Reaction mass of 2-hydroxy-3-[(1-oxodocosyl) oxy]propyltrimethylammonium chloride and 2-methylpentane-2,4-diol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15-29 November 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted in compliance with OECD Guideline No. 429: read-across substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

The Swiss GLP Monitoring Authorities (inspected on April 22, 25 - 29, 2005 and May 09 - 13, 2005 / signed on November 2005)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(2,3-dihydroxypropyl)trimethylammonium chloride

EC Number:

251-783-2

EC Name:

(2,3-dihydroxypropyl)trimethylammonium chloride

Cas Number:

34004-36-9

Molecular formula:

C6H16NO2.Cl

IUPAC Name:

1-Propanaminium, 2,3-dihydroxy-N,N,N-trimethyl-, chloride (1:1)

Test material form:

solid: particulate/powder

Details on test material:

- Description: White crystalline powder
- Storage condition of test material: At room temperature (range of 20 ± 5 °C), desiccate.
- Stability under test conditions: Stable under specified storage conditions

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 10 weeks (beginning of acclimatization)
- Weight at study initiation: 17.4-23.5 g
- Housing: Animals were individually housed in Makrolon Type 2 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Kliba 3433, batch no. 48/06 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum
- Water: Community tap water from Itingen, available ad libitum
- Acclimation period: 7 days
- Indication of any skin lesions: None

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30-70 %
- Air changes: 10-15 air changes per hour
- Photoperiod: 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.

IN-LIFE DATES: 15-29 November 2006

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

Main test: 2.5, 5, 10, 25 and 50 % w/v in propylene glycol

No. of animals per dose:

4

Details on study design:

MAIN STUDY

TEST ITEM FORMULATIONS PREPARATION
The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle propylene glycol was quantitatively added. The weight/volume (w/v) dilutions were prepared individually using a magnetic stirrer as homogenizer. Test item formulations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears. Homogeneity of the test item in the vehicle was maintained until start of treatment using an appropriate homogenizer.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, 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 S.I.
- Second, the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) 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 the test item at concentrations of 2.5 %, 5 %, 10%, 25 % or 50 % (w/v) in propylene glycol. The application volume, 25 µL, was spread over the entire dorsal surface (diameter 8 mm) 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 phosphate-buffered saline (PBS) containing 78.66 µCi/mL 3HTdR (equal to 19.7 µCi 3HTdR) by intravenous injection via a tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanized by inhalation of CO2 (dry ice). The draining lymph nodes were rapidly excised and pooled in PBS for each experimental group (8 nodes per group). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing twice with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to glass scintillation vials with 10 ml of 'lrga-Safe Plus' scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (dpm).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

Positive control results:

The S.I. of 2.4, 2.5 and 9.0 were determined with the α-hexylcinnamaldehyde at concentrations of 5 %, 10 % and 25 %, respectively, in acetone:olive oil, 4:1 (v/v). Therefore, α-hexylcinnamaldehyde was found to be a potential skin sensitizer in the LLNA tests and an EC3 value of 11.2 % was derived.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

2.5% w/v

Results 2

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

5% w/v

Results 3

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

10% w/v

Results 4

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

25% w/v

Results 5

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

50% w/v

Cellular proliferation data / Observations:

SIZE OF THE DRAINING LYMPH NODES
No abnormal findings were observed in the size or other physical features of the draining lymph nodes.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation Index (S.I.): 1.2, 0.9, 1.0, 1.3 and 1.4 at 2.5, 5, 10, 25 and 50 % w/v in propylene glycol, respectively.

EC3 CALCULATION
Calculation of the EC3 value was not performed because no test concentrations produced a S.I. of 3 or higher.

CLINICAL OBSERVATIONS:
VIABILITY / MORTALITY: No deaths occurred during the study period.
CLINICAL SIGNS: No clinical signs on the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS:
The body weights of the animals, recorded prior to the first application and prior to sacrifice, were within the range commonly recorded for animals of the strain and age.

Any other information on results incl. tables

Table 7.4.1/1: Results of skin sensitisation

Test item Concentration %(w/v)	Group	Measurement DPM	Calculation			Result
			DPM-BG^a	number of lymph nodes	DPM per lymph node^b	S.I.
-	BG I	11	-	-	-	-
-	BG II	14	-	-	-	-
-	CG 1	1763	1750	8	219	-
2.5	TG 2	2150	2137	8	267	1.2
5	TG 3	1536	1523	8	190	0.9
10	TG 4	1849	1836	8	230	1.0
25	TG 5	2345	2332	8	292	1.3
50	TG 6	2380	2367	8	296	1.4

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

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

a) =The mean BG was calculated from the average of BG I and BG II values.

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

The EC3 Value could not be calculated, since all SI’s were below 3.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, test substance is not classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP).

Executive summary:

A study was performed to assess the skin sensitisation potential of test substance in the CBA/CaOlaHsd strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

 

Five groups each of four female mice were treated daily with the test substance at concentrations of 2.5 %, 5 %, 10 %, 25 % and 50 %(w/v) in propylene glycol by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four female mice was treated with the vehicle propylene glycol only.

 

Five days after the first topical application the mice were injected intravenously into a tail vein with radio- labelled thymidine (3H-methyl thymidine, 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3HTdR measured in a β-scintillation counter.

All treated animals survived the scheduled study period. Neither clinical signs on the ears of the animals nor systemic findings were observed during the study period. No abnormal findings were observed in the size or other physical features of the draining lymph nodes.

 

The Stimulation Index (S.I) of 1.2, 0.9, 1.0, 1.3 and 1.4 were determined with the test substance at concentrations of 2.5 %, 5 %, 10 %, 25 % and 50 % (w/v), respectively, in propylene glycol.

 

The historical positive control, α-Hexylcinnamaldehyde was found to be a potential skin sensitizer in the LLNA tests and an EC3 value of 11.2 % was derived. The test system was therefore considered to be valid. 

 

Under the test conditions, test substance is not classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP).