Ammonium sodium vanadium oxide

Endpoint summary

Description of key information

An in vivo skin irritation study was performed with ammonium sodium vanadium oxide (Hansen, 2013), and results indicate that it is not irritant to skin.
An acute in vivo eye irritation / corrosion test according to OECD 405 (Hansen, 2013) was performed withammonium sodium vanadium oxide, and results indicate that it is  irritant to eyes (category II). 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013-03-01 to 2013-03-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted 2002-04-24

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-11-12

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Branch Löhndorf, 24601 Löhndorf/Post Wankendorf, Germany
- Age at study initiation: approximately 8 months
- Weight at study initiation: 2.80 - 3.25 kg
- Housing: before and after the 4-hour exposure period, the animals were kept singly in cages measuring 380 mm x 425 mm x 600 mm (manufacturer: Dipl. Ing. W. EHRET GmbH, 16352 Schönwalde, Germany). During the exposure period, the animals were kept singly in restrainers which allowed free movement of the head but prevented a complete body turn. The cages excluded irritation of the skin by excrements and urine.
- Diet (ad libitum; before and after exposure period): commercial diet, ssniff® K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum; before and after exposure period): drinking water
- Acclimation period: at least 20 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C ± 3°C (maximum range)
- Relative humidity: 30% - 70% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 mg of the test item (moistened with the vehicle) was applied to the test site.

Duration of treatment / exposure:

4 hours

Observation period:

prior to the administration and 60 minutes, 24, 48 and 72 hours after the exposure period

Number of animals:

3 female rabbits

Details on study design:

TEST SITE
- Area of exposure: approximately 24 hours before the test, the fur was removed by closely clipping the dorsal area of the trunk of the animals. Care was taken to avoid abrading the skin. Only animals with healthy intact skin were used.
The test item moistened with the vehicle was applied to the test site (area: approx. 6 cm^2) and then covered with a gauze patch. The patch was held in contact with the skin with non-irritating tape for the duration of the exposure period. The surrounding untreated skin served as a control.

INITIAL TEST AND CONFIRMATORY TEST
As it was expected that the test item would not produce any severe irritancy or corrosion, the test was started using at first only one animal, receiving a single patch for an exposure period of 4 hours.
As neither a corrosive effect nor a severe irritant effect was observed after a four-hour exposure, the test was completed using two additional animals, each with one patch only, for an exposure period of 4 hours.

SCORING SYSTEM: according to the Draize scale

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1

Time point:

other: 24, 48 and 72 horus

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: The skin of the application area of the rabbit appeared to be dry.

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #2

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Remarks on result:

other: The skin of the application area of the rabbit appeared to be dry.

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #2

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #3

Time point:

other: 24, 48 and 72 hours

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: The skin of the application area of the rabbit appeared to be dry.

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #3

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritant / corrosive response data:

Three rabbits exposed for 4 hours to 500 mg ammonium sodium vanadium oxide showed following effect:
An very slight (barely perceptible) erythema (grade 1) was observed in two animals 48 hours after patch removal but not anymore after 72h.
The skin of the application area of the three rabbits appeared to be dry.

Other effects:

There were not any systemic intolerance reactions.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Ammonium trioxovanadate is not irritating to the skin.
According to the EC-Commission directive 67/548/EEC, ammonium sodium vanadium oxide is not classified as irritating to the skin.
According to the EC-Regulation 1272/2008, ammonium sodium vanadium oxide is not classified as irritating to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2013-01-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted 2009-09-07

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

, 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Details on test animals or tissues and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Vehicle:

other: 0.9% (w/v) NaCl in deionised water

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
Prior to the test a 20% (w/v) suspension of the test item in the vehicle was prepared using ultrasonic technique.

Duration of treatment / exposure:

240 ± 15 minutes

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

not applicable

Details on study design:

COLLECTION OF BOVINE EYES
Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in Hank’s Ballanced Salt Solution (HBSS) supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 (© Biochrom) supplemented with L-glutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran was added to the medium.

PREPARATION OF CORNEAE
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors.
Each isolated cornea was mounted in a specially designed cornea holder as described in OECD guideline 437, annex III, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0). Each cornea with a value of the basal opacity > 7 was discarded and not used in the test.

