summary

  E0 E1 E2 E3 E4 O0 O1 O2 O3 O4 % animals diseased
T24 10 - - - - 10 - - - - 0
T48 10 - - - - 10 - - - - 0
T72 10 - - - - 10 - - - - 0
C24 5 - - - - 5 - - - -  
C48 5 - - - - 5 - - - -  
C72 5 - - - - 5 - - - -  

E = Erythema

O = Oedema

0-4 = Grade

T = Test group

C = Control group

24, 48, 72 hours after end of challenge treatment

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test material was not found to be a skin-sensitiser under the conditions of the test.
Executive summary:

On the basis of the test results given below and in conformity with the criteria given in Annex VI of the Directive, the substance should be not classified.

Species/strain: Hsd Poc : DH — guinea pigs (Full-Barrier)

Number of animals in test group: 10

Number of animals in negative control group: 5

Maximum concentration not giving rise to irritating effects in the preliminary test: 100%

Concentrations of test material and vehicle used at each stage of induction:

- First induction: 2.5% of the test itern, diluted in Cotton Seed Oil

- Second induction: 100% of the test item, moistened with Cotton Seed Oil

Concentrations of test material and vehicle used at each stage of challenge:

- 100% of the test itein, moistened with Cotton Seed Oil

Signs of irritation during the induction:

Induction 1: After 24 hours, at injection site 2 erythema grade 1 in 6 of 10, at injection site 3 erythema grade 1 in 4 of 10 animals. After 48 hours, at injection site 2 erythema. grade 1 in 8 of 10, at injection site 3 erythema grade 1 in 6 of 10 animal . No other signs of irritations were observed in any of the animals.

Induction 2: No signs of irritations were observed in any of the animals.

Number of animals showing skin reactions after
Challenge concentrations of test substance (%) 1st challenge 2nd challenge
  1st ch. 2nd ch. 24 h 48 h 72 h 24 h 48 h 72 h
Test group 10 - 0 0 0 - - -
Negative control group 10 - 0 0 0 - - -

Evidence of sensitization at each challenge: No treatment related effects were observed.

Other observations: No treatment related effects were observed.

Method (type of test): OECD 406 & 96/54/EC B.6

Considering the reported data of this sensitization test it can be stated that the test item LFC 2098 at the tested concentration caused no reactions identified as sensitization.