Zinc, [1,2,3-propanetriolato(2-)-.kappa.O1,.kappa.O2]-, homopolymer, stereoisomer

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 September 2008 - 01 October 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

zinc(2+) 3-hydroxypropane-1,2-bis(olate)

EC Number:

700-107-9

Cas Number:

87189-25-1

Molecular formula:

(C3H6O3Zn)n

IUPAC Name:

zinc(2+) 3-hydroxypropane-1,2-bis(olate)

Details on test material:

- Substance type: Organic
- Physical state: Powder
- Storage condition of test material: At room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L'Arbresle Cedex France
- Age at study initiation: approximately 11 weeks
- Weight at study initiation: preliminary test: 20 g, main study: 22-25 g (not exceed 20% of the mean weight)
- Housing: individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment. The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.
- Diet ad libitum: pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water ad libitum: tap water
- Acclimation period: at least 5 days before the start of the experiment
- other information: all females were nulliparous and non-pregnant

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5-23.6°C
- Humidity (%): 43-85%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h artificial fluorescent light / 12h darkness per day

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Remarks:

Merck, Darmstadt, Germany

Concentration:

0% (negative control with propylene glycol), 10% , 25%, 50%

No. of animals per dose:

5 animals per concentration

Details on study design:

TEST ARTICLE PREPARATION:
The test substance formulations (w/w) for topical application on the ears were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels. The formulations showed as white suspensions. Homogeneity with the vehicle was accomplished to a visually acceptable level. Prior to dosing the preparations were mixed thoroughly.

RANGE FINDING TESTS
Concentrations: 25% and 50%
Animals: 2 (one for each concentration)
Age: 8-14 weeks
Body weight: 20 g
Each animal was treated with 1 concentration on 3 consecutive days. The dorsal surface of both ears was epidermally treated (25 microliters/ear) with the test article, at approximately the same time per day. 3-4 hours after the last exposure, the irritation of the ears was assessed. Ears of the animal at 50% were cleaned of residual test substance with tap water. Body weights were determined on day 3 (19 and 20 g). The animals were sacrificed after the final observation and no necropsy was performed. No skin reaction occurred during this preliminary irritation study.

TREATMENT AND ADMINISTRATION (MAIN STUDY)
INDUCTION EXPOSURE
- No. of exposures: 3 (1 per day)
- Exposure period: 3 consecutive days
- Test groups: 3 (10%, 25%, 50% of test article)
- Control group: 1 (propylene glycol only)
- Site: dorsal surface of both ears (25 µl/ear)
- Frequency of applications: 1/day

TREATMENT/MEASUREMENT
-Treatment: 3 days after the last exposure
- Injection: via the tail vein with 0.25 ml of sterile buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine
- After 5 hours, animals were killed by intraperitoneal injection with pentobarbital Euthesate (0.2 ml/animal) and the draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to control) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 ml PBS.
- Tissue processing for radioactivity: A single cell suspension of lymph node cells (LNC) was prepared in PBS by gently separation through stainless steel gauze (diameter 125 µm). LNC were washed twice with an excess of PBS. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) at 4°C during the night.
- Radioactivity measurements: Precipitates were recovered by centrifugation, resuspended in TCA and transferred to Ultima Gold cocktail as the scintillation fluid. Radioactive measurements were performed using a scintillation counter. Counting time was to statistical precision of +/-0.2% or a maximum of 5 minutes whichever comes first. The scintillation counter was programmed to automatically subtract background and convert Counts per Minute (CPM) to Disintegrations per Minute (DPM).

CRITERIA USED TO CONSIDER A POSITIVE RESPONSE:
If the results indicate a SI (Simulation Index, which is the ratio of the DPM/group compared to DPM/vehicle control group) is equal or higher than 3, the test substance may be regarded as skin sensitizer, based on the test guideline and recommendations done by ICCVAM.

Positive control substance(s):

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

Results and discussion

Positive control results:

Concentrations of 0, 5, 10 and 25% of alpha-hexylcinnamicaldehyde in acetone:olive oil (4:1) were tested under conditions comparable to the study design. The SI values calculated for the positive control in concentrations of 0, 5, 10 and 25% were 1.0, 1.7, 2.8 and 3.6 respectively. An EC3 (estimated concentration that will give a SI = 3) value of 13.8% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 2 and 20%. Based on these results, it can be concluded that the LLNA as performed is an appropriate model.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 10, 25 and 50% were 1.4, 1.2 and 1.2 respectively. For further information see table 1.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 643, 555 and 533 respectively. The mean DPM/animal value for the vehicle contol group was 460. For further information see table 1.

Any other information on results incl. tables

No irritation of the ears was observed in any of the animals examined. Test substance remnants at 50% prevented scoring for erythema, no oedema was observed in this dose group (see table 2). All nodes of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted.

No changes in body weights were observed (see table 3). Individual radioactivity measures are given in table 4.

Table 1: Disintegrations Per Minute (DPM) and Stimulation Index

Group	% Test Substance	mean DPM +/- SEM	SI +/- SEM
1	0%	460 +/- 60	1.0 +/- 0.2
2	10%	643 +/- 104	1.4 +/- 0.3
3	25%	555 +/- 122	1.2 +/- 0.3
4	50%	533 +/- 61	1.2 +/- 0.2

Table 2: Skin reactions and relative size auricular lymph nodes

Concentration	Animal No.	Skin reactions (day 3)				Lymph node size (day 6)
		Left ear		Right ear		Left ear	Right ear
		erythema	edema	erythema	edema
0	1	0	0	0	0	n	n
	2	0	0	0	0	n	n
	3	0	0	0	0	n	n
	4	0	0	0	0	n	n
	5	0	0	0	0	n	n
10	6	0	0	0	0	n	n
	7	0	0	0	0	n	n
	8	0	0	0	0	n	n
	9	0	0	0	0	n	n
	10	0	0	0	0	n	n
25	11	0	0	0	0	n	n
	12	0	0	0	0	n	n
	13	0	0	0	0	n	n
	14	0	0	0	0	n	n
	15	0	0	0	0	n	n
50	16	G	0	G	0	n	n
	17	G	0	G	0	n	n
	18	G	0	G	0	n	n
	19	G	0	G	0	n	n
	20	G	0	G	0	n	n
n: considered to be normal, G: Scoring for erythema was not possible due to white test substance remnants.

Table 3: Mean body weights in grams

	Concentration (%)
	0	10	25	50
Day 1	23.6	23.4	24.6	24
Day 6	23	22.6	23.2	23.2

Table 4: Radioactivity measurements (individual animals)

group	% test substance	animal	DPM / animal
1	0%	1	310
	(Vehicle)	2	388
		3	644
		4	545
		5	413
2	10%	6	576
		7	976
		8	774
		9	385
		10	505
3	25%	11	297
		12	269
		13	613
		14	916
		15	678
4	50%	16	578
		17	427
		18	402
		19	741
		20	517

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met