Neodymium trinitrate

Endpoint summary

Description of key information

The test item showed toxicity (IC30 values of 46 μM and 51 μM and IC50 values of 1352 μM and 1101 μM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.13-fold and 1.10-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 March 2020 to 2 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: NDNC-191202-BZ
- Expiration date of the lot/batch: 12 December 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under storage conditions: Assumed to be stable
- Stability under test conditions: Assumed to be stable
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Soluble in DMSO

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: stock solution prepared in DMSO or Milli-Q. Two-fold dilution series prepared in DMSO or Milli-Q.
- Final dilution of a dissolved solid, stock liquid or gel: Final dilution carried out in culture medium

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
Cells will be subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells will be grown for more than one passage from the frozen stock, and will not be cultured for more than 25 passages from the frozen stock.
For testing, cells should be 80-90% confluent, and care should be taken to ensure that cells are grown to full confluence. One day prior to testing cells are harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. One plate is used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells are incubated overnight.
The medium is removed and replaced with fresh culture medium (150 μl culture medium containing serum but without Geneticin) to which 50 µL of the 25 fold diluted test chemical and control substances are added. At least one well per plate should be left empty (no cells and no treatment) to assess background values. The treated plates are then incubated for
48 hours ± 1 h at 37±1.0oC in the presence of 5% CO2.
200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) is added to each well. The plates are then shaken for at least 5 minutes at room temperature. Plates with the cell lysates are then placed in the luminometer to assess the quantity of luciferase
For the KeratinoSensTM cell viability assay, medium is replaced after the 48 hour exposure time with fresh medium containing MTTand cells incubated for 3 - 4 hours at 37°C ± 1.0°C in the presence of 5% CO2. The MTT medium is then removed and cells are lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption is measured at 570 nm

Positive control results:

The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
The EC1.5 of the positive control was within two standard deviations of the historical mean (114 μM and 52 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold in the second experiment (2.88-fold). Although, the induction at 250 μM was not higher than 2-fold in the first experiment (1.99-fold), a clear dose-response was observed with increasing luciferase activity induction at increasing concentrations, and therefore the experiment is considered acceptable.

Key result

Run / experiment:

other: Expt 1

Parameter:

other: Imax

Value:

1.13 µM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Expt 2

Parameter:

other: Imax

Value:

1.1 µM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Expt 1

Parameter:

other: IC30

Value:

46 µM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Expt 1

Parameter:

other: IC50

Value:

1 352 µM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Excpt 2

Parameter:

other: IC30

Value:

51 µM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Expt 2

Parameter:

other: IC50

Value:

1 101 µM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: Yes

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed toxicity (IC30 values of 46 μM and 51 μM and IC50 values of 1352 μM and 1101 μM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.13-fold and 1.10-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.

Executive summary:

The objective of this study was to evaluate the ability of Neodymium Trinitrate to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

The study procedures described in this report were based on the most recent OECD guideline. Batch NDNC-191202-BZ of the test item was a purple to blue-pink solid. The test item was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98–2000 μM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. Precipitate was observed at test concentrations of 250 μM and upwards at the start and end of the incubation period in both experiments. Two independent experiments were performed.

Both tests passed the acceptance criteria:The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration; The EC1.5of the positive control was within two standard deviations of the historical mean (114 μM and 52 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold in the second experiment (2.88-fold). Although, the induction at 250μM was not higher than 2-fold in the first experiment (1.99-fold), a clear dose-response was observed with increasing luciferase activity induction at increasing concentrations, and therefore the experiment is considered acceptable; Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (10% and 9.1% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly. The test item showed toxicity (IC30values of 46 μM and 51 μM and IC50values of 1352 μM and 1101 μM in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.13-fold and 1.10-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSensTMassay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.

In conclusion, Neodymium Trinitrate is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes)

under the experimental conditions described in this report.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Principles of method if other than guideline:

The objective of this study was to generate a weight of evidence assessment on the endpointof skin sensitization for Neodymium trinitrate. A KeratinoSensTM assay was performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a. Only the KeratinoSensTM assay, assessing key event (KE) 2 of the skin sensitization adverse outcome pathway (AOP), could be performed. Testing for KE 1 of the skin sensitization AOP with the in chemico Direct Peptide Reactivity Assay (DPRA) assay was not applicable as Neodymium trinitrate is a metal compound and testing for KE 3 with the U-SENSTM assay was considered not applicable due to the cytotoxic properties of Neodymium trinitrate. The KeratinoSensTM assay was negative as no luciferase induction was observed after keratinocytes were exposed to Neodymium trinitrate. Additionally, the publicly available data on the metal Neodymium and its analogue salts, and the nitrate salts indicate that it is unlikely that Neodymium trinitrate is a skin sensitizer. Based on these considerations, sufficient evidence has been provided to conclude that Neodymium trinitrate is unlikely to be a skin sensitizer and does not need to be classified for skin sensitization according to Regulation (EC) No 1272/2008 and related amendments.

GLP compliance:

yes

Type of study:

ARE-Nrf2 luciferase LuSens test method

Specific details on test material used for the study:

Identification: Neodymium trinitrate
IUPAC name Neodymium(3+) trinitrate
CAS number 10045-95-1
EC number 233-153-9
Molecular formula Nd(NO3)3
Purity ≥ 99.9%
Molecular weight 438.35 g/mol

Details of test system:

Lusens transgenic cell line [442D]

Group:

test chemical

Run / experiment:

run/experiment 1

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

An in vitro study showed this substance to be non-sensitising

Executive summary:

The objective of this study was to generate a weight of evidence assessment on the endpoint of skin sensitization for Neodymium trinitrate.

A KeratinoSensTM assay was performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.

Only the KeratinoSensTM assay, assessing key event (KE) 2 of the skin sensitization adverse outcome pathway (AOP), could be performed. Testing for KE 1 of the skin sensitization AOP with the in chemico Direct Peptide Reactivity Assay (DPRA) assay was not applicable as Neodymium trinitrate is a metal compound and testing for KE 3 with the U-SENSTM assay was considered not applicable due to the cytotoxic properties of Neodymium trinitrate.

The KeratinoSensTM assay was negative as no luciferase induction was observed after keratinocytes were exposed to Neodymium trinitrate. Additionally, the publicly available data on the metal Neodymium and its analogue salts, and the nitrate salts indicate that it is unlikely that Neodymium trinitrate is a skin sensitizer. Based on these considerations, sufficient evidence has been provided to conclude that Neodymium trinitrate is unlikely to be a skin sensitizer and does not need to be classified for skin sensitization according to Regulation (EC) No 1272/2008 and related amendments.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Two in vitro studies on luciferase activity resuted in negative results. Therefore this substance is not classified as a skin sensitiser.