Reaction mass of 1-O-α-D-glucopyranosyl-D-fructose and 6-O-α-D-glucopyranosyl-D-fructose and fructose and glucose and sucrose

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

26 Sep - 01 Oct 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP-Guideline study, tested with the source substance Isomaltulose (Cas# 13718-94-0). According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: L´Oréal Standard Operating Procedure: “EpiSkinTM SKIN IRRITATION TEST METHOD 15 min – 42 hours” – ECVAM Skin Irritation Validation Study – VALIDATION OF THE EpiSkinTM TEST METHOD 15 min – 42 hours FOR THE PREDICTION OF ACUTE SKIN IRRITATION OF CHEMICALS.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(BAYERISCHES LANDESAMT FÜR GESUNDHEIT UND LEBENSMITTELSICHERHEIT, LANDESINSTITUT FÜR ARBEITSSCHUTZ UND PRODUKTSICHERHEIT, München, Germany)

Test material

Test material information

Constituent 1

Reference substance name:

6-O-α-D-glucopyranosyl-D-fructose

EC Number:

237-282-1

EC Name:

6-O-α-D-glucopyranosyl-D-fructose

Cas Number:

13718-94-0

Molecular formula:

C12H22O11

IUPAC Name:

6-O-alpha-D-glucopyranosyl-D-fructose

Details on test material:

- Name of test material (as cited in study report): Isomaltulose
- Analytical purity: ≥ 98%
- Physical state: fine white powder
- Lot/batch No.: L121291500
- Expiration date of the lot/batch: 8 April 2014
- Storage condition of test material: at room temperature

Test animals

Species:

human

Strain:

other: EPISKIN-SM (TM): reconstructed three-dimensional human epidermis model

Details on test animals or test system and environmental conditions:

TEST SKIN MODEL
- Source: SkinEthic laboratories, Lyon, France
- Test system:
The model represents a reconstructed three-dimensional skin model based on normal human-derived epidermal keratinocytes which have been cultured on a collagen matrix for 13 days in cell culture inserts to form a multilayered epidermis including basal, spinous and granular layers, and a multi-layered stratum corneum. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.
- Adaptation to cell culture conditions: Upon receipt, tissues were transferred into 12-well plates containing 2 mL prewarmed maintenance medium per well and preincubated in a humidified incubator for at least 24 h (37 ± 1 °C, 5% CO2) before use.

ENVIRONMENTAL CONDITIONS (Incubator)
- Temperature (°C): 37 ± 1
- CO2 gas concentration (%): 5
- Humidity: maximum

Test system

Type of coverage:

open

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent control tissues treated with phosphate buffered saline served as negative controls, positive controls were exposed to 5% SDS.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 10 mg (26.3 mg/cm²)

POSITIVE CONTROL SUBSTANCE
- SDS, 5% (v/v) in aqua dest.

Duration of treatment / exposure:

15 ± 0.5 min

Observation period:

Not applicable. Post-treatment incubation period: 42 ± 1 h

Number of animals:

Not applicable.The test was performed in triplicates for each treatment and control group.

Details on study design:

TEST SITE
- Area of exposure: 0.38 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: The test item was washed from the epidermal surface with phosphate buffered saline (PBS). Excess PBS was removed by blotting bottom with blotting paper.
- Time after start of exposure: 15 ± 0.5 min
- Post-treatment incubation period: 42 ± 1 h


CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed 42 ± 1 h after the incubation period. Therefore, tissues were incubated in 2 mL prewarmed MTT solution for 3 h ± 5 min at 37 ± 1 °C and 5% CO2. Afterwards, tissues were dried on blotting paper and a total biopsy of the epidermis was performed. After separation of the epidermis and the collagen matrix, extraction of the formazan product from both parts was carried out in 500 µL acidic isopropanol. At the end of the extraction, extracts from the epidermis and collagen part were mixed and the optical density of 2 x 200 µL aliquots per sample was measured at 550 nm wave length in a plate spectrophotometer (Tecan Infinite 200, Tecan Austria).

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: cell viability (%)

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: mean value of negative controls (PBS). Time point: 15 min. Reversibility: other: not applicable. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

7

Remarks on result:

other:

Remarks:

Basis: other: mean value of positive controls (5% SDS). Time point: 15 min. Reversibility: other: not applicable. (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

95

Remarks on result:

other:

Remarks:

Basis: other: mean value of the test item. Time point: 15 min. Reversibility: other: not applicable. (migrated information)

In vivo

Irritant / corrosive response data:

Topical application of Isomaltulose did not result in a decreased cell viability in the EPISKIN-SM model compared to the negative control tissues treated with PBS. In contrast, exposure to 5% SDS decreased the cell viability to 7% of the negative control tissues.

Any other information on results incl. tables

Table 1: MTT assay after 15 min exposure

	Negative Control			Positive Control			Test Item			Blank
Tissue	1	2	3	1	2	3	1	2	3
OD550	0.901	0.827	0.707	0.113	0.092	0.092	0.742	0.816	0.757	0.043
	0.899	0.817	0.697	0.107	0.088	0.087	0.741	0.813	0.762
OD550(mean, blank corrected)	0.857	0.779	0.659	0.067	0.047	0.047	0.698	0.771	0.717
SDOD550	0.089			0.011			0.034
OD550(mean values of triplicates)	0.765			0.053			0.729
SD tissue viability (%)	13.1			1.5			4.9
Viability (%)	100			7			95

According to the Test Acceptance Criteria, the validity criteria are fulfilled:

1.   mean OD550 nm of the 3 negative control tissues is ≥ 0.6 and ≤ 1.5

2.   OD₅₅₀of the blank is < 0.1

3.   mean relative tissue viability of the 3 positive control tissues is ≤ 40%

4.   the standard deviation (SD) obtained from the three concurrently tested tissues is

< 18%.

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified