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Description of key information

LLNA (WoE): Skin sensitisation was studied using Local Lymph Node Assay (LLNA) carried for the target substance. The study result for the test substance was inconclusive.

HIRPT (WoE): In this study the test substance was not shown to have sensitisation potential.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
Human Repeated Insult Patch (HRIPT)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13/09/2006-05/12/2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
The LLNA study conducted resulted in non-conclusive outcome.
Qualifier:
no guideline available
Principles of method if other than guideline:
Marzulli-Mailbach method was used to carry out Human Repeated Insult Patch test to study sensitization potential of a product in human volunteers. The test item was applied in maximized conditions (occlusion or semi-occlusion) during 3 weeks (Induction phase). After two-week rest phase, the products are reapplied on the same sites (Challenge phase). If the product is allergenic, it induces a cutaneous reaction during the challenge phase.
GLP compliance:
yes
Remarks:
Standards of Good Clinical Practice Guidelines according to guidelines of ICH
Type of study:
other: Marzulli-Maibach method
Reading:
other: Day 36
Hours after challenge:
0
Group:
test chemical
No. with + reactions:
0
Remarks on result:
no indication of skin sensitisation
Reading:
other: Day 37
Hours after challenge:
24
Group:
test chemical
No. with + reactions:
0
Remarks on result:
no indication of skin sensitisation
Reading:
other: Day 38
Hours after challenge:
48
Group:
test chemical
No. with + reactions:
0
Remarks on result:
no indication of skin sensitisation
Reading:
other: Day 39
Hours after challenge:
72
Group:
test chemical
No. with + reactions:
0
Remarks on result:
no indication of skin sensitisation
Reading:
other: Day 40
Hours after challenge:
96
Group:
test chemical
No. with + reactions:
0
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
In the Human Repeated Insult Patch (HRIPT) study no signs of sensitizing potential of test item, Cyperus scariosus, ext was detected.
Executive summary:

Marzulli-Mailbach method was used to carry out  Human Repeated Insult Patch test to study sensitization potential of test item in 100 human volunteers.The application of 15% of the Cyperus scariosus, ext. item did not cause signs of sensitization.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
21 February 2003 - 9 April 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Nulliparous, non-pregnant females. 28 animals. 9 weeks old. Fed with A04 C pelleted diet. Free access to filtered tap water.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 5, 10, 25, 50 and 100%
No. of animals per dose:
Four
Details on study design:
Administration of the dosage forms:

On days 1, 2 and 3, a dose-colume of 25 ul of the control or dosage form preparations was apllied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to esure an optimized application of the test material, the animals were placed under light isoflurane anesthezia during administration.

Clinical examinations:

The animals were observed at least once a day during the study for clinical signs, signs of morbidity or mortality. Animals were weighed individually on the first day and on the day of sacrifice. Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responces.
On days 1, 2 and 3 (before application) as well as on day 6 (after sacrifice), the thickness of the left ear of each animal of the negative control and treated groups was measured using micrometer. Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (coloration, presence of residual test item etc.) was noted.

Lymp node cell proliferative responses were measured after intravenous injection of 3H-TdR and sampling of auricular lymph nodes as described by Kimber and Dearman (1991) (referred also in the OECD 429 test guideline).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
In the positive control group given HCA at the concentration of 25%, an increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI=4.82) were noted.
Key result
Parameter:
SI
Value:
0.48
Test group / Remarks:
Concentration 5%
Key result
Parameter:
SI
Value:
0.75
Test group / Remarks:
Concentration 10%
Key result
Parameter:
SI
Value:
2.66
Test group / Remarks:
Concentration 25%
Parameter:
SI
Value:
7.72
Test group / Remarks:
Concentration 50%
Parameter:
SI
Value:
7.09
Test group / Remarks:
Concentration 100%
Key result
Parameter:
EC3
Value:
27
Test group / Remarks:
Concentrations 5, 10, 25, 50, 100%
Cellular proliferation data / Observations:
The quantitiy of cells obtained in each group was satisfactory and the cellularity correlated with incorporation of 3H-TdR. The cell vaibiility was higher than 80% in each group.

In the positive control group given HCA at the concentration of 25%, an increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI=4.82) were noted, The study was therefore valid.

In the treatment group, a dose-related increase in hte stimulation index was observed and the threshold positive value of 3 for the SI was reached from the concentration of 50%, shown to be moderately irritant. The EC3 value for the test item, Cyperus scariosus, ext., is equal to 27%.

As It is well known that irritancy may be involved in false positive lymphoproliferative response, the positive results observed at the concentrations of 50 and 100% could not be taken into account to conclude on the potential of the test item to induce delayed contact hypersensitivity.

