Bisguanidinium phosphate

Endpoint summary

Description of key information

OECD 442C (DPRA), GLP, negative

Read-across: OECD 429 (LLNA), GLP, negative

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018/09/28 - 2018/10/04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

4 February 2015

Deviations:

yes

Remarks:

date of draft report delayed. No effect on study

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

The reactivity of a test chemical and synthetic cysteine or lysine containing peptides was evaluated by combining the test chemical with a solution of the peptide (reaction samples) and monitoring the remaining concentration of the peptide following 24 ± 2 hours of interaction time at room temperature (25 ± 2.5°C). The peptide is a custom material containing phenylalanine to aid in detection and either cysteine or lysine as the reactive centre. Relative concentrations of the peptide following the 24 hour reaction time were determined by HPLC with gradient elution and UV detection at 220 nm. Samples were prepared and analysed in triplicates in batches to keep the total HPLC analysis time less than 30 hours.

Cinnamaldehyde dissolved in acetonitrile was used as positive control. 100 mM solutions of the positive control chemical in acetonitrile were prepared just before use. 100 mM solutions of the test chemical in ultrapure water were prepared just before use.

Positive control results:

The acceptance criteria were met in case of the positive control with the cysteine peptide depletion value of 70.09 % and a mean lysine peptide depletion value of 57.24 %. The SD of the percent peptide depletions of the positive control was 0.65 % and 0.84 % for the cysteine and lysine depletion respectively.

Run / experiment:

other: 1

Parameter:

other: Cysteine peptide depletion [%]

Value:

6.55

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: 2

Parameter:

other: Cysteine peptide depletion [%]

Value:

7.76

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: 3

Parameter:

other: Cysteine peptide depletion [%]

Value:

12.74

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: 1

Parameter:

other: Lysine peptide depletion [%]

Value:

0.23

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: 2

Parameter:

other: Lysine peptide depletion [%]

Value:

0.53

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

other: 3

Parameter:

other: Lysine peptide depletion [%]

Value:

-0.29

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Negative depletion values are substituted with zero when calculating mean peptide depletion

Run / experiment:

mean

Parameter:

other: Cysteine peptide depletion [%]

Value:

9.02

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

mean

Parameter:

other: Lysine peptide depletion [%]

Value:

0.26

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Run / experiment:

mean

Parameter:

other: Peptide depletion [%]

Value:

4.64

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method, TOXI-COOP ZRT. demonstrated technical proficiency in a separate study (Study number.: 392-442-2996) by correctly obtaining the expected DPRA prediction for 10 proficiency substances as recommended in the OECD TG 442C guideline.

ACCEPTANCE OF RESULTS:
Two individual tests were conducted with the cysteine peptide, since the first one did not meet the acceptance criteria and had to be rejected. The second run was valid and accepted. Only one individual run was needed with the lysine peptide, which has fulfilled all the acceptance criteria. All in all, the results of the two valid runs were used for the classification of the test item.

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

The mean percent peptide depletion value of the test itemwas 4.64 %, which corresponded to a negative outcome. Results obtained from this in chemico Direct Peptide Reactivity Assay with the test item bisguanidinium phosphate indicated that the test item has no or minimal reactivity towards the synthetic peptides, thus is not a potential skin sensitiser.

Executive summary:

