Lithium Nickel Cobalt Aluminium Oxide

Endpoint summary

Description of key information

The test material was classified as Corrosive Category 1B to rabbit skin (based on one rabbit only). This classification was confirmed by the MeClas calculation on generic NCA..
Skin corrosive substances shall be considered as leading to serious damage to the eyes as well (Category 1).

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:

key study

Study period:

The study was performed between 16 July 2008 and 17 July 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994))

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: One New Zealand White rabbit was supplied by Harlan UK Limited, Bicester, Oxon, UK
- Age at study initiation: 12 to 20 weeks old.
- Weight at study initiation: 3.41 kg
- Housing: The animal was housed in a suspended cage.
- Diet (e.g. ad libitum): Free access to food (Certified Rabbit Diet) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains drinking water was allowed throughout the study
- Acclimation period: at least five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23°C
- Humidity (%): 30 to 70%
- Air changes (per hr): at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

Type of coverage:

semiocclusive

Preparation of test site:

other: clipped free of fur

Vehicle:

water

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g of the test material, moistened with 0.5 ml of distilled water

Duration of treatment / exposure:

A single 3-minute semi-occluded application of the test material and a single 1-hour semi-occluded application.

Observation period:

24 hours

Number of animals:

1

Details on study design:

Procedure:
On the day before the test the rabbit was clipped free of fur from the dorsal/flank area using veterinary clippers.
Two suitable sites were selected on the back of the rabbit. At each test site a quantity of 0.5 g of the test material, moistened with 0.5 ml of distilled water, was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the shorn skin. Each patch was secured in position with a strip of surgical adhesive tape. To prevent the animal interfering with the patches, the trunk of the rabbit was wrapped in an elasticated corset for the duration of the exposure period.
One patch was removed at each of two time points: 3 minutes and 1 hour after application. Any residual test material was removed by gentle swabbing with cotton wool soaked in distilled water.
Due to the corrosive reactions following an exposure period of 1-hour, the 4 hour exposure period was not investigated.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: 1 hour

Score:

1

Max. score:

1

Reversibility:

not fully reversible within: 24 hours

Remarks on result:

other: 3- minute exposure

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24 h

Score:

2

Max. score:

2

Reversibility:

not fully reversible within: within 24 hours

Remarks on result:

other: 3-minute exposure

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

48 h

Remarks on result:

other: test was terminated after 24h

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

72 h

Remarks on result:

other: test was terminated after 24h

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 1 hour

Score:

0

Max. score:

0

Remarks on result:

other: 3-minute exposure

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

not fully reversible within: 24 hours

Remarks on result:

other: 3-minute exposure

Irritation parameter:

edema score

Basis:

animal #1

Time point:

48 h

Remarks on result:

other: test was terminated after 24h

Irritation parameter:

edema score

Basis:

animal #1

Time point:

72 h

Remarks on result:

other: test was terminated after 24h

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: 1 hour

Score:

4

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: 1-hour exposure

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24 h

Score:

>= 0 - <= 4

Reversibility:

not reversible

Remarks on result:

other: 1-hour exposure - adverse reaction prevents accurate evaluation of erythema

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 1 hour

Score:

2

Max. score:

2

Reversibility:

not reversible

Remarks on result:

other: 1-hour exposure

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24 h

Score:

>= 0 - <= 4

Reversibility:

not reversible

Remarks on result:

other: 1-hour exposure - Adverse reaction prevents accurate evaluation of oedema

Irritant / corrosive response data:

3-Minute Exposure Period:
The individual scores for erythema/eschar and oedema are given in Table 1.
Very slight erythema was noted at the treated skin site one hour after patch removal. A small area of petechial haemorrhage was also noted at the left and right edges of the treated skin site at this observation. Well defined erythema and very slight oedema were noted at the treated skin site at the 24 hour observation. A small dark coloured scab, approximately 4 mm x 4 mm in size, was also noted on the right edge of the treated skin site at this observation.

1-Hour Exposure Period:
The individual scores for erythema/eschar and oedema are given in Table 1.
Reactions noted at the treated skin site one hour after patch removal were green coloured dermal necrosis (attributed a Draize score of 4 for injuries in depth), surrounded by dermal haemorrhage, and slight oedema. A sunken, hardened dark brown/black coloured scab, preventing accurate evaluation of erythema and oedema, was noted at the treated skin site at the 24 hour observation.

Other effects:

None.

Table1               Individual Skin Reactions Following 3-Minute and 1-Hour Exposures

Skin Reaction	Observation Time	Individual Scores - Rabbit Number and Sex
		67601 Male
		3-Minute Exposure	1-Hour Exposure
Erythema/Eschar Formation	1 Hour	1Hd	4NHd*
	24 Hours	2St	?eSu
Oedema Formation	1 Hour	0	2
	24 Hours	1	?od

 

 

N= Green coloured dermal necrosis

Hd = Small area of petechial haemorrhage on left and right edges of test site

Hd* = Dermal haemorrhage surrounding dermal necrosis

St = Small dark coloured scab, approximately 4 mm x 4 mm, on the right edge of test site

Su = Sunken, hardened dark brown/black coloured scab

?e = Adverse reaction prevents accurate evaluation of erythema

?od = Adverse reaction prevents accurate evaluation of oedema

Due to the corrosive reactions following an exposure period of 1-hour, the 4‑hour exposure period was not investigated.