A noteworthy lymphoproliferative response (SI=2.66), but which did not reach the threshold positive value of 3, was noted at the concentration of 25%, showing non-irritant. Therefore, it is not possible to definitely conclude on the potential of the test item to induce delayed contact hypersensitivity. Further assessments of concentrations around 25% would be necessary to conclude on this potential.

In evaluation of cutaneous reactions, a discrete erythema was recorded on day 6 in 3/4 animals received test item at concentration of 100%. Dryeness of the skin, sometimes together with crusts, was noted on day 6 in all animals give the test item at concentrations of 50 and 100%. A moderate increase in ear thickness was also observed in these animals, showing the irritant potential of the test item at the concentrations ≥ 50%.

Interpretation of results:
study cannot be used for classification
Conclusions:
A noteworthy lymphoproliferative response (SI=2.66) was noted at the concentration of 25%, showing no irritant effect. A dose-related increase in the stimulation index (SI) was observed and the threshold positive value of 3 for the SI was reached from the concentration of 50% shown to be moderately irritant. It is well known that irritancy may be involved in false positive lymphoproliferative response. Thus, under the experimental conditions, it is not possible to definitely conclude on the potential of the test item, Cyperus scariosus, ext. to induce delayed contact hypersensitivity.
Executive summary:

The potential of Cyperus scariosus, ext. to induce delayed contact hypersensitivity was evaluated in murine Local Lymph Node Assay (LLNA).

Twenty-eight female CBA/J mice were allocated to seven groups of four animals each:

  • five treated groups received test item at the concentrations of 5, 10, 25, 50 and 100%
  • one negative control group of four animals received the vehicle (mixture acetone/olive oil) /4/1, v/v)
  • one positive control group received alpha-hexylcinnamic aldehyde (HCA), a moderate sensitizer, at the concentration of 25%

The test item, vehicle or control item were applied over the ears (25 ul per ear) for three consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of the lymph node cells in the lymph node draining the application site was measure by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI).

The irritation potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

No mortality and no clinical signs were observed during the study.

A discrete erythema was recorded on day 6 in 3/4 animals received test item at concentration of 100%. Dryeness of the skin, sometimes together with crusts, was noted on day 6 in all animals give the test item at concentrations of 50 and 100%. A moderate increase in ear thickness was also observed in these animals, showing the irritant potential of the test item at the concentrations≥ 50%.

The EC3value for the test item is equal to 27%. In the treated groups, a dose-related increase in the stimulation index (SI) was observed and the threshold positive value of 3 for the SI was reached from the concentration of 50% shown to be moderately irritant.

As it is well known that irritancy may be involved in false positive lymphoproliferative response, the positive results observed at the concentrations of 50 and 100% could not be taken into account to conclude on the potential of the test item to induce delayed contact hypersensitivity.

A noteworthy lymphoproliferative response (SI=2.66), but which did not reach the threshold positive value of 3, was noted at the concentration of 25%, showing non-irritant. Therefore, it is not possible to definitely conclude on the potential of the test item to induce delayed contact hypersensitivity. Further assessments of concentrations around 25% would be necessary to conclude on this potential.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In vitro studies to study sensitisation of Cyperus scariosus, ext. were not conducted, since a murine Local Lymph Node Assay (LLNA) was available (Klein, E. 2004).

In LLNA, the test item was applied for three consecutive days. After 2 days of resting, the proliferation of the lymph node cells in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine. The obtained values were used to calculate stimulation indices (SI).

The irritation potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

The EC3 value for the test item is equal to 27%. A noteworthy lymphoproliferative response (SI=2.66), but which did not reach the threshold positive value of 3, was noted at the concentration of 25%, showing no irritant effect.

A dose-related increase in the stimulation index (SI) was observed and the threshold positive value of 3 for the SI was reached from the concentration of 50%, shown to be moderately irritant. 

In summary, based on the LLNA assay it is not possible to conclude on the potential of the test item to induce delayed contact hypersensitivity.

For a second study, Human Repeated Insult Patch test to study sensitization potential of a product in human volunteers using Marzulli-Mailbach method. The test item was applied in maximized conditions (occlusion or semi-occlusion) during 3 weeks (Induction phase). After two-week rest phase, the products are reapplied on the same sites (Challenge phase).

In the Human Repeated Insult Patch (HRIPT) study no signs of sensitizing potential of test item, Cyperus scariosus, ext was detected.

Justification for classification or non-classification

The substance is not classified for skin sensitisation according to the CLP Regulation No.1272/2008.