N-(cyclohexylthio)phthalimide

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Remarks:

Statement of GLP compliance No. G-026

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

N-(cyclohexylthio)phthalimide

EC Number:

241-774-1

EC Name:

N-(cyclohexylthio)phthalimide

Cas Number:

17796-82-6

Molecular formula:

C14H15NO2S

IUPAC Name:

N-(cyclohexylthio)phthalimide

Details on test material:

Duslin, product of Duslo, a.s.
Test batch No.: 041/07
Active substance: 99,42 % weight
Weight loss: 0,08 % weight
Toluene insolubles: 0,05 % weight
Ash: 0,008 % weight
Melting point: first liquid 92,3°C
complete liquid 92,5 °C
Sieve retention: 2,0 mm 0 %
0,5 mm 0 %

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

The animals (8-12 weeks old, 18-20 g) were housed individually in plastic cages T I (Velaz Praha). The animals (5 pre group) were marked by serial numbers 1-25. These numbers were placed on the cage together with the marking of group.
Housing
The mice were housed according to SOP 002/53204/07 Mice.
Diet
The standard diet MP (TOP DOVO) was served. The supplier of the diet is approved by State Veterinary and Food Administration SR.
Water
Ad libitum.
Environment
Environmental controls for the animal room were set to maintain 22±2°C, a relative humidity of 55 ±10% a minimum of 10 air changes/hour, and a regulated light regime 12/12 (SOP 013/53204/07 Climatic condition in exp. animal house).

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

epicutaneous, open

Vehicle:

other: acetone/olive oil

Induction 2

Concentration / amount:

The test substance in concentrations of 5, 1 and 0,5% was applicated in volume of 25 µl to the dorsum of each ear. as positive control α-Hexylcinnamaldehyde in concentration 25%, in the same volume was used. The vehiculum as a control in volume 25 µl was applicated.

Challenge

Challenge 1

Route:

epicutaneous, open

Vehicle:

other: acetone/olive oil

Challenge 2

Concentration / amount:

The test substance in concentrations of 5, 1 and 0,5% was applicated in volume of 25 µl to the dorsum of each ear. as positive control α-Hexylcinnamaldehyde in concentration 25%, in the same volume was used. The vehiculum as a control in volume 25 µl was applicated.

No. of animals per dose:

15 females - test substance
5 females - control
5 females - positive control

Details on study design:

Test procedure
Day 1:
Each animal was indetified and the body weight was recorded (SOP G 008/52000/07). To the dorsum of each ear was applicated 25 αl of the appropriate dilution of the test substance, or the vehicle alone.
Days 2 and 3:
The application procedure carried out on day 1 was reeated.
Days 4 and 5:
No treatment.
Day 6:
The body weight of each animal was recorded. 25 µl of phosphate-buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine into all test and control mice via the tail vein was injected.
Five hours later, the animals were killed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach).
Clinical observations
Animals were carefully observed once daily for any clinical signs, either of local irritation at the application site or of systemic toxicity. All observations were systematically recorded with individual records being maintained for each animal.
The clinical observation were scored as o (no effect), + (weak effect), ++ (moderate effect), +++ (strong effect).
Preparation of cell suspensions
Cell suspension of lymph node cell from pooled treatment groups was prepared by gentle mechanical desagregation by glass homogenizer. Lymph node cells were washed with an excess of PBS and centrifuged (SOP 056/211/03) by 600 g at 4°C for 10 min. Suspension of cells were precipitated with 5% trichloroacetic acid (TCA) at 4 °C for 18-20 h. Pellets were centrifuged by 2000 g at 4°C for 5 min., re-suspended in 1 ml TCA and transfered to scintillation vials containing 10 ml of scintillation fluid for 3H-counting.
Determination of cell proliferation (incorporated radioactivity)
Incorporation of 3H-methyl thymidine into the lymph node cells was measured by β-scintillation counting on Liquid scintillation analyzer TRI-CARB, 2000CA, Packard (SOP 023/53202/07) as disintegrations per minute (DPM). The incorporation was expressed as DPM/treatment group.
Calculation of SI and EC3 values
The SI value was obtained by dividing the pooled radioactive incorporation for each treatment group by the incorporation of the pooled vehicle control group.
The concentration eliciting a SI of 3 is identified as the EC3 value and is calculated by linear interpolation of points on the dose-response curve, immediately above and below threefold the threshold having the coordinates (a,b) and (c,d) according to the equation : EC3 = c+[(3-d)/(b-d)]x(a-c).

Challenge controls:

5 females - control
5 females - positive control

Positive control substance(s):

yes

Remarks:

α-Hexylcinnamaldehyde

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0,5%, 1% and 5% Duslin

No. of animals per dose:

5 females

Positive control substance(s):

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

Results and discussion

Positive control results:

Clinical observations
Animals were carefully observed for any clinical signs, either of local irritation at the application site or of systemic toxicity.
The daily clinical observation of the animals did not show any visible clinical symptoms.
Body Weights
The animal body weights were measured at the start of the test and at the scheduled kill. In course of the experiment the increase of mean body weight was observed in group treated at concentration 5% (from 24,04 to 24,49 g) and slight decrease were observed in animal groups which were treated at concentrations 1 and 0,5% of tested substance (difference between initial mean body weight and terminal body weight was 0,04 and 0,28 g resp.) This suggested, that the test substance slightly affected the food intake of animals only at lowest used concentration. The individual and mean values of animal body weights of the control group, positive control group and three test substance groups are shown in tab.1
Lymph node proliferation
After application of Duslin the dose dependent increase of the lymph node weights was registered. The pooled lymph node weights of treated groups were 0,0394 g for 0,5% concentration, 0,042 g for 1% concentration and 0,0855 g for 5% concentration of tested substance. The lymph node weight of control group was 0,0288 g and for positive control group was 0,0388 g. The DPM values for treated groups were 923.1, 1166,3 and 3858,9. The positive responsens, with SI value 3,27 and 10,83 resp. were registered after application of test substance at concentration 1 and 5 % resp. The calculated EC3 value was 0,8 %. The lymph node weight, SI values and EC3 value are shown in table 2.

In vivo (LLNA)

Results

Results 1

Parameter:

other: Migrated information from in vivo LLNA study

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 5.0. Group: positive control.

Results 2

Parameter:

other: Migrated information from in vivo LLNA study

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 5.0. Group: test group. Dose level: 1,0%, Duslin. No with. + reactions: 5.0. Total no. in groups: 5.0. Clinical observations: The daily clinical observation of the animals did not show any visible clinical symptoms.

Results 3

Parameter:

other: Migrated information from in vivo LLNA study

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 5.0. Group: test group. Dose level: 5% Duslin. No with. + reactions: 5.0. Total no. in groups: 5.0. Clinical observations: The daily clinical observation of the animals did not show any visible clinical symptoms..

Results 4

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

SI EC3 Control: - - Pos. Control: 3,15 - Duslin 5%: 10,83 0,80 Duslin 1%: 3,27 - Duslin 0,5%: 2,59 -

Results 5

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Control: 356,2 Pos. Control: 1122,4 Duslin 5%: 3858,9 Duslin 1%: 1166,3 Duslin 0,5%: 923,1

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information sensitising potency, moderate allergen