Cyclohexanaminium, N,N,N-trimethyl-, sulfate (2:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

bis(N,N,N-trimethylcyclohexanaminium) sulfate

EC Number:

807-085-0

Cas Number:

1004297-30-6

Molecular formula:

2 C9 H20 N .O 4S

IUPAC Name:

bis(N,N,N-trimethylcyclohexanaminium) sulfate

Constituent 2

Reference substance name:

Trimethylcyclohexylammonium sulfate, aqueous solution 50 wt.%

IUPAC Name:

Trimethylcyclohexylammonium sulfate, aqueous solution 50 wt.%

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Trimethylcyclohexylammonium sulfate, aqueous solution 50 wt.%
- Physical state: colorless, clear liquid
- Analytical purity: 51.8%
- Lot/batch No.: DENECKE00120

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V.; Host /The Netherlands
- Age at study initiation: 1st pre-test: 10 - 11 weeks (beginning of treatment)
2nd pre-test: 8-9 weeks (beginning of treatment) Main study: 11 - 12 weeks (beginning of treatment)
- Housing: group (Makrolon Type II / III)
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days prior

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): approx. 45-65 %
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

2, 5 and 10% (As the purity of the test item was approx. 50%, the actual tested concentrations were approx. 1, 2.5 and 5% (w/w))

No. of animals per dose:

5 females

Details on study design:

Methode:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2, 5, and 10% in DMF (actual concentration of 1, 2.5 and 5%). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 19.8 μCi of 3H-methyl thymidine (equivalent to 79.2 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.

Positive control substance(s):

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

Statistics:

A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations custom made statistical program ´R` Decision Tree was used. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test and the Grubb’s test were used for detection of possible outliers (performed with custom made statistical program ´R` Decision Tree).
However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in April 2014.

In vivo (LLNA)

Results

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

In this study Stimulation Indices (S.I.) of 1.11, 1.46, and 2.64 were determined with the test item at concentrations of 2, 5, and 10% (w/w) in DMF, respectively (actual concentration of approx. 1, 2.5 and 5% (w/w)). A clear dose response was observed.

Any other information on results incl. tables

Lymph Node Weights and Cell Counts

The measured lymph node weights and -cell counts of all animals treated were recorded after sacrifice. A statistically significant increase in lymph node weight was observed in the high dose group in comparison to the vehicle control group. A statistically significant increase in lymph node cell count was observed in the mid and high dose group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was exceeded in the high dose group (index of 1.72). The statistically significant increases were considered not to be biologically relevant, as none of the affected dose groups produced an S.I. above 3.

Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups exceeded this threshold.

Test item concentration*	Group Calculation
	Mean DPM per animal (2 lymph nodes)^a)	SD	S.I.
Vehicle Control Group (DMF)	957.0	191.3	1.00
2% Trimethylcyclohexylammonium sulfate, aqueous solution 50 wt.%	1064.2	279.8	1.11
5% Trimethylcyclohexylammonium sulfate, aqueous solution 50 wt.%	1400.8	255.0	1.46
10% Trimethylcyclohexylammonium sulfate, aqueous solution 50 wt.%	2528.0	449.8	2.64^S

a) Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

^S Mean DPM value for the group was according to the ANOVA (Dunnett-test) significantly higher than the corresponding control value.

* As the purity of the test item was approx. 50%, the actual tested concentrations were approx. 1, 2.5 and 5% (w/w).

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Applicant's summary and conclusion

Executive summary:

The test item Trimethylcyclohexylammonium sulfate, aqueous solution 50 wt.% was not a skin sensitiser under the test conditions of this study.