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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 22 April to 14 May, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, with OECD guidelines
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The lymph nodes were resuspended in 1mL 5% trichloroacetic acid instead of 3mL 5% TCA on the day of preparation. Nevertheless, all macromolecules have been precipitated since values obtained for the vehicle controls are well within the historical control
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The lymph nodes were resuspended in 1mL 5% trichloroacetic acid instead of 3mL 5% TCA on the day of preparation. Nevertheless, all macromolecules have been precipitated since values obtained for the vehicle controls are well within the historical controls
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 17.5-21.0 g
- Housing: single
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

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


IN-LIFE DATES: From: To:
Vehicle:
propylene glycol
Concentration:
10, 25, 50 and 100% (w/v) in propylene glycol
No. of animals per dose:
4 per dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility:
- Irritation:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was 100% of the undiluted test item. The solutions were formulated in propylene glycol.To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 10, 25, 50, and 100 % on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index and data are compatible with a conventional dose response (allowing for either local toxicity or immunological suppression).


TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 10, 25, 50, and 100% (w/v) in propylene glycol. The application volume, 25 µl, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application, all mice were administered with 250 µl of 78.9 µCi/ml 3H-methyl thymidine (corresponds to 19.7 µCi 3HTdR per mouse) by intravenous injection via a tail vein.
Prior to the first application of the test item and prior to treatment with 3HTdR.the ear thickness was determined using a micrometer .
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital Sodium. The draining lymph nodes were rapidly excised and pooled per group. Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze. After washing two times with phosphate buffered saline, the lymph node cells were resuspended in 5 % trichloroacetic acid and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid and transferred to plastic scintillation vials with 10, 25, 50, and 100% ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
After the lymph nodes have been excised, both ears of mice were punched at the apical area using a biopsy punch. For each animal both punches were immediately weighed per animal using an analytical balance.
The proliferative response of lymph node cells is expressed as DPM/node and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations were calculated.
Positive control results:
Experiment performed in February 2008 gave the following results for alpha-Hexylcinnamaldehyde in acetone/olive oil 4:1:
Stimulation Index:
1.78 at 5%
1.84 at 10%
4.87 at 25%

EC3=15.7% (w/v)
Parameter:
SI
Remarks on result:
other: 0.93 at 10% 1.12 at 25% 0.72 at 50% 1.42 at 100%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 606.2 for control 565.1 at 10% 681.8 at 25% 435.1 at 50% 861.9 at 100%

Calculation and Results of Individual Data

Vehicle: propylene glycol

Test item concentration % (w/v)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

29

---

---

---

---

---

BG II

30

---

---

---

---

---

1

4879

4850

8

606.2

10

2

4550

4521

8

565.1

0.93

25

3

5484

5455

8

681.8

1.12

50

4

3510

3481

8

435.1

0.72

100

5

6925

6896

8

861.9

1.42

BG  =   Background (1 ml 5% trichloroacetic acid) in duplicate

1     =   Control Group

2-5 =   Test Group

S.I.  =   Stimulation Index

a)     =   The mean value was taken from the figures BG I and BG II

b)     =   Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
In this study the stimulation indices (S.I.) of 0.93, 1.12, 0.72 and 1.42 were determined with the test item at concentrations of 10, 25, 50 and 100% in propylene glycol. The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.The test item Triquat Monomer was found to be not a skin sensitiser under the described conditions.

Triquat Monomer is not classified as a skin sensitiser according to the criteria of Annex VI Directive 67/548/EEC and EU GHS
Executive summary:

In this study the test item Triquat Monomer dissolved in propylene glycol was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, 50, and 100%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. Local signs of irritation of the ear skin were not detected. Ear thickness or ear weights were not affected.

In this study Stimulation Indices (S.I.) of 0.93, 1.12, 0.72 and 1.42 were determined with the test item at concentrations of 10, 25, 50, and 100% in propylene glycol, respectively. The test item was not a skin sensitiser in this assay under the described conditions.

Triquat Monomer is not classified as a skin sensitiser according to the criteria of Annex VI Directive 67/548/EEC and EU GHS.

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

Concentrations of 10, 25, 50% in propylene glycol and the substance applied neat induced no increase of the SI above 3. The substance is not considered a sensitiser.


Migrated from Short description of key information:
A reliable LLNA study (OECD 429) showed no signs of sensitisation when tested at up to 100%.

Justification for selection of skin sensitisation endpoint:
Study in compliance with currtent EC/OECD guidelines, and GLP standards.

Respiratory sensitisation

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

There is no data from human observation indicating respiratory sensitisation among the worker population in the plant.

Furthermore, no significant inhalation exposure is expected to the substance during manufacture and use, either as vapours or aerosols. The substance is expected to have a limited volatility potential (Vapour pressure estimated to be 3062 Pa at 25 degree C, boiling point of 106.4 degree Celsius). No cutaneous sensitisation was observed which would trigger an alert for respiratory sensitisation.


Migrated from Short description of key information:
No specific data are available, and no observation report, and there are no alerts indicating a respiratory sensitisation potential.

Justification for classification or non-classification

Skin sensitisation:

A reliable LLNA study showed no cutaneous sensitisation.

Respiratory sensitisation:

No classifiation due to the lack of data. There are no specific alerts. Also, no significant exposure is expected to aerosols or vapours during manufacture and use. There has been no report of observations in human.