The animal was killed for humane reasons immediately after the 24-hour observation, due to the irreversible effects, in accordance with Company policy and current UK Home Office guidelines.

The test material was classified as CORROSIVE to rabbit skin (based on one rabbit only).

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test material was classified as CORROSIVE to rabbit skin (based on one rabbit only).

Executive summary:

Introduction. 

The study was performed to assess the irritancy potential of the test material to the skin of the New Zealand White rabbit. The method was designed to meet the requirements of the following:

§        OECD Guidelines for the Testing of Chemicals No. 404 “Acute Dermal Irritation/Corrosion” (adopted)

§        Method B4 Acute Toxicity (Skin Irritation) of Commission Directive 2004/73/EC

Results. 

A single 3-minute semi-occluded application of the test material to the intact skin of one rabbit produced no corrosive effects.

A single 1-hour, semi-occluded application of the test material to the intact skin of one rabbit produced reactions indicative of dermal corrosion. The reactions included green coloured dermal necrosis and scabbing.

The animal was killed for humane reasons immediately after the 24-hour observation, due to the irreversible effects, in accordance with Company policy and current UK Home Office guidelines.

Due to the corrosive reactions following an exposure period of 1-hour, the 4-hour exposure period was not investigated.

Conclusion. 

The test material was classified as corrosive to rabbit skin (based on one rabbit only).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

other: SkinEthic Reconstituted Human Corneal model

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The study was performed between 08 July 2008 and 10 July 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

SkinEthic Reconstituted Human Corneal Ephitelium model

Deviations:

not specified

Principles of method if other than guideline:

The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994))

Species:

other: The SkinEthic RHC model consists of transformed human keratinocytes

Strain:

other: The SkinEthic RHC model consists of transformed human keratinocytes of the cell line HCE

Details on test animals or tissues and environmental conditions:

TEST ANIMALS: Not applicable.

Tissue: The purpose of this study was to determine the eye irritation potential of the test materials using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France)
The SkinEthic RHC model consists of transformed human keratinocytes of the cell line HCE that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye.



ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature
- Humidity (%): Not stated.
- Air changes (per hr): Not stated.
- Photoperiod (hrs dark / hrs light): Not stated.


IN-LIFE DATES: Not applicable.

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg of test material.
- Concentration (if solution): Not applicable.

Duration of treatment / exposure:

Triplicate tissues were treated with 30 mg of the test material for 10 minutes.

Observation period (in vivo):

Not applicable.

Number of animals or in vitro replicates:

Not applicable.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the relevant exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco’s Phosphate Buffered Saline (DPBS). Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated ‘holding plate’ containing 300 µl of maintenance medium (at room temperature) until all the tissues were rinsed.
- Time after start of exposure: 10 minutes.


SCORING SYSTEM:
The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues.
After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured (OD540). Data are presented in the form of % viability (MTT conversion relative to negative controls).



TOOL USED TO ASSESS SCORE:
The test material was classified according to the following criteria:
i) If the % relative mean tissue viability was ≥ 60% the test material was considered to not likely to be a severe ocular irritant.
ii) If the % relative mean tissue viability was < 60% the test material was considered to be irritant.

Irritation parameter:

other: % viability

Remarks:

mean value

Run / experiment:

10 minutes exposure period

Value:

7

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other:

Other effects / acceptance of results:

According to the protocol followed the test material was considered to be Irritant (I). It was considered unnecessary to proceed with tissue histopathology.

Assessment of Eye Irritation Potential

        

The mean OD₅₄₀ values and mean viabilities for each treatment group are given in Table 1.

The relative mean viability of the test material treated tissues after a 10 minute exposure was 7.0%.

It was considered unnecessary to proceed with tissue histopathology.

Qualitative Evaluation of Tissue Viability (MTT Uptake Visual Assessment)

The qualitative evaluation of tissue viability is presented in Table 2

The test material treated tissues appeared white and were considered to be indicative of dead tissue. The negative control treated tissues appeared blue and were considered to be indicative of viable tissue. The positive control material treated tissues appeared blue/white and were considered to be indicative of semi‑viable tissue.

Table1               Assessment of Eye Irritation Potential – Viability of RHC Tissues

Material	Mean OD540	% Viability
Negative Control	0.981	100*
Positive Control	0.454	46.3
Test Material	0.069	7.0

* = The mean viability of the negative tissues is set at 100%

Table2               Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material	Score
	Tissue 1	Tissue 2
Negative Control	-	-
Positive Control	+	+
Test Material	++	++

MTT Visual Scoring Scheme of SkinEthic Tissues

-       =   Blue tissue (viable)

+      =   Blue/White tissue (semi viable)

++    =   Tissue completely white (dead)

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

According to the protocol followed the test material was considered to be Irritant (I).

