Tricobalt bis(orthophosphate)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

LLNA:BrdU

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 January 2023 - 17 January 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

adopted 22 July 2010

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

Tricobalt bis(orthophosphate)

EC Number:

236-655-6

EC Name:

Tricobalt bis(orthophosphate)

Cas Number:

13455-36-2

Molecular formula:

Co3(PO4)2

IUPAC Name:

tricobalt bis(orthophosphate)

impurity 1

Reference substance name:

Dicobalt diphosphate

EC Number:

238-689-7

EC Name:

Dicobalt diphosphate

Cas Number:

14640-56-3

Molecular formula:

Co2P2O7

IUPAC Name:

dicobalt diphosphate

impurity 2

Reference substance name:

cobalt phosphate octahydrate

Cas Number:

10294-50-5

Molecular formula:

Co3(PO4)2.8H2O

IUPAC Name:

cobalt phosphate octahydrate

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Remarks:

CBA/J (CBA/JRj)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
Female CBA/J strain mice (SPF caw) were supplied by Charles-River (69210 Saint-Germain-Nuelles).
On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant.
After an acclimatisation period of at least five days under stabling and nutritional conditions identical to those of the test, the animals were selected at random and given a unique number within the study by indelible ink-marking on the tail and a number written on a cage card.
At the start of the main study the animals were 8 or 11 weeks old.
The animals were weighed at the beginning and at the end of the study

HOUSING
The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
The temperature and relative humidity were controlled to remain within target ranges of 19°C to 25°C and 30% to 70%, respectively.
The rate of air exchange was at least ten changes per hour and the lighting was controlled by a time
switch to give twelve hours continuous light (07.00 to 19.00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

FOOD AND DRINK
The drinking water (tap water from public distribution system) and food (ENVIGO 2916) were supplied ad libitum.
Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas – Eurofins (FRANCE).

ANIMAL WELFARE
The standard study plan related to this study was approved by the registered Ethics Committee No. 76.
The study was performed in accordance with the guidelines regarding the care and use of animals for experimental procedures:
- the European Communities Council Directive 2010/63/UE of 22 September 2010,
- the French Decree No. 2013-118 of 01 February 2013 and French Decree No. 2020-274 of 17 March 2020 amending certain provisions relating to the protection of animals used for scientific purposes.
The animals were provided with suitable environmental enrichment (Tunnel).
The study was designed and was conducted to cause the minimum suffering or distress to the animals, according to the guidelines and to our internal animal welfare’s procedure.
At the end of the test, the animals were euthanized with sodium pentobarbital (Dolethal®).

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

50%; 25% and 10% in PG

No. of animals per dose:

4 female mice per test group
4 female mice for negative control (vehicle)

Details on study design:

PRELIMINARY STUDY:
Regarding the irritant potential of the test item predicted in the safety data sheet, and in accordance with the animal welfare, a preliminary screening test was performed using one mouse at the tested concentration of 60%. The mouse was treated by daily application of 25 µL of the test item diluted at 60% in PG to the dorsal surface of each ear for three consecutive days (Day 1, Day 2 and on Day 3).
The animal was observed daily from Day 1 to Day 6. Any signs of toxicity or excessive local irritation noted during this period were recorded.
Ear thickness was recorded on Day 1, Day 3 and on Day 6.

