Iodine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2010-07-20 to 2010-08-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

yes

Remarks:

The test article characterization was not conducted according to the Good Laboratory Practices. This is not expected to have any impact on the study.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Iodine

EC Number:

231-442-4

EC Name:

Iodine

Cas Number:

7553-56-2

Molecular formula:

I2

IUPAC Name:

iodine

Specific details on test material used for the study:

Batch No.: Pallets 38361-38380
Purity: 99.8%

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratory
- Age at study initiation: Screening (9 wks old), Quantitative Irritation Testing (10 wks old) and Main test (11 wks old)
- Weight at study initiation: 20.4 - 21.2 g for screen animals, 20.3 - 23.1 g for quantitative irritation test (QIT) animals, 21.2 - 25.5 g formain study animals
- Housing: Individual suspended wire-bottom cages
- Diet (e.g. ad libitum): PMI Rodent Chow ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: at least 5 days prior to start

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 44-76
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

1, 0.5, and 0.25%

No. of animals per dose:

5 mice per group

Details on study design:

Dosing:
Preliminary Dermal Irritation Screen: Three groups of CBA/J mice (2 animals per group) were treated with increasing concentrations of test item, (5%, 10% and 25%) in DMSO. Treatment was made by topical application of the test article concentrations to the dorsum of each ear once daily for three consecutive days (at approximately the same time each day). The test substance was spread over the entire dorsal surface of the ear using a micropipette at 25μL/ear.
Quantitative Irritation Test (QIT): Six groups of two CBA/J mice per group were treated with six concentrations (0.05%, 0.1 %, 0.25%, 0.5%, 1 % and 2.5%) of the test article in DMSO in the same manner as in the irritation screening study.
Main Test: Five groups of CBA/J mice (5 animals per group) were treated by topical application of the test article concentrations (0.25%, 0.5% and 1.0%), vehicle control or positive control in the same manner as in the screen.
Type and Frequency of Observations:
AII animals in the study were observed once daily throughout the study for clinical signs, either of local irritation at the application site or systemic toxicity, and for mortality
Measurements:
Body weights were recorded on Day 1, immediately prior to dosing, and on Day 6 (prior to sacrifice in the screen, and prior to BrdU injection in the main test). Ear thickness measurements were performed on Day 1 prior to dosing, on Day 3 before the third test article application (approximately 48 hours after the first test article application), and on Day 6 before sacrifice (approximately 120 hours after the first dose and 72 hours after the third dose). Changes in ear thickness on Day 3 and Day 6 relative to Day 1 were expressed as a percent of the Day 1 pre-dose values. Ear thickness increases of 25% or more were considered biologically significant (based on the scientific literature and historical laboratory data) and deemed indicative of a greater than moderate local dermal irritation response.
Node Isolation and Processing:
At 5 hours pre-sacrifice, mice were intraperitoneally injected 200μL of phosphate-buffered saline (PBS) containing BrdU. At sacrifice, each mouse lymph node set were excised and a single-cell suspension of lymph node cells was prepared.

Positive control substance(s):

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

Statistics:

For each test group, the individual animal stimulation index (SI) values, along with the mean group SI and standard deviation were calculated. Student's t-Test were run to statistically compare each group to the control group.

Results and discussion

Positive control results:

alpha-hexyl cinnamic aldehyde stimulation index (SI): 6.4

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Test group / Remarks:

0.25% group

Results 2

Parameter:

SI

Value:

1.8

Test group / Remarks:

0.5% group

Results 3

Parameter:

SI

Value:

2.2

Test group / Remarks:

1% group

Cellular proliferation data / Observations:

Test Article Formulation and Dosing: The test article formed a homogenous solution in the vehicle, DMSO. No difficulties were experienced with the application of the test article to the ears or with the retention of test article by the ear surface. However, in the screen, test material appeared to stain the ears and hair at the dose sites beginning on Day 2. This did not affect ear measurements.
Body Weights: Body weight changes were noted but were not significant (<2 g).
Mortality and Systemic Observations: AII animals survived the in-life phase of the study and were observed to be normal.

