Ammonium trivanadium octaoxide

Endpoint summary

Description of key information

An in vitro skin irritation study (EpiSkinTM) was performed with ammonium trivanadium octaoxide (Heppenheimer, 2010), and results indicate that it is not irritant to skin.

An acute eye irritation / corrosion test according to OECD 405 (Leuschner, 2010) was performed with ammonium trivanadium octaoxide, and results indicate that it has the potential to seriously damage the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-03-03 to 2010-03-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008 B.46 (draft)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals; Draft proposal for a new guideline, in vitro skin irritation: Reconstructed Human Epidermis (RhE) Test method, 11 December 2009, Vers. 4.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Details on test animals or test system and environmental conditions:

Not applicable - This was a in vitro study without any test animals.

Vehicle:

water

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approx. 15 mg of the test item were applied to each of triplicate tissues.
No further information on the amount/concentration applied was stated.

Duration of treatment / exposure:

15+/- 1 min

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

CELL CULTURE
EpiSkin TM kits (Lot No.: 10-EKIN-007) are purchased from Skinethic Laboratories (06000 Nice, France). The EpiSkin TM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin TM tissues (surface 0.38 cm^2) are cultured on specially prepared cell culture inserts.
EpiSkin TM tissues were shipped with ice packs on medium-supplemented agarose gels in a 12-well plate. On day of experiment EpiSkin TM tissues were transferred to 12-well plates with maintenance medium.

TREATMENT:
The negative control (deionised water (lot no. 1.3.10, produced in-house; 15 µL were applied to each of triplicate tissues) and positive control (5% sodium lauryl sulphate (lot no. 1353471 51508322; Fluka, Sigma-Aldrich, 89555 Steinheim, Germany) solution in deionised water, prepared freshly prior to the performance of the experiment; 15 µL were applied to each of triplicate tissues), and the test item were added into the insert atop the concerning EpiSkin TM triplicate tissues. Additionally, the test item tissues were wetted with 15 µl of deionised water. The plates were placed into the incubator for 15+/- 1 min at 37 +/- 1.5 °C, 5 +/- 0.5 % CO2.
After the end of the treatment interval the inserts were removed immediately from the plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for 42 +/- 1 hour at 37 +/- 1.5 °C, 5 +/- 0.5 % CO2.

MTT ASSAY:
Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues (Faller, C., Bracher, M., Dami, N., Roguet, R., 2002. Predictive ability of reconstructed human epidermis equivalents for assessment of skin irritation of cosmetics. Toxicology in vitro 16 (5), 557-552.).The percent reduction of cell viablity in comparison of untreated negative controls is used to predict skin irritation potential.
After the treatment procedure (42 hours) was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to plates containing 2 mL assay medium containing 0.3 mg/mL MTT per well. After a 3 hour incubation period (37 +/- 1.5 °C, 5 +/- 0.5 % CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissues samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for approx. 72 hours in the refrigerator.
Per each tissue sample 2 X 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) at 570 nm without reference filter. Mean values were calculated from the 2 wells per tissue sample.

EVALUATION OF RESULTS:
The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [OD test item/ OD negative control] * 100
For the test item and the positive control the mean relative viability +/- standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model:
For the current test, an irritation potential of a test item according to EU classification R38 is predicted if the mean relative tissue viablity of three individual tissues is reduced below 50 % of the negative control.

ACCEPTABILITY OF THE ASSAY:
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is ≥0.6 till ≤ 1.5.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 40%.
The standard deviations in between tissues of the same treatment group should be ≤ 18%.

The data of the quality control (determined by Skinethic Laboratories, 06000 Nice, France) of the respecitve EpiSkin TM lot is mentioned below under "Attached background material" (the acceptance limit of the IC50 should be in the between 1.0 and 3.0 mg/L after 18 hours treatment with SLS).

TEST FOR DIRECT MTT REDUCTION.
For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 15 mg of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
A colour change was not observed.

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Value:

82.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

After treatment with the test item ammonium trivanadium octaoxide, the relative absorbance values decreased to 82.9 %. This value is well above the threshold for irritancy of ≤ 50 %. Therefore, the test item is not considered to possess an irritant potential.

