By-product from Guanidinoacetic acid manufacturing

Endpoint summary

Description of key information

Three GLP-Studie according to the OECD Guidelines 442C, 442D and 442E have been conducted to assess the skin sensitizing potential of By-product from Guanindinoacetic acid manufacturing.

In the DPRA-Study accoridng to OECD 442C, protein depletion could be observed.

In the KeratinoSens(r)-Study (442D) and in the h-CLAT-Study (442E) no adverse effect could be observed.

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:

2021-11-24 - 2021-07-29

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 KeratinoSens™ test method)

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Justification for non-LLNA method:

The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. The ARE-Nrf2 luciferase test method makes use of an immortalised adherent cell line (e.g. KeratinoSens™) derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances. Therefore, this test method is considered to be able to detect chemicals that cause skin sensitisation when used within Integrated Approaches to Testing and Assessment (IATA), together with other relevant complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as non-testing methods, including read-across from chemical analogues. Depending on the regulatory framework, positive results generated with these methods may be used on their own to classify a chemical into UN GHS category 1.

Specific details on test material used for the study:

201009MA2 solid

Details of test system:

Keratinoses transgenic cell line [442D]

Vehicle / solvent control:

water

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

1.07 %

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and
non-sensitisers.

In the present study By-product from Guanidinoacetic acid manufacturing was dissolved in dist. water.

Based on a calculated molecular weight of 77.20 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (I_max) induction of 1.53 was determined at a test item concentration of 2000 µM, but increase was not statistically significant (p <0.05) compared to the negative control. The corresponding cell viability was 71.9%. At the next lower concentration of 1000 µM, a statistically significant luciferase activity of 1.52 was determined. The corresponding cell viability was 82.1%. No further induction about the threshold of 1.5 was observable. Microscopically, slight cytotoxic effects were observed at the two highest test item concentrations. The calculated EC_1.5 was <1000 µM (850.55). Due to the microscopically observed cytotoxic effects, the not statistically significant I_max and the missing dose-response relationship, the test material is considered to be a non-sensitiser in the first experiment.

In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range and no EC_1.5 value could be calculated. Therefore, the test material is considered to be a non-sensitiser in the second experiment.

Under the condition of this study the test item is therefore considered as non-sensitiser

The controls confirmed the validity of the study.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Justification for non-LLNA method:

The correlation of upregulation of immunologically relevant cell surface markers with the skin sensitising potential of a chemical has been reported and represents the third key event of the skin sensitisation process as described by the AOP for skin sensitisation. This method, which measures the markers of DC activation, using the DC-like cell line THP-1 is considered relevant for the assessment of the skin sensitisation potential of chemicals. Therefore, this test method is considered to be able to detect chemicals that cause skin sensitisation when used within Integrated Approaches to Testing and Assessment (IATA), together with other relevant complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as non-testing methods, including read-across from chemical analogues. Depending on the regulatory framework, positive results generated with these methods may be used on their own to classify a chemical into UN GHS category 1.

Specific details on test material used for the study:

Batch No.: 201009MA2 solid

Details of test system:

THP-1 cell line [442E]

Vehicle / solvent control:

water

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC200, CD54 [442E]

Value:

110 %

Cell viability:

> 93 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC150, CD86 [442E]

Value:

100 %

Cell viability:

> 93 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs. Therefore, the test item might be considered as non-sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.

In the present study By-product from Guanindinoacetic acid manufacturing was dissolved in water. For the dose finding assay stock solutions with concentrations ranging from 100 mg/mL to 0.78 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:

1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL

In all experiments no precipitation or turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 96.6% (CD86), 96.3% (CD54) and 96.1% (isotype IgG1 control) in the first experiment and to 96.1% (CD86), 93.0% (CD54) and 97.1% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item can be considered as non-sensitiser.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-01-12 - 2021-07-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

According to REACH regulation Annex VII, 8.3.2 column 2 an in vivo test (LNNA is preferred) will only be performed, when the in chemico / in vitro
methods according to 8.3.1, column 1 are not applicable for the test substance or the are not convincing.

The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP. It is therefore a crucial step for the sensitising potential of a chemical.

This test method is able to detect chemicals that cause skin sensitisation and allows for hazard identification in accordance with UN GHS “Category 1”.

