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Administrative data

Description of key information

In two in vitro reconstructed human epidermis (EpiSkin) assays, conducted in accordance with OECD Test Guidelines 431 and 439 (for assessing skin corrosion and irritation, respectively) and to GLP, diammonium sodium hexakis(nitrito-N)rhodate was considered to be neither corrosive nor irritating to skin (Kiss, 2012a,b).

 

In an OECD Test Guideline 405 study, to GLP, diammonium sodium hexakis(nitrito-N)rhodate (0.1 g) caused some conjunctival effects in the eyes of three male rabbits within 1 hr of instillation, but these effects were fully reversible within 1 week (Kiss, 2012c).

 

No relevant respiratory tract irritation data were identified.

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:
22-24 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Conducted according to GLP
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, Section 4, No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” adopted 22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals. Available at: [http://ecvam.jrc.ec.europa.eu]
Deviations:
no
Qualifier:
according to
Guideline:
other: IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS MODEL TEST in relation to Regulation (EC) No 440/2008 (as amended) and Regulation (EC) No 1907/2006 on REACH (Annex III, B.46).
Deviations:
no
Principles of method if other than guideline:
The test is designed to predict and classify the skin irritant potential of chemicals according to chemical safety regulations, using the reconstructed human epidermis model EPISKIN-SM and parameters related to skin irritation.
EPISKIN-SM is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability. Cell viability determination is based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and conversion into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of cell viability in treated tissues is compared to negative controls and expressed as a %. The % reduction in viability is used to predict the irritation potential.

The EPISKIN-SM has been found scientifically valid for reliably predicting no label and R38 (irritant) substances in respect to the previous EU classification scheme and has been confirmed in April 2009 by ESAC for use under the UN GHS system as "applicable to all authorities". It is approved by international regulatory agencies as a replacement for the identifcation of irritants/corrosives in the in vivo rabbit skin assay (OECD 404).
GLP compliance:
yes (incl. certificate)
Species:
human
Strain:
other: Reconstructed human epidermis model (see details below)
Details on test animals and environmental conditions:
EPISKIN-SM (Source: SkinEthic, France, Batch No.:11-EKIN-047, Expiry date: 26 December 2011) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Type of coverage:
other: Applied evenly to the epidermal surface following the application of 10 ul distilled water to this surface
Preparation of test site:
other: in vitro cell culture
Vehicle:
water
Remarks:
distilled
Controls:
other: negative control skin unit tested in triplicate
Amount / concentration applied:
20 mg to each of three test skin units.
10 µl PBS (phosphate buffered saline) was added to each of the three negative control skin units and 10 µl SDS (sodium dodecyl sulfate, 5% aqueous solution) was added to each of the three positive control skin units.
For additional control for staining effects of the test item, 10 µl distilled water was applied to the epidermal surface of a single skin unit to ensure good contact with the epidermis, then 20 mg of the test item was applied evenly to the epidermal surface.
Duration of treatment / exposure:
Exposure for 15 minutes (± 0.5 min) at room temperature (20-37°C).
Then incubated with fresh “maintenance medium” for 42 hours (± 1h) at 37°C.
Observation period:
Not applicable to this test system
Number of animals:
Not applicable to this test system
Details on study design:
EPISKIN-SM assay plate contained reconstructed epidermis units (area: 0.38 cm2); each was attached to the base of a tissue culture vessel and maintained on nutritive agar.

After test substance exposure and subsequent incubation, preparations for cell viability determination were: incubation with MTT solution (at 37 degrees C for 3 hours) followed by incubation with acidified isopropanol for formazan extraction (around two hours at room temperature with gentle agitation).

For cell viability measurements, the OD (Absorbance / Optical Density) of the samples in a spectrophotometer was read at 540 nm using acidified isopropanol solution blank (6×200 µL). (The validity of the microplate reader was verified with a standard verification plate daily before use. The standard plate was calibrated yearly by the manufacturer.)

For each treated tissue, OD (as adjusted for colouring potential of the test substance) was calculated and the tissue viability was expressed as a % relative to negative control.

Criteria for classification as irritant/non-irritant: If the resulting mean relative viability (as adjusted for intrinsic colour) is less than or equal to 50% of the negative control, the test substance is considered to be irritant to skin.

