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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
Bühler test
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1973
Report Date:
1973

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
not applicable
Principles of method if other than guideline:
- Principle of test: Male and female guinea pigs were exposed to the test item (at the intended maximum concentration) and a vehicle control for 5 days (Monday through Friday) for 2 consecutive weeks (total of 10 treatments), a positive control for 5 days (Monday through Friday) for 1 week, and an untreated control. The induction period was followed by a 2 week observation period. After the observation period, a challenge test was performed with 1/10 of the induction dose for the test item and positive control groups. Previously untreated animals were also challenge tested in the same manner as the test item, vehicle control and positive control groups.
- Short description of test conditions: Experimental conditions were maintained at 23 to 33 °C with 45 to 73% relative humidity.
- Parameters analysed / observed: Systemic intoxiction symptoms, body weight, mortality, local changes
GLP compliance:
no
Type of study:
Buehler test
Justification for non-LLNA method:
The REACH Annex VII information requirements were revised in 2016 to endorse a battery of in vitro assays for skin sensitisation. Conducted prior to the validation and regulatory acceptance of alternative methods, the reliable and GLP compliant in vivo Buehler test was considered sufficient to fulfil the information requirement.

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
Pirbright-Hartley
Remarks:
White-EMD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 215 to 430 g (mean 333 g)
- Housing: Individually caged in Makrolon cages Type III.
- Diet: Altromin MS, ad libitum.
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 to 33 °C
- Humidity (%): 45 to 73 %

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, open
Vehicle:
peanut oil
Remarks:
DAB 7
Concentration / amount:
3 g test item + 100 mL vehicle
Day(s)/duration:
5 times per week (Monday through Friday) for 2 consecutive weeks, total of 10 times
Adequacy of induction:
other: intended maximum concentration
Route:
epicutaneous, open
Vehicle:
other: ether
Concentration / amount:
2% dinitrochlorobenzene
Day(s)/duration:
5 days (Monday through Friday)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, open
Vehicle:
peanut oil
Remarks:
DAB 7
Day(s)/duration:
5 times per week (Monday through Friday) for 2 consecutive weeks, total of 10 times
Adequacy of induction:
other: vehicle control
Route:
other: no treatment
Vehicle:
unchanged (no vehicle)
Adequacy of induction:
other: untreated control
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, open
Vehicle:
peanut oil
Remarks:
DAB 7
Concentration / amount:
0.3 g test item + 100 mL vehicle
Day(s)/duration:
Single application
Adequacy of challenge:
other: 1/10 of the previously used concentration
No.:
#1
Route:
epicutaneous, open
Vehicle:
other: ether
Concentration / amount:
0.2% dinitrochlorobenzene
Day(s)/duration:
Single application
Adequacy of challenge:
other: 1/10 of the previously used concentration
No.:
#1
Route:
epicutaneous, open
Vehicle:
peanut oil
Remarks:
DAB 7
Day(s)/duration:
Single application
Adequacy of challenge:
other: vehicle control
No.:
#2
Route:
epicutaneous, open
Vehicle:
peanut oil
Remarks:
DAB 7
Concentration / amount:
0.3 g test item + 100 mL vehicle
Day(s)/duration:
Single application
Adequacy of challenge:
other: 1/10 of the previously used concentration, applied on previously untreated animals
No.:
#2
Route:
epicutaneous, open
Vehicle:
other: ether
Concentration / amount:
0.2% dinitrochlorobenzene
Day(s)/duration:
Single application
Adequacy of challenge:
other: 1/10 of the previously used concentration, applied on previously untreated animals
No.:
#2
Route:
epicutaneous, open
Vehicle:
peanut oil
Remarks:
DAB 7
Day(s)/duration:
Single application
Adequacy of challenge:
other: applied on previously untreated animals
No. of animals per dose:
5 males and 5 females per treatment, positive control and vehicle control.
7 males and 8 females for the untreated control.
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 10 for test item and vehicle control, 5 for positive control.
- Exposure period: 2 weeks for test item and vehicle control, 1 week for positive control.
- Test groups: Test item and positive control.
- Control group: Vehicle control and untreated control.
- Site: Left flank.
- Frequency of applications: 5 times per week (Monday through Friday) for 2 consecutive weeks for test item and vehicle control, 5 times per week (Monday through Friday) for 1 week for positive control.
- Duration: Induction exposure was followed by 2 weeks of no treatment for the test item and vehicle control and 1 week of no treatment for the positive control.
- Concentrations: 3 g test item + 100 mL vehicle