Specific details on test material used for the study:

Batch No.: 201009MA2 solid

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Vehicle / solvent:

water

Positive control:

cinnamic aldehyde

Key result

Run / experiment:

run/experiment 1

Parameter:

cysteine depletion

Value:

0.73 %

At concentration:

100 mM

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Key result

Run / experiment:

run/experiment 1

Parameter:

cysteine depletion

Value:

13.7 %

Vehicle controls validity:

not examined

Negative controls validity:

not specified

Positive controls validity:

valid

Key result

Run / experiment:

run/experiment 2

Parameter:

cysteine depletion

Value:

16.52 %

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Key result

Run / experiment:

run/experiment 1

Parameter:

lysine depletion

Value:

0 %

At concentration:

100 mM

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Key result

Run / experiment:

run/experiment 1

Parameter:

lysine depletion

Value:

0 %

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Key result

Run / experiment:

run/experiment 2

Parameter:

lysine depletion

Value:

0.34 %

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Interpretation of results:

other: According to the key results of this study "By-product from Guanidinoacetic acid manufacturing" has to be classified as skin sensitiser. As the other studies gave a negative result, the Substance does not have to be classified.

Conclusions:

In this study under the given conditions the 100 mM stock solution of the test item showed minimal reactivity towards both peptides in the first experiment. Due to the observed precipitation the prediction model does not apply and a prediction cannot be made.
In this study under the given conditions the maximum solubility stock solution of the test item showed low reactivity towards both peptides in both experiments. The test item is considered as “sensitiser”.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

In the present study By-product from Guanindinoacetic acid manufacturing was dissolved in water, based on the results of the pre-experiments. Based on a molecular weight of 77.20 g/mol a 100 mM stock solution was prepared (first experiment) and additionally the neat test item was tested to its maximum solubility (first and second experiment). The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

 

All test item solutions were freshly prepared immediately prior to use.

 

Experiment 1 (concentrations: 100 mM and maximum solubility):

In the cysteine experiment, for the 100 mM stock solution and the maximum solubility of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item including the co-elution control at both concentrations, 100 mM and maximum solubility. Samples were centrifuged prior to the HPLC analysis. The observed precipitates may be residual single suspended solids, which were not completely removed by the filtration step.

In the lysine experiment, for the 100 mM stock solution and the maximum solubility of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the
24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item (100 mM and maximum solubility). Precipitation was observed for the samples of the positive control (including the co-elution control). Samples of the positive control were centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitations were regarded as not relevant.

 

Experiment 2 (concentration: maximum solubility):

In the cysteine experiment, for the maximum solubility of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

In the lysine experiment, for the maximum solubility of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Turbidity and phase separation was observed for the samples of the positive control. Samples of the positive control were centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed turbidity and phase separation were regarded as not relevant.

 

In the first experiment, no co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C_water).

In the first cysteine experiment, precipitation was observed at both concentrations, 100 mM and maximum solubility. The observed precipitates may be residual single suspended solids, which were not completely removed by the filtration step. Since it cannot be determined if the precipitate resulted from the test item or the peptide, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was < 6.38% (0.37%). According to the evaluation criteria in the guideline, if a precipitation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. Due to the observed precipitation in the cysteine experiment no prediction can be made.

The maximum solubility stock solution of the test item (100 mg/mL) showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (6.85%). Even though a precipitate was observed a positive result can still be used. According to the evaluation criteria in the guideline, for test items with a combined cysteine/lysine peptide depletion between 3% and 10% a second run should be considered. Thus, no prediction can be made.

 

Therefore, a second experiment with the maximum solubility concentration (100 mg/mL) was performed.

In the second experiment, no co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C_water).

The 100 mg/mL stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (8.43%). Based on the prediction model 1 the test item can be considered as sensitiser.

 

Therefore, in the second experiment the results of the first experiment were confirmed and the test item can be considered as sensitiser.

 

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 65.83% (first experiment) and 67.02% (second experiment).

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

In three in vitro studies to assess the skin sensitising potential of By-product from Guanidinoacetic acid manufacturing the three key events (KE) of skin sensitisation were addessed:

• KE1: protein binding


• KE2: keratinocyte activation


• KE3: dendritic cell activation

According to the OECD Guideline 497 (2021) "Guideline on Defined Approaches for Skin Sensitisation" a substance does not have to be classified as skin sensitiser if at least two assessed key events showed a negative result.