Irritation / corrosion parameter:
other: other:
Value:
68
Remarks on result:
other:
Remarks:
Basis: mean Cell/tissue viability. Time point: 42 hours. Reversibility: other: Not applicable. Remarks: Score is a percentage (%) of negative control. Mean relative viability <=50% the test substance is considered to be irritant to skin.
Irritant / corrosive response data:
Mean cell viability (as adjusted for intrinsic colour) was 68% of the negative control (range 60%-76%).
Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro reconstructed human epidermis (EpiSkin) assay, conducted in accordance with OECD Test Guideline 439 and to GLP, diammonium sodium hexakis(nitrito-N)rhodate was considered to be non-irritating to skin.
Executive summary:

Diammonium sodium hexakis(nitrito-N)rhodate was tested for skin irritation potential in an in vitro reconstructed human epidermis model (EpiSkin assay) conducted in accordance with OECD Test Guideline 439, and to GLP.

 

EpiSkin is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability. Cell viability determination is based on cellular mitochondrial dehydrogenase activity, measured by MTT reduction and conversion into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of cell viability in treated tissues is compared to negative controls and expressed as a %. If the resulting mean relative viability (as adjusted for intrinsic colour) is less than or equal to 50% of the negative control, the test substance is considered to be irritant to skin.

 

Following a 15-minute exposure to the test substance, the test system skin cell viability was calculated to be greater than 50% (the average was 68% and the range was 60 -76%), and it was therefore considered to be non-irritating to skin.

 

Under the conditions of this assay, diammonium sodium hexakis(nitrito-N)rhodate does not require classification for skin irritation according to EU CLP criteria (EC 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:
26 March - 2 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Conducted according to GLP
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Remarks:
Minor humidity deviations were expected to have no impact on the study outcome
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Remarks:
Minor humidity deviations were expected to have no impact on the study outcom
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
Remarks:
Minor humidity deviations were expected to have no impact on the study outcom
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: S&K-LAP Kft., 2173 Kartal, Császár road 135, Hungary
- Age at study initiation: ~12 weeks
- Body weight range at the beginning of the in-life phase: 3167 – 3204 g
- Body weight range at the end of the in-life phase: 3402 – 3416 g
- Housing: individually in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages
- Diet (e.g. ad libitum): Purina and UNI diet (Lot number: 00701211 and 0030 03 12) for rabbits produced by AGRIBRANDS Europe Hungary PLC, H-5300 Karcag, Madarasi road, Hungary, ad libitum.
- Water (e.g. ad libitum): municipal tap water, as for human consumption, ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3°C
- Humidity (%): 24 – 52 %
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g

Duration of treatment / exposure:
Following instillation into the conjunctival sac of one eye, the eyelids were held closed for several seconds. No rinsing was performed.
Observation period (in vivo):
1 week (The eyes were examined at 1, 24, 48 and 72 hours, and 1 week after treatment).
Number of animals or in vitro replicates:
3 (males)
Details on study design:
Rinsing of treated eyes was not performed.

The other eye remained untreated and was used for control purposes.

The eyes were examined at 1, 24, 48 and 72 hours, and 1 week after treatment. The nature, severity and duration of all lesions observed were described.

SCORING SYSTEM: The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (24 April 2002) shown in Appendix 1 of the attached study report.

TOOL USED TO ASSESS SCORE: none reported; presumably subjective observation by an experienced evaluator
Irritation parameter:
chemosis score
Remarks:
Conjunctival chemosis
Basis:
mean
Remarks:
of all 3 animals
Time point:
other: Mean of 24, 48 and 72-hour timepoints
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Remarks on result:
other: Slight chemosis was seen in one animal at one hour; this had resolved by 24 hours
Irritation parameter:
other: Conjunctival discharge
Basis:
mean
Time point:
other: Mean of 24, 48 and 72-hour timepoints
Score:
0
Max. score:
3
Reversibility:
other: Not applicable
Remarks on result:
other: Considerable discharge was seen in one animal, and slight discharge in two animals, at 1 hour; this had resolved to normal by 24 hours in all cases
Irritation parameter:
other: Conjunctival redness
Basis:
mean
Time point:
other: Mean of 24, 48 and 72-hour timepoints
Score:
0.56
Max. score:
3
Reversibility:
fully reversible
Remarks:
1 week
Remarks on result:
other: One animal was symptom-free by 24 hours, one by 72 hours and one by 1 week
Irritation parameter:
cornea opacity score
Remarks:
opacity, degree of density
Basis:
mean
Time point:
other: Mean of 24, 48 and 72-hour timepoints
Score:
0
Max. score:
4
Reversibility:
other: Not applicable
Remarks on result:
other: No corneal opacity was seen in any animal at any time point
Irritation parameter:
iris score
Basis:
mean
Time point:
other: Mean of 24, 48 and 72-hour timepoints
Score:
0
Max. score:
2
Reversibility:
other: Not applicable
Remarks on result:
other: The iris was not affected in any animal at any time point
Irritant / corrosive response data:
Initial Pain Reaction (IPR) (score 2) was observed in all animals.