Any other information on results incl. tables

Irritation Screening

Table 1: Results of the initial test irritation

Ear Measurements			Day 1		Day 3		Day 6		Mean Ear Thickness (mm)			%Difference
		AN #	Left	Right	Left	Right	Left	Right	Pre-Dosing (Day 1)	48 Hr (Day 3	End In-Life (Day 6)	Day 3- Day 1		Day 6- Day 1
5% Test item		1	0,18	0,18	0,23	0,23	0,44	0,48	0,18	0,23	0,46	28%^a		156%^a
		2	0,19	0,18	0,23	0,24	0,48	0,44
10%  Test item		3	0,20	0,20	0,31	0,34	0,50	0,48	0,21	0,32	0,47	52%^a		124%^a
		4	0,21	0,21	0,31	0,32	0,41	0,50
25%  Test item		5	0,21	0,20	0,36	0,38	0,54	0,57	0,20	0,36	0,55	80%^a		175%^a
		6	0,19	0,20	0,35	0,36	0,55	0,55
a = an increase in ear thickness of 25% indicates a positive dermal irritation response

Table 2: Results of the quantitative irritation screening

Ear Measurements			Day 1		Day 3		Day 6		Mean Ear Thickness (mm)			%Difference
		AN #	Left	Right	Left	Right	Left	Right	Pre-Dosing (Day 1)	48 Hr (Day 3	End In-Life (Day 6)	Day 3- Day 1		Day 6- Day 1
0.05%  Test item		1	0,19	0,21	0,21	0,21	0,21	0,23	0,20	0,22	0,22	10%		10%
		2	0,20	0,21	0,21	0,23	0,21	0,23
0.1%  Test item		3	0,21	0,20	0,22	0,22	0,20	0,24	0,21	0,22	0,23	5%		10%
		4	0,21	0,22	0,22	0,23	0,24	0,24
0.25%  Test item		5	0,19	0,21	0,19	0,18	0,21	0,22	0,20	0,19	0,23	-5%		15%
		6	0,18	0,20	0,18	0,21	0,23	0,24
0.5%  Test item		7	0,20	0,21	0,21	0,21	0,21	0,22	0,20	0,20	0,22	0%		10%
		8	0,20	0,20	0,21	0,18	0,22	0,22
1%  Test item		9	0,19	0,18	0,19	0,20	0,23	0,22	0,19	0,19	0,22	0%		16%
		10	0,19	0,19	0,19	0,19	0,21	0,23
2.5%  Test item		11	0,20	0,19	0,22	0,21	0,34	0,33	0,20	0,22	0,35	10%		75%^a
		12	0,20	0,19	0,21	0,23	0,35	0,38
a = an increase in ear thickness of 25% indicates a positive dermal irritation response

Main Study

Table 3: Irritation as Measured by Ear Swelling (% Change Day 1 to Day 6) 

DMSO	25% HCA	0.25%  Test item	0.5% Test item	1%  Test item
10.5%	63.2%	15%	15.8%	15%

The EC3, the concentration at which the stimulation index is equal to 3 (calculated to determine skin sensitization potency) could not be calculated for test item.

Applicant's summary and conclusion

Interpretation of results:

other: not sensitising

Conclusions:

To determine the sensitising potential of topically applied test item (iodine), a Local Lymph Node Assay was performed in female mice. The study followed guideline EPA OPPTS 870.2600 (2003) and OECD guideline for the Testing of Chemicals No. 429 (2002). The stimulation index of the substance was 1.0 at 0.25%, 1.8 at 0.5% and 2.2 at 1%. The test item (iodine) showed no skin sensitiser potential.

Executive summary:

To determine the sensitising potential of topically applied test item (iodine), a Local Lymph Node Assay was performed in female mice. The study followed guideline EPA OPPTS 870.2600 (2003) and OECD guideline for the Testing of Chemicals No. 429 (2002).

In order to select the doses for the main study, an irritation screening was performed with solutions of 25%, 10%and 5% of test item in dimethyl sulfoxide (DMSO) using two animals per concentration. As irritation was observed (ear swelling > 25%) in the animals, a quantitative irritation test was carried out using 6 concentrations of test item (2 animals per concentration).

For the main study, solutions of 0.25%, 0.5% and 1% test item iodine) in DMSO were applied to the dorsum of both ears one time per day three consecutive days. Control mice were treated with the vehicle (alone) and an additional group with 25% alpha-hexyl cinnamic aldehyde (as positive control). Test item was negative for excessive local irritation (<25% increase in ear thickness) suggesting to be non irritant at the concentrations tested. The stimulation index of the substance was 1.0 at 0.25%, 1.8 at 0.5% and 2.2 at 1%.

Based on these results, test item (iodine) is not a considered a dermal sensitizer agent under testing conditions. The EC 3 concentration could not be calculated.