This study was undertaken to evaluate the skin sensitisation potential of the test item bisguanidinium phosphate in chemico. The Direct Peptide Reactivity Assay (DPRA) proposed the molecular initiating event of the skin sensitisation Adverse Outcome Pathway (AOP), namely protein reactivity, by quantifying the reactivity of the test chemical towards cysteine and lysine model synthetic peptides. At the beginning of the assay the solubility of the test chemical was assessed and ultrapure water was chosen as the appropriate solvent. Two individual tests were conducted with the cysteine peptide, since the first one did not meet the acceptance criteria and had to be rejected. The second run was valid and accepted. Only one individual run was needed with the lysine peptide, which has fulfilled all the acceptance criteria. All in all, the results of the two valid runs were used for the classification of the test item. The positive control replicates showed the expected percent peptide depletion values within acceptable limits with 70.09 % cysteine peptide depletion and 57.24 % lysine peptide depletion. The back-calculated values of the reference control replicates were within the expected molarity concentration range for the cysteine and lysine peptides, as well. The percent cysteine peptide depletion value of the test item was 9.02 % while the percent lysine peptide depletion was 0.26 %.The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides, therefore the Cysteine 1:10 / Lysine 1:50 prediction model was used for the discrimination between sensitisers and non-sensitisers. The mean peptide depletion of the test item was 4.64 %, which did not exceed the 6.38 % threshold of the applicable prediction model. The mean percent peptide depletion value of the test item was 4.64 %, which corresponded to a negative outcome. Results obtained from this in chemico Direct Peptide Reactivity Assay with the test item bisguanidinium phosphate indicated that the test item has no or minimal reactivity towards the synthetic peptides, thus is not a potential skin sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
please refer also to Read Across Statement attached in Section 13

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In this read-across approach data on guanidinium phosphate (CAS 5423-22-3, EC 226-551-9) is used to fill data gaps for bisguanidinium phosphate (CAS 5423-23-4, EC 226-552-4), in accordance with Regulation No 1907/2006 (REACH), Annex XI. The basis for this read-across is the “Read-Across Assessment Framework” (RAAF) (ECHA 2017). The read-across hypothesis for the analogue approach is that both salts bisguanidinium phosphate and guanidinium phosphate result in the identical cat- and anion in a water based environment and have consequently similar (eco)toxicological and fate properties. According to the RAAF this approach is covered by scenario 2: “Different compounds have the same type of effect(s)”.
“This scenario covers the analogue approach for which the read-across hypothesis is based on different compounds with qualitatively similar properties. For the REACH information requirement under consideration, the property investigated in a study conducted with one source substance is used to predict properties that would be observed in a study with the target substance if it were to be conducted. Qualitatively similar properties or absence of effect are predicted. The predicted property may be similar or based on a worst-case approach” (ECHA 2017).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
In this read-across approach data on guanidinium phosphate (CAS 5423-22-3, EC 226-551-9) is used to fill data gaps for bisguanidinium phosphate (CAS 5423-23-4, EC 226-552-4).
The target substance bisguanidinium phosphate and the source substance guanidinium phosphate are phosphate salts of the base guanidine.
With a pKb of 0.4, guanidine is a strong base. Therefore, in neutral water guanidine occurs fully protonated as its conjugate acid guanidinium cation. Most guanidine derivatives are in fact salts containing guanidinium cation as the conjugate acid. The source and the target substances are phosphate salts of guanidine consisting of guanidinium cation as conjugate acid and phosphate anion as conjugate base. The relation between guanidinium cation and the phosphate anion is 2:1 in the target substance and 1:1 in the source substance. As a consequence, the conjugate base in the target substance is a hydrogenphosphate (HPO4), whereas in the source substance it is a dihydrogenphosphate (H2PO4).

3. ANALOGUE APPROACH JUSTIFICATION
Based on the structural similarity, the similarity in physical chemical properties and the QSAR predicted similarity in the (eco)toxicological profile, the read-across approach is justified. Thus, the available studies for the source substance guanidinium phosphate were used to fill data gaps for bisguanidinium phosphate for several environmental and toxicological endpoints. Since phosphates are endogenous compounds occurring in nature in considerable amounts, the guanidinium ions are considered to trigger the (eco)toxicity of bisguanidinium phosphate.
To underline the negative results of the DPRA (OECD Guideline 442C), read-across was performed to the LLNA (OECD Guideline 429) available for the source substance guanidinium phosphate. This confirms that bisguanidinium phosphate is not skin sensitising.

4. DATA MATRIX
The data matrix is included in the Read Across Statement attached in Section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

assessment report

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research is an appropriate model for testing for contact hypersensitivity.

Key result

Parameter:

SI

Value:

0.5

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

50%

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 246, 442 and 388 DPM respectively. The mean DPM/animal value for the vehicle control group was 456 DPM.

Results Pre-screen test:

No irritation and no signs of systemic toxicity were observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from day 1 pre-dose values.White test substance remnants were present on the dorsal surface of the ears of all animals at 50% (days 1, 2 and 3), which did not hamper scoring of the skin reactions.