Executive summary:

Introduction. 

The purpose of this study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.

Methods. 

The experimental design of the study consists of a test for Direct Reduction of MTT by the test materials, followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test material for 10 minutes. Triplicate tissues treated with 30 µl of Solution A served as the negative control and triplicate tissues treated with 30 µl of 1% w/v Sodium Dodecyl Sulphate served as the positive control.

At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured (OD₅₄₀). Data are presented in the form of % viability (MTT conversion relative to negative controls).

The test material was classified according to the following criteria:

i)                   If the % relative mean tissue viability was ≥ 60% the test material was considered to not likely to be a severe ocular irritant.

ii)                  If the % relative mean tissue viability was < 60% the test material was considered to be irritant.

Results. 

The relative mean viability of the test material treated tissues after a 10 minute exposure was 7.0%.

It was considered unnecessary to proceed with tissue histopathology.

Quality criteria. 

The quality criteria required for acceptance of results in the test were satisfied.

Discussion. 

Under the conditions of the test the test material was considered to be an irritant (I). However, a subsequent in vivo skin irritation test identified the test material as being corrosive to the skin. Therefore the corrosive results of the in vivo skin irritation test should also be taken into consideration when concluding the eye irritation potential of the test material.

Conclusion. 

According to the protocol followed the test material was considered to be Irritant (I).

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation/Corrosion

A study was performed to assess the irritancy potential of the test material to the skin of the New Zealand White rabbit in accordance with the following standardised guidelines: OECD Guidelines for the Testing of Chemicals No. 404 “Acute Dermal Irritation/Corrosion” (adopted) and Method B4 Acute Toxicity (Skin Irritation) of Commission Directive 2004/73/EC.

A single 3-minute semi-occluded application of the test material to the intact skin of one rabbit produced no corrosive effects.

A single 1-hour, semi-occluded application of the test material to the intact skin of one rabbit produced reactions indicative of dermal corrosion. The reactions included green coloured dermal necrosis and scabbing. The animal was killed for humane reasons immediately after the 24-hour observation, due to the irreversible effects

 

The test material was classified as corrosive Category 1B to rabbit skin (based on one rabbit only).

 

Supporting Information

The skin irritation potential of the test material was investigated in vitro using the EPISKIN reconstituted human epidermis model. Under the conditions of the test, the test material was considered to be non irritant. However, the in vivo skin irritation test identified the test material as being corrosive. The in vitro skin irritation test is designed to identify irritant substances by their ability to induce reversible damage to the skin and cause an inflammatory response which can be quantified by measuring the release of inflammatory mediators. The test was not designed to identify corrosive materials and therefore the corrosive results of the in vivo skin test should be considered to override the non irritant result in this study.

 

Eye Irritation/Corrosion

An in vivo eye irritation study, as requested in REACH Annex VIII, was not conducted as it was considered unjustified. This was based upon:

- an in vitro eye irritation study which considered the substance to be an eye irritant.

- an in vivo skin irritation study which classified the substance as corrosive to the skin

 

REACH Annex VIII; column 2 states for in vivo eye irritation:

The study does not need to be conducted if:

- the substance is classified as irritating to eyes with risk of serious damage to eyes, or

- the substance is classified as corrosive to the skin and provided that the registrant classified the substance as eye irritant.

 

The eye irritation potential of the test material was investigated using the SkinEthic Reconstituted Human Corneal model (HCE). According to the protocol followed the test material was considered to be Irritant (I).

However, skin corrosive substances shall be considered as leading to serious damage to the eyes as well (Category 1).

Justification for selection of skin irritation / corrosion endpoint:
The key study was conducted in accordance with the standardised guidelines OECD Guidelines for the Testing of Chemicals No. 404 “Acute Dermal Irritation/Corrosion” (adopted) and Method B4 Acute Toxicity (Skin Irritation) of Commission Directive 2004/73/EC. It complies with GLP and was awarded a reliability score of 1 in accordance with the principles laid out by Klimisch (1997).

Justification for selection of eye irritation endpoint:
The key study was conducted using the SkinEthic Reconstituted Human Corneal model (HCE). It complies with GLP and was awarded a reliability score of 1 in accordance with the principles laid out by Klimisch (1997).
No in vivo study was performed as the test material was classified as Corrosive Category 1B to skin. Skin corrosive substances shall be considered as leading to serious damage to the eyes as well (Category 1).

Effects on skin irritation/corrosion: corrosive

Effects on eye irritation: irritating

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation 1272/2008, the test material is classified as Corrosive Category 1B to skin. As a result of this, it shall be considered as leading to serious damage to the eyes as well (Category 1).