OUTLINE OF STUDY
Complete medium was completely removed from the anterior compartment and replaced by the test item, positive control (10% (w/v) Benzalkonium chloride (Sigma, 89555 Steinheim, Germany, lot no. 036K0208) in 0.9% (w/v) NaCl solution in deionised water (saline, produced in-house, lot no. 180113)) or negative control (0.9% (w/v) NaCl in deionised water (produced in-house, lot no. 180113)).
The anterior compartment received the test item suspension or negative or positive control at a volume of 0.75 mL each on the surface of the corneae.
Since the test item could not be suspended homogeneously, it was taken care that each 0.75 mL of the prepared stock suspension was distributed evenly to the corneae.
The test item, positive control and negative control were tested in triplicate.
The anterior compartment was then plugged again. The corneae were turned into a horizontal position and slightly rotated to ensure uniform covering of the corneae with the test or control items and were incubated in a water-bath in horizontal position at 32 ± 1 °C for 240 minutes.
After the incubation, the test item or control items, respectively, were rinsed off from the application side with 0.9% (w/v) NaCl in deionised water at least three times or until no visual evidence of the test substance was observed. Fresh cMEM was added into the anterior compartment and opacity was measured (t240).
In the second step of the assay, permeability of the cornea was determined. 1 mL of a Na-fluorescein solution, 0.5 % (w/v) dissolved in HBSS (Hank’s buffered salt solution), was placed in the anterior compartment, replacing the cMEM. Corneae were incubated again in a horizontal position for an additional 90 minutes at 32 ± 1 °C in the water-bath. The optical density of an aliquot of the mixed complete medium from the posterior chamber was measured spectrophotometrically at 490 nm (OD490).

CRITERIA FOR DETERMINATION OF A VALID TEST
According to the OECD 437 protocol, the test its considered acceptable if the positive control gives an In Vitro Irritation Score (IVIS) that falls within two standard deviations of the current historical mean. The negative or solvent/vehicle control responses should result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control. Accordingly and based on historical control data, the test was acceptable if the in vitro irritation score of the positive control was ≥ 30 and the in vitro irritation score of the negative control was ≤ 3.

EVALUATION OF RESULTS
- Opacity: the change of opacity value of each treated cornea or positive and negative control corneae was calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae was calculated and this value was subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Permeability: the corrected OD490 value of each cornea treated with positive control and test item was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IN VITRO IRRITATION SCORE CALCULATION
The following formula was used to determine the in vitro irritation score of the negative control:
In vitro Irritation Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro irritation score of the positive control and the test item:
In vitro Irritation Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The in vitro irritation score was calculated for each individual treatment and positive control cornea. The mean in vitro irritation score irritation value of each treated group was calculated from the individual in vitro irritation score values.
Depending on the score obtained, the test item was classified into the following category according to OECD guideline 437 (table 1 in the field "Any other information on materials and methods incl. tables" below).

Irritation parameter:

other: In vitro irritation score

Basis:

mean

Time point:

other: 240 minutes

Score:

2.49

Remarks on result:

other: Standard deviation: 1.71

Irritant / corrosive response data:

Relative to the negative control, exposure of the test item ammonium sodium vanadium oxide to the corneae did not induce an increase of the corneal permeability. Only a very slight increase of the opacity values occurred. The calculated mean in vitro irritation score was 2.49 (threshold for corrosivity / severe irritancy: ≥ 55.1). According to OECD guideline 437, the test item is not classified as corrosive / severe irritant to the eye.

Table 1: Results after 240 Minutes Incubation Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		In vitro Irritation Score	Mean in vitro irritation score	Proposed in vitro Irritation Scale
		Mean		Mean
Negative Control	0	0.00	0.063	0.060	0.95	0.90 ± 0.05	Non corrosive / non severe irritant
	0		0.060		0.90
	0		0.056		0.84
Positive Control	248.00		- 0.012*		247.83	207.93 ± 40.34	Corrosive / severe irritant
	208.00		0.053*		208.80
	167.00		0.010*		167.16
Ammonium sodium vanadium oxide	2.00		- 0.005*		1.93	2.49 ± 1.71	Non corrosive / non severe irritant
	1.00		0.009*		1.14
	4.00		0.027*		4.41

*corrected values (subtraction of average negative control from measured permeability of each replicate)

- With the negative control (0.9% (w/v) NaCl in deionised water) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro irritation score 0.90). The score is well within the concurrent negative control range, i.e. within two standard deviations of the current historical mean.

