[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Remarks:

The substance is used exclusively in cosmetic products which fall within the scope of the Cosmetics Regulation. Hence, an in vitro study was conducted to build weight of evidence.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 Feb 2021 - 14 Apr 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

in vitro method. No standard guidelines available. Method sufficiently documented. Reliability 2

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test
The neutral red uptake (NRU) assay is used to determine cell viability as an indicator of acute toxicity. Neutral red penetrates cellular membranes, entering cells via non-ionic diffusion and accumulates intracellularly in lysosomes. Viable cells take up and retain the neutral red dye, while damaged or dead cells do not, therefore, the neutral red uptake assay can be employed as a direct measure of cell viability, using membrane integrity as the measured endpoint. Incorporated NR is released from the cells using a solubilization solution. The absorbance of the NR is quantified using a spectrophotometer.

- Short description of test conditions:
Neonatal human dermal fibroblast cultures (HDFn-XF) were obtained commercially as cryopreserved primary cells (Lifeline Cell Technology. Carlsbad, USA). They were originally derived from donor tissue with informed consent for the tissue to be used for research purposes, in adherence with the Human Tissue Act (UK) 2004. Xeno-free Fibrolife culture medium and sub-culture reagents (Cell Systems, Germany) are free of animal-derived components providing a fully human cell culture system.

- Parameters analysed / observed:
Viability

GLP compliance:

no

Remarks:

Work conducted in the spirit of the GLP and in line with the lab's status as a GLP laboratory approv ed by the UK Medicines and Health Regulatory Authority (MHRA).

Limit test:

no

Species:

other: in vitro; neonatal human dermal fibroblast (HDFn-XF)

Route of administration:

other: cell culture

Vehicle:

other: No vehicle. cell culture medium was used as solvent

Details on oral exposure:

A single application of 8 concentration of the test item (n=6) was applied in cell culture medium (d
ilution factor of 5 in the range finder, 1.3 in the main test). The top concentration was previously
determined by solubility testing.

Doses:

Range finder: 200,000; 40,000; 8000; 1600; 320; 64; 12.8; 2.56 ug/mL
Main test: 8000; 6153.85; 4733.73; 3641.33; 2801.022; 2154.63; 1657.41; 1274.93 ug/mL

Details on study design:

A single application of 8 concentration of the test item (n=6) was applied in cell culture medium (dilution factor of 5 in the range finder, 1.3 in the main test). A single application of 8 concentrations of the positive control (n=6) was applied in cell culture medium (dilution factor 1.2). A single application of culture medium was applied as negative control (n=12).

Statistics:

Standard IC50 regression based calculation method.

Preliminary study:

None

Dose descriptor:

other: IC50

Effect level:

723.28 other: ug/mL

Based on:

test mat.

Remarks:

Exposure duration 24+-1 hours

Remarks on result:

other: Main experiment 1: Invalid results due to high variability

Key result

Dose descriptor:

other: IC50

Effect level:

2 273.84 other: ug/mL

Based on:

test mat.

Remarks:

Exposure duration 24+-1 hours

Remarks on result:

other: Main experiment 2

Dose descriptor:

other: IC50

Effect level:

1 189.98 other: ug/mL

Based on:

test mat.

Remarks:

Exposure duration 24+-1 hours

Remarks on result:

other: Main experiment 3. Test item was below 50% viable at all concentrations tested and IC50 was calculated by extrapolation. These results were not considered in the interpretation.

Key result

Dose descriptor:

other: IC50

Effect level:

2 867.11 other: ug/mL

Based on:

test mat.

Remarks:

Exposure duration 24+-1 hours

Remarks on result:

other: Main experiment 4

GHS criteria used for comaprison of results:

IC50 (ug/mL)	Corresponding LD50 (mg/kg)	Statement	GHS criteria
ND	5-50	Fatal if swallowed	Cat 1 and 2
< 10	50-300	Toxic if swallowed	Cat 3
10-1000	300-2000	Harmful if swallowed	Cat 4
>1000	2000-5000	Potentially harmful if swallowed	Cat 5

 

Interpretation of results:

other: Not classified under CLP based on corresponding LD50 values.

Conclusions:

The test substance was not classified under CLP based on corresponding LD50 values.

Executive summary:

The neutral red uptake (NRU) assay is used to determine cell viability as an indicator of acute toxicity. Neutral red penetrates cellular membranes, entering cells via non-ionic diffusion and accumulates intracellularly in lysosomes. Viable cells take up and retain the neutral red dye, while damaged or dead cells do not, therefore, the neutral red uptake assay can be employed as a direct measure of cell viability, using membrane integrity as the measured endpoint. Incorporated NR is released from the cells using a solubilization solution. The absorbance of the NR is quantified using a spectrophotometer.

 

A single application of 8 concentration of the test item (n=6) was applied in cell culture medium (dilution factor of 5 in the range finder, 1.3 in the main test). A single application of 8 concentrations of the positive control (n=6) was applied in cell culture medium (dilution factor 1.2). A single application of culture medium was applied as negative control (n=12).

 

The endpoint was cell viability. IC50 was determined based on regression. In the 5 main experiments, 2 results were ignored because of the high variability in main experiment 1 and less than 50% viability at all concetrations in the main experiment 3. Based on the results in the other main experiments, the test subtance is not classified under CLP based on corresponding LD50 values.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

>= 2 000 - <= 5 000 mg/kg bw

Quality of whole database:

No study available on acute oral toxicity. Substance is used in cosmetics. In vitro study was conducted to build weight of evidence.

Justification for classification or non-classification

The endpoint was cell viability. IC50 was determined based on regression. In the 5 main experiments, 2 results were ignored because of the high variability in main experiment 1 and less than 50% viability at all concetrations in the main experiment 3. Based on the results in the other main experiments, the test subtance is not classified under CLP based on corresponding LD50 values.