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Toxicological information

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:
08 October 2013 to 12 November 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as an unpublished report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This was an exception with regard to GLP and was reflected in the GLP compliance statement.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: soild
Details on test material:
- Name of test material (as cited in study report): Potassium dicyanoaurate
- Physical state: White solid.
- Storage condition of test material: Room temperature in the dark.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd., Oxon, UK
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: Individually housed in suspended soild floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): Food available ad libitum (2014C Tekland Global Rodent diet, Harlan Laboratories Ltd., Oxon, UK)
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h of light (6.00 to 18.00)/ 12 h of dark

IN-LIFE DATES: From: 08 October 2013 To: 12 November 2013

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
Preliminary test: 25, 10 and 5 % w/w
Main test: 5, 2.5 and 1 % w/w
No. of animals per dose:
Prelimirary test: 3 female mice, one per test concentation
Main test: 4 females per test item concentration
Details on study design:
RANGE FINDING TESTS:
- Design: The mice were treated by daily application of the test item at a concentrations of 25, 10 or 5 % w/w in dimethyl formamide, to the dorsal surface of each ear (25 µL per ear) for 3 consecutive days. The mice were observed twice daily on days 1-3 and once daily on days 4-6. Local skin irritation was scored daily. Any clinical signs of toxicity were recorded. The bodyweight was recorded on day 1 and on day 6. The thickness of each ear was measured using a Mitutoyo 547-300S gauge, pre-dose on day 1, post-dose on day 3 and 6. Mean ear thickness changes were calculated between time periods day 1 and 3 and days 3 and 6.
- Irritation: A mean ear thickness increase of equal to of greater than 25% was considered to indicate excessive irritation an limited biological relevance to the endpoint of sensitisation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT:
Groups of 4 mice were treated with the test item at concentrations of 5, 2.5 and 1 % w/w in dimethyl formamide. The mice were treated by daily application of the test item to the dorsal surface of each ear (25 µL per ear) for 3 consecutive days. Five days after the first topical application all mice were injected via the tail vein with 250 µL of PBS containing 3H-methyl thymidine (80 µCi/mL, specific activity 2.0 Ci/mmol) giving a total of 20 µCi to each mouse.
- Name of test method: 3H-methyl thymidine incorporation
- Observations: clinical observations performed twice daily on days 1-3 and daily on days 4-6. Bodyweight of each mouse was recorded on day 1 (prior to dosing) and day 6 (prior to termination).
- Terminal procedures: 5 hours after administration of 3HTdR all mice were sacrificed and the a single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish and the lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm for 10 min. The pellet was resuspended in 10 mL PBS and repelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5% Trichloroacetic acid (TCA). After approx. 18 h of incubation at 4 °C, the precipitates were recovered by centrifugation at 2100 rpm for 10 min., resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. the number of radioactive disintegrations per minute was measured using the Beckmann LS6500 scintillation system.
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if at least one concentration of the test item results in a three fold or greater increase in 3H-methyl thymidine incorporation compared to control values.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was freshly prepared as a solution in dimethyl formamide within 2 hours of being applied to the test system. It was assumed that the formulation was stable for this duration. The test item was applied once daily to the dorsal surface of each ear for three consecutive days. The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
ɑ-Hexylcinnamaldehyde tech., 85% was considered to be a sensitiser under the condition of the test. The simultation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group was 7.91.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control were 7.98, 8.61 and 9.88 for 1, 2.5 ans 5 % w/w, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegration per minute were 6695.68, 53399.16, 57665.65 and 66160.86 dpm for vehicle control, 1, 2.5 and 5 % w/w, respectively.

Any other information on results incl. tables

Table 1. Local skin irritation - Preliminary Screening Test

Concentration (% w/w) in dimetyhl formamide

Animal number

Local skin irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

left

right

left

right

left

right

left

right

left

right

left

right

25

S-1

0

0

0

0

0

0

-

-

-

-

-

-

10

S-2

0

0

0

0

0

0

0

0

0

0

0

0

5

S-3

0

0

0

0

0

0

0

0

0

0

0

0

Table 2. Measurement of Ear Thickness and Mean Ear Thickness Changes - Preliminary Screening Test

Concentration (% w/w) in dimethyl formamide

Animal number

Ear thickness measurement (mm)

Day 1

Day 3

Day 6

Pre-dose

Post-dose

left

right

left

right

left

right

25

S-1

0.21

0.20

0.23+

0.25+

-

-

Overall mean (mm)

0.21

0.24

-

Overall mean ear thickness change (%)

na

17.07

-

Concentration (% w/w) in dimethyl formamide

Animal number

Ear thickness measurement (mm)

Day 1

Day 3

Day 6

Pre-dose

Post-dose

left

right

left

right

left

right

10

S-2

0.23

0.23

0.22

0.24

0.31

0.31

Overall mean (mm)

0.23

0.23

0.31

Overall mean ear thickness change (%)

na

0.00

34.78

Concentration (% w/w) in dimethyl formamide

Animal number

Ear thickness measurement (mm)

Day 1

Day 3

Day 6

Pre-dose

Post-dose

left

right

left

right

left

right

5

S-3

0.21

0.23

0.21

0.22

0.22

0.24

Overall mean (mm)

0.22

0.22

0.23

Overall mean ear thickness change (%)

na

-2.27

4.55

+ - ear thickness increase possibly due to residual test item on the ears.

- - no data, animal dead

na-not applicable

Table 3. Disintegration per Minute, Disintegration per Minute/Node and Stimulation Index

Concentration (% w/w) in dimethyl formamide

dpm

Dpm/Node

Stimulation Index

Result

Vehicle control

6695.68

836.96

na

na

1

53399.16

6674.90

7.98

positive

2.5

57665.65

7208.21

8.61

positive

5

66160.86

8270.11

9.88

positive

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item, potassium dicyanoaurate, was considered to be a sensitizer under the conditions of the test.
Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of potassium dicyanoaurate in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The study was conducted according to the OECD Guideline 429 ('Skin sensitization: Local Lymph Node Assay') and EC method B42. The test was carried out according to GLP.

Materials and Methods

Following a preliminary test, the main test was conducted with groups of 4 mice treated with the test item at concentrations of 1, 2.5 and 5 % w/w in dimethyl formamide. The mice were treated by daily application of 25 µL of the test item to the dorsal surface of each ear for three consecutive days. A further group of four mice received the vehicle alone. Five days after the first topical application all mice were injected with PBS containing 3H -methyl thymidine.

Results

The radioactive disintegration per minute were 6695.68, 53399.16, 57665.65 and 66160.86 dpm for vehicle control, 1, 2.5 and 5 % w/w, respectively. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control were 7.98, 8.61 and 9.88 for 1, 2.5 and 5 % w/w, respectively. The test item, potassium dicyanoaurate, was considered to be a sensitizer under the conditions of the test.