- The positive control (10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl in deionised water) induced clear opacity of the corneae (mean in vitro irritation score 207.93) corresponding to a classification as corrosive /severe irritant to the eye (CLP/EPA/GHS (Cat 1)). The score is well within the concurrent positive control range, i.e. within two standard deviations of the current historical mean.

- Thus, based on historical positive and negative control data, this test meets the acceptability criteria in accordance with OECD 437.

Table 2: Historical data

	Positive Control	Negative Control
Meanin vitro Irritation Score	176.71	1.78
Standard Deviation	42.65	0.75
Range of in vitro Irritation Scores	99.4 - 292.3	0.41 - 2.99

Values of 138 studies with solid test items performed February 2007 until November 2012

Interpretation of results:

other: not corrosive / not severely irritating to the eye

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

According to the test, ammonium sodium vanadium oxide is not corrosive / not severely irritating to the eye.
Ammonium sodium vanadium oxide should not be classified and labelled as severe eye irritant according to regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation: Three studies on skin irritation were conducted. An in vitro study on skin corrosion (OECD 435) with a negative outcome and a positive in vitro skin irritation study (OECD 439) which indicates an irritation potential of ammonium sodium vanadium oxide.

However, a third study in skin irritation/corrosion was conducted in vivo (OECD 404). The study shows no irritation/corrosion potential of ammonium sodium vanadium oxide. The erythema and edema scores at all relevant time points were “0” (24, 48, 72 h). Results of in vivo studies should take precedence over in vitro studies. Hence, ammonium sodium vanadium oxide is not irritation to skin and should not be labelled as such.

Rationale for in vivo testing: Ammonium sodium vanadium oxide tested positive in vitro, i.e. in the human skin model test according to OECD 439 as the mean relative absorbance decreased to 36% and below the threshold for irritancy of ≤ 50%. However, long-term experience of the Sponsor in manufacture and handling indicates that the substance is not a skin or eye irritant. Furthermore, ammonium sodium vanadium oxide did not cause an increase of the corneal permeability or opacity in thein vitroBCOP test according to OECD 437 (meanin vitroirritation core of 6.44) and thus is not corrosive and not a severe irritant to the eye.

Eye irritation:

Two studies on eye irritation / corrosion were conducted. The first study was initiated in vitro to show that ammonium sodium vanadium oxide has no corrosive potential. Since no fully validated in vitro eye irritation study exist at time of study initiation, an in vivo eye irritation study was conducted and results indicated an irritating potential of ammonium sodium vanadium oxide to eyes.

Justification for selection of skin irritation / corrosion endpoint:
The study selected is conducted in vivo. Since in vivo studies take presedence over in vitro studies, this study was selected for endpoint conclusion. For more information please refer to the discussion below.

Justification for selection of eye irritation endpoint:
This study is selected as key study, since the study was conducted in vivo. In vivo studies should take presedence over in vitro studies.

Effects on eye irritation: irritating

Justification for classification or non-classification

Skin irritation:

Ammonium sodium vandium oxide does not possess an irritation potential and does not require classification as skin irritantaccording to Directive 67/548/EECandRegulation (EC) 1272/2008.

Eye irritation:

Ammonium sodium vanadium oxide possesses an irritation potential and requires classification as eye irritantaccording to Directive 67/548/EEC (R36)andRegulation (EC) 1272/2008 (category 2).

Respiratory irritation:

Ammonium sodium vanadium oxide is neither irritating/corrosive to skin nor corrosive to eyes. Only mild, reversible effects were observed in the in vivo eye irritation test (Hansen, 2013). Furthermore, local reversible or irreversible adverse health effects were not observed below lethal levels (i.e. no pathological findings) in the relevant acute inhalation toxicity tests (Weniger, 2006 a,b). Hence, ammonium sodium vanadium oxide does not possess an irritation potential in the respiratory tract and does not require classification as respiratory irritant according to Directive 67/548/EEC and Regulation (EC) 1272/2008.