One hour after the application, conjunctival redness (score 1) and conjunctival discharge (score 1 or score 3) were seen in all animals. Conjunctival chemosis (score 1) was observed in one rabbit.

At 24 and 48 hours after treatment, conjunctival redness (score 1) was observed in two animals.

At 72 hours after treatment, conjunctival redness (score 1) was observed in one animal.

At 1 week after treatment no signs of eye irritation or other clinical signs were observed.

As there were no clinical signs observed, the study was terminated after the 1 week observation.

During the study, the control eye of each animal was symptom-free.

The individual mean scores for each animal (considering readings at 24, 48 and 72 hours after the treatment) were as follows:
chemosis : 0.00, 0.00, 0.00
discharge : 0.00, 0.00, 0.00
redness : 1.00, 0.67, 0.00
cornea opacity : 0.00, 0.00, 0.00
iris : 0.00, 0.00, 0.00
Other effects:
There were no clinical signs observed that could be related to treatment.
Interpretation of results:
GHS criteria not met
Conclusions:
In an OECD Test Guideline 405 study, to GLP, diammonium sodium hexakis(nitrito-N)rhodate (0.1 g) caused some conjunctival effects in the eyes of three male rabbits within 1 hr of instillation, but these effects were fully reversible within 1 week.
Executive summary:

In an in vivo eye irritation study carried out in accordance with OECD Test Guideline 405, and to GLP, 0.1 g of powdered diammonium sodium hexakis(nitrito-N)rhodate was instilled into the conjunctival sac of one eye of 3 male New Zealand White rabbits. Following instillation the eyelids were held closed for several seconds; no rinsing was performed. The other eye remained untreated and was used for control purposes. The eyes were examined at 1, 24, 48, 72 hours, as well as 1 week after treatment, and scored according to the Draize system.

 

Mild conjunctival effects (including redness, chemosis and discharge) occurred within 1 hour of application. These were either reduced or absent at 24 hours and were fully reversible within 1 week.

 

Based on the results of this study, diammonium sodium hexakis(nitrito-N)rhodate does not require classification for eye irritation, according to EU CLP criteria (EC 1272/2008).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No relevant human irritation/corrosion data were identified.

 

Diammonium sodium hexakis(nitrito-N)rhodate was tested for skin corrosion and irritation potential in two EpiSkin (in vitro reconstructed human epidermis model) assays conducted in accordance with OECD Test Guidelines 431 and 439, respectively, and to GLP. EpiSkin is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability, as determined by cellular mitochondrial dehydrogenase activity. The reduction of cell viability in treated tissues is compared to negative controls and expressed as a %. In the test for skin corrosion, following a 4-hour exposure to the test substance, the test system skin cell viability was calculated to be greater than 35% (the average was 72% and the range was 69 -75%), and it was therefore considered to be non-corrosive to skin. In the skin irritation test, following a 15-minute exposure to the test substance, the test system skin cell viability was calculated to be greater than 50% (the average was 68% and the range was 60-76%), and it was therefore considered to be non-irritating to skin (Kiss, 2012a,b).

 

In an in vitro eye irritation study in isolated chicken eyes, conducted in accordance with OECD Test Guideline 438, diammonium sodium hexakis(nitrito-N)rhodate appeared to be not severely irritating/corrosive and does not require a classification as a severe eye irritant. However, this test substance remained adhered to the corneal surface after the post-treatment rinse (Kiss, 2012d). As such, an in vivo study is required to determine classification (see below).

 

In an in vivo eye irritation study carried out in accordance with OECD Test Guideline 405, and to GLP, 0.1 g of powdered diammonium sodium hexakis(nitrito-N)rhodate was instilled into the conjunctival sac of one eye of 3 male New Zealand White rabbits. Following instillation the eyelids were held closed for several seconds; no rinsing was performed.The other eye remained untreated and was used for control purposes.The eyes were examined at 1, 24, 48, 72 hours, as well as 1 week after treatment, and scored according to the Draize system. Mild conjunctival effects (including redness, chemosis and discharge) occurred within one hour of application. These were either reduced or absent at 24 hours and were fully reversible within 1 week (Kiss, 2012c).

 

No respiratory tract irritation data were identified. A new study was not conducted as it is not a REACH Standard Information Requirement.

Justification for classification or non-classification

Based on the results of the available (in vitro) skin corrosion and irritation studies and the (in vivo and in vitro) eye irritation studies, diammonium sodium hexakis(nitrito-N)rhodate does not require classification for skin or eye irritation according to EU CLP criteria (EC 1272/2008).