Based on these results, the highest test substance concentration selected for the main study was a 50% concentration.

Other results - main study:

No irritation of the ears was observed in any of the animals examined. White test substance remnants were present on the dorsal surface of the ears of all animals at 25 and 50% (days 1, 2 and/or 3), which did not hamper scoring of the skin reactions.

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

 

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, guanidinium phosphate (1:1) was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 50%. Based on the structural and toxicological similarity, this result is also considered to be valid for the target substance bisguanidinium phosphate.

Executive summary:

An LLNA skin sensitisation study was performed according to OECD/EC test guidelines with guanidinium phosphate (1:1). Mice were exposed up to 50% of the test substance. No irritation of the ears was observed in any of the animals examined. White test substance remnants were present on the dorsal surface of the ears of all animals at 25 and 50% (days 1, 2 and/or 3), which did not hamper scoring of the skin reactions. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals. The SI values calculated for the substance concentrations 10, 25 and 50% were 0.5, 1.0 and 0.9 respectively. Since there was no indication that the test substance elicited an SI≥3 when tested up to 50%, guanidine phosphate (1:1) was considered not to be a skin sensitiser. Based on the structural and toxicological  similarity, this result is also considered to be valid for the target substance bisguanidinium phosphate. Thus, it is not classified according to Regulation (EC) 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

OECD 442C (DPRA)

This study was undertaken to evaluate the skin sensitisation potential of the test item bisguanidinium phosphate in chemico. The Direct Peptide Reactivity Assay (DPRA) proposed the molecular initiating event of the skin sensitisation Adverse Outcome Pathway (AOP), namely protein reactivity, by quantifying the reactivity of the test chemical towards cysteine and lysine model synthetic peptides. At the beginning of the assay the solubility of the test chemical was assessed and ultrapure water was chosen as the appropriate solvent. Two individual tests were conducted with the cysteine peptide, since the first one did not meet the acceptance criteria and had to be rejected. The second run was valid and accepted. Only one individual run was needed with the lysine peptide, which has fulfilled all the acceptance criteria. All in all, the results of the two valid runs were used for the classification of the test item. The positive control replicates showed the expected percent peptide depletion values within acceptable limits with 70.09 % cysteine peptide depletion and 57.24 % lysine peptide depletion. The back-calculated values of the reference control replicates were within the expected molarity concentration range for the cysteine and lysine peptides, as well. The percent cysteine peptide depletion value of the test item was 9.02 % while the percent lysine peptide depletion was 0.26 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides, therefore the Cysteine 1:10 / Lysine 1:50 prediction model was used for the discrimination between sensitisers and non-sensitisers. The mean peptide depletion of the test item was 4.64 %, which did not exceed the 6.38 % threshold of the applicable prediction model. The mean percent peptide depletion value of the test item was 4.64 %, which corresponded to a negative outcome. Results obtained from this in chemico Direct Peptide Reactivity Assay with the test item bisguanidinium phosphate indicated that the test item has no or minimal reactivity towards the synthetic peptides, thus is not a potential skin sensitiser.

OECD 429 (LLNA) - Read-across

An LLNA skin sensitisation study was performed according to OECD/EC test guidelines with guanidinium phosphate (1:1). Mice were exposed up to 50% of the test substance. No irritation of the ears was observed in any of the animals examined. White test substance remnants were present on the dorsal surface of the ears of all animals at 25 and 50% (days 1, 2 and/or 3), which did not hamper scoring of the skin reactions. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals. The SI values calculated for the substance concentrations 10, 25 and 50% were 0.5, 1.0 and 0.9 respectively. Since there was no indication that the test substance elicited an SI≥3 when tested up to 50%, guanidine phosphate (1:1) was considered not to be a skin sensitiser. Based on the structural and toxicological  similarity, this result is also considered to be valid for the target substance bisguanidinium phosphate.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the congruent negative results of a DPRA with bisguanidinium phosphate and an LLNA performed with the source substance guanidinium phosphate the target substance bisguanidinium phosphate is not classified according to Regulation (EC) 1272/2008.