Results after treatment with ammonium trivanadium octaoxide

 

Dose group	Treatment Interval	Absorbance 570 nm Tissue 1	Absor-bance 570 nm Tissue 2	Absorbance 570 nm Tissue 3	Mean Absorbance of 3 Tissues	Absorbance [%] Tissue 1, 2 + 3	Rel. Standard Deviation	Rel. Absorbance [% of Negative Control]
Negative Control	15 min	0.878	0.819	0.873	0.857	102.5 95.6 101.9	3.8	100.0
Positive Control	15 min	0.293	0.209	0.203	0.235	34.2 24.4 23.7	5.9	27.4
Ammonium trivanadium octaoxide	15 min	0.812	0.619	0.701	0.711	94.8 72.3 81.8	11.3	82.9

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Historical data:

Positive Control		Negative Control
Number of Studies	73	Number of Studies	73
Period	July 2007 - March 2010	Period	July 2007 - March 2010
Mean Viability	16.5 %	Mean OD	1.081
Standard Deviation	11.0%	Standard Deviation	0.262
Range of Viabilities	3% - 36%	Range of ODs	0.7 – 1.6*

* The upper OD value is outside of the range of 0.6 - 1.5 recommended by the OECD guideline. Nevertheless since the OD value is only slightly above the required range, the historical data can still be considered as valid.

Interpretation of results:

GHS criteria not met

Conclusions:

After treatment with ammonium trivanadium octaoxide, the relative absorbance values decreased to 82.9 %. This value is well above the threshold for irritancy of ≤ 50 %. Therefore, the test item is not considered to possess an irritant potential.
The test item is not classified as skin irritant according to Regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-05-18 to 2010-05-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

, 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 tissues and environmental conditions:

TEST ANIMALS
- Source: LPT Laboratory of Pharmacologyand Toxicology GmbH & Co. KG, Branch Löhndorf, 24601 Löhndorf/Post Wankendorf, Germany
- Age at study initiation: Approx. 4 - 5 months
- Weight at study initiation: Animal 1: 2.4 kg; Animal 2: 2.3 kg; Animal 3: 2.3 kg
- Housing: For 8 hours following test item application, the animals were kept singly in restrainers which allowed free movement of the head but prevented a complete body turn, wiping of the eyes with the paws and excluded irritation of the eye by excrements and urine. During the acclimatisation period and after the 8-hour period in restrainers, the animals were kept singly in cages with dimensions of 380 mm x 425 mm x 600 mm (manufacturer: Dipl.Ing. W. EHRET GmbH, 16352 Schönwalde, Germany).
- Diet (ad libitum): Commercial diet, ssniff7 K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum before and after the exposure period): Tap water
- Acclimation period: At least 20 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 °C +/- 3 °C (maximum range)
- Relative humidity: 30% - 70% (maximum range; aim was 50% - 60%)
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg of the test item were administered into one eye each of three animals. The test item was placed into the conjunctival sac of the right eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second in order to prevent loss of the material. The left eye, which remained untreated, served as a control.
No further information on the amount/concentration applied was stated.

Duration of treatment / exposure:

1 hour (One hour after application the eyes were rinsed.)

Observation period (in vivo):

1, 24, 48 and 72 hours and 4 and 5 days after the administration

Number of animals or in vitro replicates:

3 male rabbits

Details on study design:

It is explicitly noted and in accordance with the guideline that the test was performed initially using one animal. As no corrosive or severe irritant effects were observed in this animal, 2 further animals were employed 24 hours after start of the initial test.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The eyes were rinsed with portions of 20 mL 0.9% aqueous NaCl solution, each.
- Time after start of exposure: 1 hour after administration

SCORING SYSTEM:

TOOL USED TO ASSESS SCORE: The eyes were examined ophthalmoscopically with a slit lamp prior to the administration and 1, 24, 48 and 72 hours and 4 and 5 days after the administration. The eye reactions were observed and registered.
24 hours after administration, fluorescein (Fluorescein SE Thilo drops (ALCON PHARMA GmbH, 79108 Freiburg, Germany) was applied to the eyes before being examined to aid evaluation of the cornea for possible lesions.

