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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-09-21 to 2016-09-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
06 July 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-piperazine-1,4-diyldiethanesulfonic acid
EC Number:
227-057-6
Cas Number:
5625-37-6
Molecular formula:
C8H18N2O6S2
IUPAC Name:
2,2'-piperazine-1,4-diyldiethanesulfonic acid
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/Ca Ola Hsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI COOP ZRT. Cserkesz u. 90., 1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals: SPF at arrival, good conventional hygienic level during test
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 16.9 - 20.4 g
- Fasting period before study: no
- Housing: group caging (4 mice/cage)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 30 - 70
- Air changes (per hr): not available
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
other: Aqueous 1 % (w/v) Pluronic®PE 9200 (solution of the test item in 1N NaOH followed by dilution with aqueous 1 % (w/v) Pluronic®PE 9200)
Concentration:
2.5, 5 and 10 %
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: No or not adequate solubility was observed with seven recommended vehicles at 2.5 % (w/v) concentration and above. Based on information originated from another test performed with the test item, the maximum solubility in 1N NaOH was evaluated. The test item was completely soluble in 1N NaOH at a maximum concentration of 25 % (w/v). Due to extreme pH (base) 1N NaOH alone cannot be applied on the ears of animals hence formulations by dilution with Plu (not pH adjusted), was evaluated. The highest adequate concentration (using the recommended concentration series) resulting in acceptable pH and adequate solubility of the test item was 10 % (w/v).
- Irritation: No sign of significant irritation (indicated by an erythema score ≥ 3) or any other local effect were observed for the three concentrations 2.5, 5 and 10 %.
- Systemic toxicity: No mortality, significant treatment related effect on the body weights or any other sign of systemic toxicity were observed for the three concentrations 2.5, 5 and 10 %.
- Ear thickness measurements: No increase of ≥ 25 % of ear thickness observed on any day of measurement were observed for the three concentrations 2.5, 5 and 10 %.
- Erythema scores: all 0 for all concentrations, mice and time points (days 1, 2, 3, 4, 5, 6)

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: animals were assigned randomly achieving body weight homogeneity between groups
- Criteria used to consider a positive response: The test item is considered as a skin sensitizer, if exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.

TREATMENT PREPARATION AND ADMINISTRATION:
Test item: see above for preparation.
Vehicle control for the test item: Based on results of the formulation evaluation 1N NaOH and Plu (not pH adjusted) mixed in an appropriate ratio (1:4) cannot be applied on the ears of animals due to unacceptable pH (approximately 12). According to this the vehicle control group animals were treated with Plu adjusted with 1N NaOH to a pH similar to the test item formulations (pH 6.59, 6.47, 6.22).
Vehicle for the positive control: AOO
Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance (25 % (w/v)) or the vehicles using a pipette, on the dorsal surface of each ear. After the treatments animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not specified.

Results and discussion

Positive control results:
The positive control group animals were treated with 25 % (w/v) HCA solution (formulated
in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.
Significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 9.3). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed validity of the assay.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
2.4
Test group / Remarks:
2.5 %
Key result
Parameter:
SI
Value:
0.5
Test group / Remarks:
5 %
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
10 %
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Disintegration per minute (per mouse (average)):
vehicle control: 450.8
2.5%: 1078.5
5.0%: 227.8
10%: 422.0


DETAILS ON STIMULATION INDEX CALCULATION: ratio of DPM/mouse of test concentrations and vehicle control

EC3 CALCULATION: Based on the results no EC3 value (dose calculated to induce a stimulation index of 3) was calculated for the test item.

CLINICAL OBSERVATIONS: No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score ≥ 3) or any other local effect were observed in any treatment group.

BODY WEIGHTS: No significant, treatment related effect on body weights was considered during the test. Body weights decreased by ≥ 5 % were observed in the AOO control group only (in 2 of 4 animals, 7 % or 5 % decrease) but it was considered not significant as no significant effect on the mean body weight has been observed.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be not a skin sensitizer.
Executive summary:

The study conducted according to OECD 429 evaluated the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. A formulation evaluation and a Dose Range Finding test (DRF) were performed to find an appropriate vehicle and the maximum applicable concentration according to the relevant guidelines. Solubility of the test item in vehicles preferred in the LLNA was evaluated. No adequate solubility was achieved with these vehicles, but the test item was completely soluble in 1N NaOH at a maximum concentration of 25 % (w/v). Due to extreme pH (base) 1N NaOH alone could not be applied on the ears of animals as vehicle control hence test item formulation was prepared by solution of the test item in 1N NaOH followed by dilution with aqueous 1 % (w/v) Pluronic®PE 9200 to achieve a concentration of 10 % (w/v). Lower test concentrations were diluted with aqueous 1 % (w/v) Pluronic®PE 9200. According to results of the DRF (where no adverse effect was observed up to this maximum concentration) the test item was examined in the main test as 10 %, 5 % or 2.5 % (w/v) formulations. Appropriate positive control (α-Hexylcinnamaldehyde, HCA) and its respective negative (vehicle) control (Acetone:Olive oil 4:1 (v/v) mixture, AOO) were also employed. Aqueous 1 % (w/v) Pluronic®PE 9200 adjusted with 1N NaOH to a pH similar to the test item formulations (Plu) was used as negative control for the test item. The positive control item (25 % (w/v) HCA in AOO) induced significant stimulation over the relevant control (SI = 9.3) thus confirming the validity of the assay. No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group. No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control was noted for the test item at the applied test concentrations. The observed stimulation index values were 0.9, 0.5 and 2.4 at test item concentrations of 10 %, 5 % and 2.5 % (w/v), respectively. No significant dose-response relationship was observed. Accordingly, the test item was considered to be not a skin sensitizer. In conclusion, under the conditions of the present assay, the test item formulated in 1 N NaOH and aqueous 1 % (w/v) Pluronic®PE 9200 and tested at the maximum feasible concentration of 10 % (w/v, based on solubility) and also at concentrations of 5 % or 2.5 % (w/v) was shown to have no skin sensitization potential in the Local Lymph Node Assay.