B. CHALLENGE EXPOSURE
- No. of exposures: Single application.
- Day(s) of challenge: 1 day.
- Exposure period: 1 day.
- Test groups: Test item and positive control.
- Control group: Vehicle control and untreated control.
- Site: Right flank (previously untreated).
- Concentrations: 0.3 g test item + 100 mL vehicle
- Evaluation (hr after challenge): 3 hours

OTHER: Prior to the first treatment, the animals were shaved at the left flank. Shaving was repeated twice (Monday and Thursday) during the treatment period. The animals were weighed twice per week (Monday and Thursday) after the start of the trial for 2 weeks, including the following Monday.
Challenge controls:
Vehicle control
Positive control substance(s):
yes
Remarks:
dinitrochlorobenzene (2% solution in ether)

Results and discussion

Positive control results:
Marked erythema was observed after three treatments of the positive control group, which persisted throughout the remaining induction period. The symptoms disappeared after the first day of the observation period. A distinctly circumscribed, very marked erythema developed about 3 hours after the challenge application at the treated skin site in all animals. Previously untreated animals tolerated the challenge application with the positive control (at 1/10 of the induction dose) without irritation and reaction.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
3
Group:
test group
Dose level:
0.3 g test item + 100 mL peanut oil DAB 7
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
3
Group:
negative control
Dose level:
peanut oil DAB 7
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
3
Group:
positive control
Dose level:
0.2% dinitrochlorobenzene
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
distinctly circumbscribed, very marked erythema
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
3
Group:
other: test group (previously untreated)
Dose level:
0.3 g test item + 100 mL peanut oil DAB 7
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
3
Group:
other: negative control (previously untreated)
Dose level:
peanut oil DAB 7
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
3
Group:
other: positive control (previously untreated)
Dose level:
0.2% dinitrochlorobenzene
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Mortality and clinical abnormalities were not observed during the treatment and observation times. All animals gained considerable weight. No differences in local changes during the induction and observation period were observed between the test item treatment and vehicle control animals. No reaction was observed in the test item or vehicle control after the challenge application. Previously untreated animals tolerated the challenge application with the test item (at 1/10 of the induction dose) and vehicle without irritation and reaction.

Table 1. Body weight

 Group  Sex  Mean body weight (g)              Difference of final weight from starting weight (g)

 

 

 Day 0

 Day 3

 Day 7

 Day 10

 Day 14

 

 Test item  Male  372  397  429  457  486  +144
 Test item  Female  330  341  360  376  402  +72
 Positive control  Male  255  298  326  361  398  +143
 Positive control  Female  249  280  319  337  371  +122
 Vehicle control  Male  401  434  471  508  547  +146
 Vehicle control  Female  324  349  385  406  438  +144
 Untreated control  Male  344  383  424  445  466  +122
 Untreated control  Female  416  448  480  506  526  +110

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
No differences in local changes during the induction, observation period or challenge test were observed in animals treated with the test item.
Executive summary:

Skin sensitisation of the test item was tested in male and female Pirbright-White-EMD guinea pigs using a Buehler test. Guinea pigs were exposed to the test item (3 g test item in 100 mL peanut oil DAB 7, intended maximum concentration) and a vehicle control (peanut oil DAB 7) for 5 days (Monday through Friday) for 2 consecutive weeks (total of 10 treatments), a positive control (2% dinitrochlorobenzene in ether) for 5 days (Monday through Friday) for 1 week, and an untreated control. The induction period was followed by a 2 week observation period. After the observation period, a challenge test was performed with 1/10 of the induction dose for the test item and positive control groups. Previously untreated animals were also challenge tested in the same manner as the test item, vehicle control and positive control groups. Mortality and clinical abnormalities were not observed during the treatment and observation times. All animals gained considerable weight. No differences in local changes during the induction and observation period were observed between the test item treatment and vehicle control animals. No reaction was observed in the test item or vehicle control after the challenge application. Previously untreated animals tolerated the challenge application with the test item and positive control (at 1/10 of the induction dose) and vehicle without irritation and reaction. In the positive control, marked erythema was observed after three treatments, which persisted throughout the remaining induction period. The symptoms disappeared after the first day of the observation period. A distinctly circumscribed, very marked erythema developed about 3 hours after the challenge application at the treated skin site in all animals. This study is considered to be reliable with restriction (Klimisch 2) as it was conducted similar to OECD Guideline 406, however there were minor limitations in experimental design and reporting.