OTHER OBSERVATIONS: Any further lesions not covered by the scoring system were recorded. Body weight of all animals was measured at the beginning of the study and at the end of the study. Behaviour and food consumption were monitored.
No further information on the study design was stated.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal# 1

Time point:

24/48/72 h

Score:

1.67

Max. score:

3

Reversibility:

fully reversible within: 5 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for this animal on test day 5.

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

2

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for this animal on test day 5.

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

2.67

Max. score:

3

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for this animal on test day 5.

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for this animal on test day 5.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for this animal on test day 5.

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

2

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for this animal on test day 5.

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

2.67

Max. score:

3

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for this animal on test day 5.

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: Due to character and severity of the damages in the eyes of the animal the study was discontinued for this animal on test day 5.

Irritant / corrosive response data:

Corneal opacity was observed in animal nos. two and three 24 hours (grade 2: Easily discernible translucent area, details of iris slightly obscured) and 48 hours to 4 days (grade 3: nacreous areas, no details of iris visible, size of pupil barely discernible) after instillation. The mean score per animal, following grading at 24, 48 and 72 h after installation of the test material was calculated ≤ 3.
The fluorescein test performed 24 hours after instillation revealed corneal staining in animal no. 2 (1/4 to 1/2 of the surface) and in animal no. 3 (up to 1/4 of the surface).
Irritation of the iris was observed in animal nos. two and three 24 hours (grade 1: Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, iris reactive to light (a sluggish reaction is considered to be an effect)) and 48 hours to 4 days (grade 2: haemorrhage, gross destruction, or no reaction to light) after instillation. The mean score per animal, following grading at 24, 48 and 72 h after installation of the test material was calculated ≤ 2; in two of these three animals ≥ 1.5
Conjunctival redness was observed in all animals:
- animal no. 1: 60 minutes, 72 hours and 4 days (grade 1: some blood vessels hyperaemic (injected)) and 24 and 48 hours (grade 2: diffuse, crimson colour; individual vessels not easily discernible) after instillation;
- animal nos. 2 and 3: 60 minutes (grade 1: some blood vessels hyperaemic (injected)), 24 hours (grade 2: diffuse, crimson colour; individual vessels not easily discernible) and 48 hours to 4 days (grade 3:diffuse beefy red) after instillation. The mean score in two of three animals, following grading at 24, 48 and 72 h after installation of the test material was calculated > 2.5.
Chemosis was observed in all animals:
- animal no. 1: 24 and 48 hours (grade 1: some swelling above normal) after instillation;
- animal nos. 2 and 3: 24 hours to 4 days after instillation. The mean score in two of three animals, following grading at 24, 48 and 72 h after installation of the test material was calculated to be 2.
In addition, secretion was observed in all animals 60 minutes to 72 hours, in animal nos. 2 and 3 until 4 days after instillation.
Due to character and severity of the damages in the eyes of animal nos. 2 and 3 the study was discontinued for these animals on test day 5.

Other effects:

There were no systemic intolerance reactions.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The observed irritation, including iris lesions, cornea opacity, could be regarded as irreversible within a time period of 21 days.
According to the EC Regulation 1272/2008 and subsequent adaptations, the test item is classified as "irreversible effects on the eye" Category 1" (H318: Causes serious eye damage).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Justification for classification or non-classification

Skin irritation:

Ammonium trivanadium octaoxide does not possess a skin irritation potential and does not require classification as skin irritant according to Regulation (EC) 1272/2008.

 

Eye irritation:

Ammonium trivanadium octaoxide possesses an eye irritation potential and requires classification as irreversible effects on the eye (Category 1) according to Regulation (EC) 1272/2008.

Respiratory irritation:

Dyspnoea and respiratory murmur were observed during the acute inhalation toxicity test, and observed post mortem findings in decedents were damages to the lungs, including subpleural haemorrhages and oedema (Werner, 2006). Nevertheless, ammonium trivanadium octaoxide has the potential to seriously damage the eyes (i.e. all observed effects were irreversible). According to EC Regulation No. 1272/2008, ammonium trivanadium octaoxide needs to be classified STOT SE 3 „Specific target organ toxicity after single exposure Category 3“ (H335: May cause respiratory irritation).