Trimethylamine, N-oxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 23 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Trimethylamine, N-oxide

EC Number:

214-675-6

EC Name:

Trimethylamine, N-oxide

Cas Number:

1184-78-7

Molecular formula:

C3H9NO

IUPAC Name:

N,N-dimethylmethanamine oxide

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, batch no.160405
- Expiration date of the lot/batch: 31 January 2019
- Purity test date: no certificate of analysis was provided in the report but the purity was described at >98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature dessicated
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in acetonitrile
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test item stock solution was prepared freshly.
For both the cysteine and lysine reactivity assay 12.37 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1647 µL ACN after vortex mixing and 1 minute of sonication to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
Any residual volumes were discarded.

FORM AS APPLIED IN THE TEST (if different from that of starting material) in solution in acetonitrile

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system)
- Details on study design:

Test system:
Synthetic peptides containing cysteine (SPCC) (Ac RFAACAA COOH) or synthetic peptides containing lysine (SPCL) (Ac RFAAKAA COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.

Source: JPT Peptide Technologies GmbH, Berlin, Germany.
Trifluoroacetic acid (TFA) >99%, Sigma Aldrich, Zwijndrecht, The Netherlands
EXPERIMENTAL DESIGN
-Preparation of Solutions for Cysteine Reactivity Assay:

Synthetic Peptide Containing Cysteine (SPCC) Stock Solution:
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.

SPCC Reference Control Solutions :
Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN.

A SPCC calibration curve was prepared as described in the table below (section "any other information of material and methods incl. tables")

-Preparation of Solutions for Lysine Reactivity Assay

Synthetic Peptide Containing Lysine (SPCL) Stock Solution:
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.

SPCL Reference Control Solutions:
Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN.

SPCL Calibration Curve:
A SPCL peptide calibration curve was prepared as described in the table below(section "any other information of material and methods incl. tables")

-For cysteine and lysine, co-elution control, test item and positive control were prepared and details in tables below (section "any other information of material and methods incl. tables")

SAMPLE INCUBATIONS:

After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 25.5 hours and 24 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours. Prior to HPLC PDA analysis the samples were visually inspected for precipitation.

-HPLC-PDA Analysis:
SPCC and SPCL peak areas in the samples were measured by HPLC PDA

The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer (according to OECD TG 442C)

Results and discussion

Positive control results:

The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 76.8% ± 1.5%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 54.1% ± 1.8%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: TMAO

Parameter:

other: SPCC depletion (%)

Value:

20.4

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: TMAO

Parameter:

other: SPCL depletion (%)

Value:

0.2

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: TMAO

Parameter:

other: Mean of SPCC and SPCL depletion (%)

Value:

10.3

Other effects / acceptance of results:

Solubility Assessment of the Test Item
At a concentration of 100 mM, TMAO anhydrous was soluble in ACN. Therefore this solvent was used to dissolve the test item in this DPRA study.

Cysteine Reactivity Assay
The correlation coefficient (r2) of the SPCC standard calibration curve was 0.994. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.

The mean peptide concentration of Reference Controls A was 0.521 ± 0.011 mM while the mean peptide concentration of Reference Controls C was 0.523 ± 0.028 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.

The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 5.8%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The mean area ratio (A220/A258) of the Reference Control samples was 19.29. The mean A220/A258 ratio ± 10% range was 17.36-21.22. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

Lysine Reactivity Assay

The correlation coefficient (r2) of the SPCL standard calibration curve was 0.996. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.

The mean peptide concentration of Reference Controls A was 0.511 ± 0.016 mM while the mean peptide concentration of Reference Controls C was 0.515 ± 0.031 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.

The CV of the peptide areas for the nine Reference Controls B and C was 3.7%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The mean area ratio (A220/A258) of the Reference Control samples was 15.26. The mean A220/A258 ratio ± 10% range was 13.73-16.78. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

Any other information on results incl. tables

Table 7 : Acceptability of theDirect Peptide Reactivity Assay (DPRA)

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.994	>0.99	0.996
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.521 ± 0.011	0.50 ± 0.05	0.511 ± 0.016
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.523 ± 0.028	0.50 ± 0.05	0.515 ± 0.031
CV (%) for RC samples B and C	<15.0	5.8	<15.0	3.7
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	76.8	40.2-69.0	54.1
SD of peptide depletion cinnamic aldehyde (%)	<14.9	1.5	<11.6	1.8
SD of peptide depletion for the test item (%)	<14.9	3.3	<11.6	0.3

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

 

Table 8: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification forthe Test Item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
TMAO anhydrous	20.4%	±3.3%	0.2%	±0.3%	10.3%	Positive: Low reactivity

SD = Standard Deviation.

 

Applicant's summary and conclusion

Interpretation of results:

other:

Conclusions:

Under the experimental condition of the study, in the cysteine reactivity assay the test item showed 20.4% SPCC depletion while in the lysine reactivity assay the test item showed 0.2% SPCL depletion. The mean of the SPCC and SPCL depletion was 10.3% and as a result the test item was considered to be positive in the DPRA and classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The objective of this in chemico GLP compliant study was to determine the reactivity of TMAO anhydrous towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) according to OECD TG 442C method.

After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm.  SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.  

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA.

In the cysteine reactivity assay the test item showed 20.4% SPCC depletion while in the lysine reactivity assay the test item showed 0.2% SPCL depletion.  The mean of the SPCC and SPCL depletion was 10.3% and as a result the test item was considered to be positive in the DPRA and classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid.  TMAO anhydrous was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.