MAIN STUDY:
Test item administration:
Groups of four mice were treated with the test item diluted at 50%, 25% and 10% in PG. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.
A fifth additional mouse (*) is systematically treated in each group in case of problem which may occur during the study, in particular during the excision of lymph nodes. As the mouse Sf4676 was found dead on day 6, the lymph nodes of the additional mouse Sf4677 were collected and the data concerning this animal were used in the study. Furthermore, the ELISA test is systematically performed with this fifth additional mouse in the control group 1. The extreme value is ruled out for the calculation of BrdU Index.
The assay has undergone extensive inter-laboratory validation and has shown to reliably detect test materials that are moderate to strong sensitizers.
The strain of mouse used in these laboratories had been shown to produce satisfactory responses using known sensitizers and non-sensitizers during the in-house validation.
The results of the study were believed to be of value in predicting the sensitisation potential of the test item to man.
Clinical observations:
- Clinical observations and mortality: All animals were observed daily on days 1, 2, 3, 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Bodyweights: The bodyweight of each mouse was recorderd on day 1 (prior to dosing) and on day 6 (prior to termination).
- Ear thickness measurements and recording of local reactions:
Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses.
On Day 1 and on Day 3 (before application) as well as on Day 6 (after sacrifice) of each experiment, the thickness of the right ear of each animal of the vehicle control and treated groups was measured by a micrometer.
Furthermore, on Day 6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighed in order to assess the irritation potential of the test item and the two lymph nodes per mouse were weighed.
Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (dryness, presence of residual test item…) was noted.

BrdU INJECTION:
Day 5
0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route.
The BrdU solution was prepared by weighing 115.10 mg of 5-bromo-2'-deoxyuridine (SIGMA – Batch No. HMBJ1524) in a glass sample bottle and adding 11.51 mL of physiological saline (Dutscher, Batch No. C0866A01). Then, the preparation was stirred by vortex and magnetically for 55 minutes just before the administration to obtain a colourless solution.

TERMINAL PROCEDURES:
- Termination: On Day 6 (end of the test), the animals were euthanized with sodium pentobarbital (Dolethal®). The draining auricular lymph nodes from the four mice were excised.
-Preparation of Single Cell Suspension:rom each mouse, a single-cell suspension of lymph node cells (LNC) excised bilaterally was prepared by gentle mechanical disaggregation through a disposable plastic pestle to crush the lymph nodes followed by passage through a #70 nylon mesh in 15 mL of DPBS (Ca2+ / Mg2+ - free) into a well of a multi-well 6. The optimized volume was based on achieving a mean absorbance of the negative control group within 0.1- 0.2.

STIMULATION INDEX DETERMINATION:
BrdU was measured by ELISA using a commercial kit (e.g. Roche Applied Science, Mannheim, Germany, Catalogue Number 11 647 229 001 – Batch No. 57750800). Briefly, 100 µL of the LNC suspension was added to the wells of a flat-bottom microplate at least in triplicate. After fixation and denaturation of the LNC, anti-BrdU antibody was added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and the substrate solution was then added and allowed to produce chromogen. After 5 to 30 min, 30 µL of 1 M H2SO4 was added in each well, then shaken for one minute. Absorbance at 450 nm with a reference wavelength of 690 nm was then measured.
The BrdU labelling index was defined as:
BrdU labelling index = (ABSem -ABSblankem) - (ABSref - ABSblankref)
em = emission wavelength and ref = reference wavelength
Determination of cell proliferation:
Results were expressed as the Stimulation Index (SI).
Results for each treatment group were expressed as the mean SI. The SI was derived by dividing the mean BrdU labelling index/mouse within each test group by the mean BrdU labelling index for the control group.
SI = Mean BrdU labelling index/mouse Treated Group/ Mean BrdU labelling index/mouse Control Group
The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.6 compared to control values.
However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result (i.e. SI value between 1.6 and 1.9) is declared positive.
Any test item failing to produce a SI > 1.6 will be classified as a "non-sensitiser".

DETERMINATION OF THE EC1.6 VALUE:
The EC1.6 value (theoretical concentration resulting in a SI value of 1.6) was determined by linear interpolation of points on the dose-response curve, immediately above and below the 1.6-fold threshold. The equation used for calculation of EC1.6 was:
EC1.6 = c+[(1.6-d)/(b-d)]*(a-c) with
a = the lowest concentration giving stimulation index > 1.6
b = the actual stimulation index caused by a
c = the highest concentration failing to produce a stimulation index of 1.6
d= the actual stimulation index caused by c

