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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:
from 1998-07-13 to 1998-10-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study performed under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

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
GLP compliance:
yes
Type of study:
Buehler test

Test material

Constituent 1
Chemical structure
Reference substance name:
Diguanidinium carbonate
EC Number:
209-813-7
EC Name:
Diguanidinium carbonate
Cas Number:
593-85-1
Molecular formula:
CH5N3.1/2CH2O3 (one guanidine species, as denoted in the ESIS database) CH5N3.CH5N3.CH2O3 (as in the crystalline from, basis for the molecular weight 180.1658 g/mol as given below)
IUPAC Name:
bis[amino(imino)methanaminium] carbonate
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Guanidine Carbonate Pure
- Substance type: Salt with organic kation and inorganic anion
- Physical state: White crystalline powder
- Analytical purity: 99.7 %
- Impurities: Sulphate ash <0.05 %, heavy metals calculated as lead, <10 ppm (mg/L), iron 1 ppm, lead <1 ppm, arsenic <3 ppm
- Purity test date: 1998-06-09
- Lot/batch No.: 1867 of Agrolinz Melamin GmbH, St.-Peter-Strasse 25, A-4021 Linz
- Expiration date of the lot/batch: Not specified but considered stable under conditions of storage for at least 5 years
- Storage condition of test material: At room temperature in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstrasse 27, Box 1161, D-33176 Borchen.
- Age at study initiation: 6 weeks
- Weight at study initiation: 329 g to 419 g
- Housing: Makrolon cages type III (23 cm x 39 cm x 18 cm) with wire mesh lids, single caging.
- Diet (e.g. ad libitum): Altromin Standard Diet No. 3022, ad libitum, offered in stainless steel containers.
- Water (e.g. ad libitum): Tap water, acidified with HCl to pH = 3, offered in Makrolon bottles with stainless steel canules, ad libitum.
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 60
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
Deionised
Concentration / amount:
Preliminary study: 40 %, 20 %, 5 % and 1 % in deionised water
Main study: 40% in deionised water
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
Deionised
Concentration / amount:
Preliminary study: 40 %, 20 %, 5 % and 1 % in deionised water
Main study: 40% in deionised water
No. of animals per dose:
Preliminary study: 3 females
Main study: 20 females for test substance group, 10 females for control group
Details on study design:
RANGE FINDING TESTS:
All concentrations were derived from a preliminary study with 3 female guinea pigs (epicutaneous exposure, exposure time 6 hours, same mode of administration as in the definitive study). The test substance concentrations were: 40 %, 20 %, 5 % and 1 % in deionised water. 40 % of the test substance in deionised water was the highest technically feasible concentration. No adverse skin reactions were observed at any of these concentrations in any animal. Therefore it was decided to use 40 % for the epicutaneous induction and for the challenge exposure.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 h
- Test groups: test substance in deionised water
- Control group: deionised water
- Site: left flanks
- Frequency of applications: day 0, 7 and 15
- Duration: 0-21 day
- Concentrations: same throughout (40% (w/v) in deionised water)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 28
- Exposure period: 6 hours
- Test groups: posterior right flank: test substance (40 % (w/v) in deionised water), anterior right flank: deionised water
- Control group: posterior right flank: test substance (40 % (w/v) in deionised water), anterior right flank: deionised water
- Site: right flank
- Concentrations: same throughout (40% (w/v) in deionised water)
- Evaluation (hr after challenge): 24, 48
Positive control substance(s):
yes
Remarks:
HEXYL CINNAMIC ALDEHYDE

Study design: in vivo (LLNA)

Statistics:
After challenge exposure, the number of animals with skin reactions on the test substance treated areas was recorded. A skin reaction was regarded as "positive", if the test substance treated area gained a higher score than the control area of the same animal. The potential of the test substance to cause skin sensitisation was established by subtracting the percentage of animals exhibiting an inflammatory reaction in the control group from the percentage of positively reacting test substance group animals. Differences in the body weights between the test substance group and the control group on Days 0 and 30 were compared by the t-test (two sided), p = 0.05.

Results and discussion

Positive control results:
17/20 animals, i.e. 85 % of the animals of the positive control group, were regarded as sensitised. The results prove the sensitivity of the strain of animals used and the reliability of the experimental technique.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
other: any reading time
Group:
negative control
Dose level:
0.5 mL of 40% (w/v) solution
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: other: any reading time. Group: negative control. Dose level: 0.5 mL of 40% (w/v) solution . No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
other: any reading time
Group:
test chemical
Dose level:
0.5 mL of 40% (w/v) solution
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
other: Reading: other: any reading time. Group: test group. Dose level: 0.5 mL of 40% (w/v) solution. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None.

Any other information on results incl. tables

Skin reactions after induction exposures

Group K1 (negative control): No adverse skin reactions in any animal at any time.

Group B (test substance): Severe erythema and/or oedema after the third induction exposure in 1/20 animals.

Skin reactions after challenge exposure

These were the reactions of interest for the grading of the allergenic potency of the test substance.

Group Kl (negative control):

Vehicle area: no positive skin reaction in any animal at any reading time.

Test substance area: no positive skin reaction in any animal at any reading time.

No animal had a "positive skin reaction".

Group B (test substance):

Vehicle area: no positive skin reaction in any animal at any reading time.

Test substance area: no positive skin reaction in any animal at any reading time.

No animal had a "positive skin reaction".

Mortality

All animals survived till the end of the study.

Body weight

There were no significant differences in mean body weights between the test substance group and the control group on Days 0 and 30.

General observations

Immediately after the beginning of all exposures (inductions, challenges) the motor activities of all animals were increased. This is due to the dressings which restrict the freedom of movement. Soon afterwards the behaviour was normal again. Except for the observations described above no abnormal behaviour or clinical signs of the animals were detected.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Classification according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and CLP (Regulation (EC) No 1272/2008) is not required.
Executive summary:

The "Buehler test" of E.V. Buehler was performed to reveal a possible sensitising potential of Guanidine carbonate.

Twenty female guinea pigs were used as a test substance group and another 10 females were used as a negative control group. There were three epicutaneous induction exposures and one epicutaneous challenge exposure. The concentration of the test substance was 40 % (w/v) in deionised water for all three induction exposures and for the challenge exposure. The areas of administration were covered occlusively for 6 hours. Investigations performed were in conformance with the OECD TG 406.

All animals survived till the end of the study. Possible skin reactions excluded, no other adverse effects were noted. The test substance did not stain the skin of the guinea pigs and scoring was not impeded by skin dyeing. The application sites of all control animals were normal at each time. In the test substance group, erythema and/or oedema were noted in 1/20 animals after the third induction exposure.

In the negative control group, the control areas of all animals were normal 24 and 48 hours after the end of the challenge exposure.

All test substance treated areas of all animals of the negative control group and of the test substance group were also unaffected. No animal was regarded as irritated or sensitised.

Classification according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and CLP (Regulation (EC) No 1272/2008) is not required.

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