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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:
6 June - 6 July 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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992-07-17
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
An appropriate Buehler test is available which would not justify conducting an additional LLNA due to animal welfare.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Reaction mass of basic (III) chromium sulfate with sodium sulfate and water
- State of aggregation: green powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratory Animal Breeders, 88353 Kißlegg, Germany
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals: only healthy animals exhibiting no clinical signs were used for the study. The animals were not vaccinated or treated against infections either before receipt, or during the adaptation or study period.
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: 331 - 397 g
- Housing (study period): groups of two or five animals per Noryl cages; bedding material: low-dust wood shavings (Rettenmaier & Söhne, GmbH & co. 73494 Rosenberg, Germany.
- Diet (ad libitum): "PROVIMI KLIBA 3420 - Maintenance Diet for Guinea Pigs" (Supplier: PROVOMI KLIBA AG)
- Water (ad libitum): tap water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Relative humidity: 40 - 70 %
- Air changes: ≥ 10/hr
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, semiocclusive
Vehicle:
physiological saline
Concentration / amount:
80 % test item formulation
Day(s)/duration:
Days 0, 7 and 14 (exposure duration: 6 hours)
Challenge
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
physiological saline
Concentration / amount:
80 % test item formulation
Day(s)/duration:
day 28 (exposure duration: 6 hours)
No. of animals per dose:
Test animals: 20 animals
Control: 10 animals
Details on study design:
RANGE FINDING TESTS:
- Induction:
In a dose range-finding test three concentrations (25, 50 and 80 %) and the vehicle were tested in each case on five guinea pigs. The suitable areas of the body were shaved 30 minutes before treatment. The patches loaded with 0.5 mL of the test item formulations or the vehicle were applied to each animal under occlusive conditions for 6 hours. At the end of the exposure period, the remaining test item was removed with sterile physiological saline solution. Twenty-one hours later the treated areas were shorn. The dermal reactions were evaluated 30 and 54 hours after the start of the application.
No signs of irritation were seen at concentrations of up to 80% (the highest tested) (6-hour occlusive application).
Based on the results of the dose range-finding study, the following concentration was selected for the inductions: 80%

- Challenge:
The challenge concentration was determined on 2 guinea pigs which were treated in the same manner as the control animals during the inductions. The concentrations (25, 50 and 80 %) used for this range-finding study were selected on the basis of the range-finding tests for the induction concentration.
The patches loaded with 0.5 mL of test item formulations or the vehicle were applied to each animal under occlusive conditions for 6 hours. At the end of the exposure period, the remaining test item was removed with sterile physiological saline solution. Twenty-one hours later the treated areas were shorn. The dermal reactions were evaluated 30 and 54 hours after the start of the application.
No signs of irritation were seen at concentrations of up to 80% (the highest tested) (6-hour occlusive application).
Based on the results of the dose range-finding study, the following concentration was selected for the challenge: 80%

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: three
- Frequency of applications: once per week
- Site: Thirty minutes or 24 hours prior to test formulation appplication the skin of the left flank was shaved.
The animals in the test item group were treated with a hypoallergic patch loaded with the test item applied to the left flank. The patches were held in place on the skin using "ORABAND"® adhesive plaster. In the case of the control group animals, a hypoallergic patch loaded only with the vehicle was applied to the left flank and fixed with a strip of "ORABAND"® adhesive tape for each of the inductions. The patches were removed after an exposure period of six hours, and any remaining test item was removed with sterile physiological saline solution.
The volume applied per animal was 0.5 mL
- Concentrations:
Test animals: 80% of test item formulation
Control animals: vehicle
The treatment areas were visually assessed 30 hours after initiation of exposure.

B. CHALLENGE EXPOSURE
- No. of exposures: one
- Site: backs and right flanks of the animals were shorn 24 hours prior to the challenge treatment. A hypoallergenic patch loaded with the 80% test item formulation was applied and fixed to the right flank of each animal in the control and test item group. As a control a patch loaded only with the vehicle was applied and fixed also to the right flank. The patches were held in place on the skin with a ORABAND self-adhesive tape for 6 hours.
The volume applied per animal was 0.5 mL.
At the end of the six-hours exposure period, the patches were removed and the remaining test item was rinsed away with sterile physiological saline solution.Twenty- one hours later the skin of the animals was shorn in the region of the treatment sites.
- Concentrations (test and control animals): 0% and 80%
- Evaluation (hr after challenge): 24 and 48 hours

C. OBSERVATIONS:
- clinical signs: at least once daily
- body weights: on day 1 before the first induction and after the last evaluation on day 31 in the control group and the test item group and at day 24 in the range-finding group.
Challenge controls:
Five control animals were challenged with 80% test material (in physiological saline) and vehicle alone.
Positive control substance(s):
yes
Remarks:
alpha hexyl cinnamic aldehyde (vehicle: physiological saline solution; epicutanous: 40 % test item; challenge: 20 % test item)

Results and discussion

Positive control results:
Challenge with 20% positive control (in saline) resulted in dermal effects in 9/20 animals (45% response).

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
80%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
80%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
80%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
80%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Reading:
other: challenge
Group:
positive control
Dose level:
20 % of the positive control
No. with + reactions:
9
Total no. in group:
20
Clinical observations:
Skin effects (grade 1) were observed in 9/20 animals.

Any other information on results incl. tables

- dermal reactions during the induction phase were limited to one animal following the second and third exposures (Grade 1 erythema, desquamation). Similar findings were not seen in this animal following challenge.

- clinical signs: appearance and behaviour of the test item group animals were not different from the control group.

- body weight: by the end of the study the mean body weight of the treatment group animals was in the same range than that of the control group.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The substance is not a skin sensitiser.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classification as skin sensitiser.
Executive summary:

The potential of the test material (a mixture of basic chromium sulphate, sodium sulphate and water) to induce delayed contact hypersensitivity (skin sensitisation) was investigated in a three-induction Buehler study using female Hartley guinea pigs. The concentrations of the test material used for induction and challenge applications were based on the results of a preliminary study. The results of an acceptable positive control study are also reported, confirming the sensitivity of the assay. Induction was performed on 20 test animals using 6 -hour semi-occlusive application of 0.5 ml test material (80% in physiological saline); a total of three applications were made over a three-week period. Ten control animals were similarly treated using vehicle. Two weeks following the final induction application, all test and control animals were challenged using a 6- hour semi-occlusive application of the test material (80%). Dermal reactions were assessed at 24 and 48 hours following patch removal. No dermal reactions were seen in test or control animals. No evidence of sensitisation was seen under the conditions of this study.

The substance tested in this study is considered to be a worse case based on its higher water solubility, dermal penetration and pH. The negative results of this study can therefore be confidently extrapolated to chromium (III) oxide.