INTERPRETATION OF THE RESULTS:
In accordance with the Regulation (EC) n° 1272/2008, the studies obtained for test item should be classified:
- If at least one concentration of the test item results in a SI ≥3, as sensitizer and characterised by the signal word 'Warning' with the hazard statement H317 'May cause an allergic skin reaction'.
The value 3 is based on the interpretation of [3H]-Thymidine incorporation in lymphocytes as descripted in the OECD test guideline n° 429.
According to the OECD test guideline n°442-B, the decision process regards a result as positive when SI ≥ 1.6. However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result (i.e. SI value between 1.6 and 1.9 ) is declared positive.
For a borderline positive response between a SI of 1.6 and 1.9, users may want to consider additional information such as dose-response relationship, evidence of systemic toxiicity or excessive irritation and where appropriate, statistical significance together with SI values to confirm that such results are positive. Consideration should be also given to various properties of the test substance, including whether it has a structural relationship to known skin sensitizers, whether it causes excessive skin irritation in the mouse, and the nature of the dose-response observed.

In accordance with the Regulation (EC) n° 286/2011, the positive test item will be classified in sub-category 1A or 1B in accordance with the following:
- Sub-category 1A: EC value ≤ 2%
- Sub-category 1B: EC value > 2%

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

In the positive control group given Hexylcinnamaldehyde (CAS n° 101-86-0 - purity 95%), the Stimulation Index expressed as the BrdU labelling idnex for each treatement group divide by the mean BrdU labelling index of the vehicle control group are as follows:
- C = 5%: SI = 1.12
- C = 10%: SI = 1.39
- C = 25%: SI = 1.95
In conclusion, in view of the results, under these experimental conditions, the substance Hexylcinnamaldehyde in accordance with the Regulation (EC) n° 1272/2008 has to be calssified in category 1 'Skin sensitization'.
The study was therefore considered valid.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

mean

Value:

2.02

Variability:

+/- 0.26

Test group / Remarks:

concentration = 10%

Results 2

Key result

Parameter:

SI

Remarks:

mean

Value:

2.81

Variability:

+/- 0.19

Test group / Remarks:

concentration = 25%

Results 3

Key result

Parameter:

SI

Remarks:

mean

Value:

2.87

Variability:

+/- 0.40

Test group / Remarks:

concentration = 50%

Results 4

Key result

Parameter:

other: EC1.6

Value:

7.92

Remarks on result:

other: %

Cellular proliferation data / Observations:

A stimulation index higher than 1.6 was recorded at the tested concentrations of 10%, 25% and 50%. The Stimulation Index (SI) calculated by individual approach was 2.02, 2.81 and 2.87 for the treated groups at 10%, 25% and 50%, respectively.
The EC1.6 determined by linear regression was 7.92%.

Any other information on results incl. tables

PRELIMINARY SCREENING TEST:

No mortality was noted at the tested concentrations of 60%.

Slight bodyweight loss was noted in the animal at the tested concentrations of 60%.

No sign of excessive irritation was noted at the tested concentration of 60%.

Therefore, 50% was chosen as the highest concentration for the main study.

MAIN TEST:

Clinical obsevations and mortality:

One animal was found dead on day 6 in animals treated at 50%.

No mortality was noted in the animals treated at 10% and 25% and control animals during the test.

A decrease in spontaneous activity (3/4) was noted in animals treated at 50%.

No signs of systemic toxicity were noted in the test animals treated at 25%, 10% and control animals during the test.

Weight evolution:

Bodyweight loss was noted in three animals of control group, one animal of  treated group at 10%, two animals of treated group at 25% and four animals of treated group at 50%.

Local irritation:

No significant increase in ear thickness and in ear weight were noted in animal treated at 10%, 25% and 50%, respectively. Therefore, the test item has to be considered as not excessively irritant at these concentrations.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The results obtained, under these experimental conditions, enable to conclude that the test item Tricobalt bis(orthophophate) has to be classified as a skin sensitizer in Category 1B, in accordance with the Regulation EC No. 1272/2008 on classification, labelling and packaging of substances and mixtures. The signal word “Warning” and hazard statement H317 “May cause an allergic skin reaction” are required.