Diethyl bis(2-hydroxyethyl)aminomethylphosphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

• Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 1535, TA 1537, TA 98 and TA 100: negative with and without metabolic activation (similar to OECD 471, 1980, rel.2, WoE)


• Gene mutation (Ames (QSAR) - Alkylphosphate mechanism via OECD Toolbox v4.5): negative (2022, rel.2, WoE)


• Gene mutation (Ames (QSAR) - Iminium ion mechanism via OECD Toolbox v4.5): negative (2022, rel.2, WoE)


• Chromosome aberration (in vitro mammalian chromosome aberration test, OECD 473 equiv.): positive with and without metabolic activation (1982, rel. 1, Key)


• Gene mutation (mammalian cell gene mutation assay, thymidine kinase gene, OECD 490 equiv.): positive with and without metabolic activation (1981, rel. 1, Key)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
OECD QSAR Toolbox

2. MODEL (incl. version number)
v4.5

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CCOP(=O)(CN(CCO)CCO)OCC

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
[[Explain how the model fulfils the OECD principles for (Q)SAR model validation. Consider attaching the QMRF and/or QPRF or providing a link]
see TPRF information below

5. APPLICABILITY DOMAIN
[Explain how the substance falls within the applicability domain of the model]
see TPRF information below

6. ADEQUACY OF THE RESULT
see TPRF information below

Prediction summary
Predicted endpoint: Gene mutation; No effect specified; No species specified; No duration specified; No guideline specified
Predicted value: Negative
Unit/scale: Gene mutation I
Data gap filling method: Read-across analysis
Summary: Prediction of potential for Ames mutagenicity in E.coli and TA 102 strains via RA to nearest neighbors to the query compound which also share the the same alerts within the general mechanistic profiler "DNA binding by OECD" profiler. The formation of reactive iminium species is the proposed mechanism. The specific strains have not been assessed for the target compound, however studies on other strains were shown to be negative. Therefore by subcategorizing for these tertiary amine compounds which have available data in the missing strains it is shown that the nearest compounds to the target are all negative, therefore lending weight to the existing argument from the study in the other strains that the compound is negative for Ames mutagenicity.

Predicted value: Negative
Predicted endpoint (OECD Principle 1 - Defined endpoint): Human Health Hazards -> Genetic Toxicity -> Gene mutation -> E. Coli WP2 UvrA
Prediction plot: (see Figure 1 below)

Calculation approach (OECD principle 2 - Unambiguous algorithm): takes the highest mode value from the 5 nearest neighbours Active descriptor: log Kow (calculated)
Data usage: Maximal value* *When multiple values are available for the same chemical, their maximal value is taken in prediction calculations

Uncertainty of the prediction (OECD principle 4 - Uncertainty of the prediction):
The prediction is based on 5 values, 5 of them (100%) equal to predicted value Prediction confidence is measured by the p-value: 0.00412
Mechanistic interpretation: manually editable field Prediction of potential for Ames mutagenicity in E.coli and TA 102 strains via RA to nearest neighbors to the query compound which also share the the same alerts within the general mechanistic profiler "DNA binding by OECD" profiler. The formation of reactive iminium species is the proposed mechanism. In the general mechanistic profiler a mechanism is proposed whereby P450 metabolism leads to a reactive iminium species potentially leading to DNA adducts via an SN1 mechanism.
Adequacy of the prediction: manually editable field The specific strains have not been assessed for the target compound, however studies on other strains were shown to be negative. By subcategorizing for these tertiary amine compounds which have available data in the missing strains it is shown that the nearest compounds to the target are all negative, therefore lending weight to the existing argument from the study in the other strains that the compound is negative for Ames mutagenicity.
Target profiles (OECD principle 5 - Chemical and biological mechanisms)
Profiles used for grouping/subcategorization
Profile criteria -:- Target profiles
SN1 >> Iminium Ion Formation >> Aliphatic tertiary amines (DNA binding by OECD) (primary grouping) -:- SN1; SN1 >> Iminium Ion Formation; SN1 >> Iminium Ion Formation >> Aliphatic tertiary amines
Organic functional groups (subcategorization) -:- (Thio)Phosphonic acid derivatives; Alcohol; Aliphatic amine, tertiary; Amine, tertiary; Dihydroxyl derivatives; Phosphonate ester

Predefined
Profile criteria -:- Target profiles
US-EPA New Chemical Categories -:- Aliphatic Amines
Substance type -:- Discrete chemical; Organic; Mono constituent (predefined)
OECD HPV Chemical Categories -:- Tertiary Amines
General Mechanistic
Profile criteria -:- Target profiles
Protein binding by OECD -:- SN2; SN2 >> SN2 reaction at sp3 carbon atom; SN2 >> SN2 reaction at sp3 carbon atom >> Phosphonates
Protein binding by OASIS -:- SN2; SN2 >> Nucleophilic substitution at sp3 carbon atom; SN2 >> Nucleophilic substitution at sp3 carbon atom >> (Thio)Phosphonate esters
DNA binding by OASIS -:- SN2; SN2 >> Alkylation; SN2 >> Alkylation >> Alkylphosphates, Alkylthiophosphates and Alkylphosphonates
DNA binding by OECD -:- SN1; SN1 >> Iminium Ion Formation; SN1 >> Iminium Ion Formation >> Aliphatic tertiary amines
Endpoint Specific
Profile criteria -:- Target profiles
Protein binding alerts for Chromosomal aberration by OASIS -:- SN2; SN2 >> Protein and/or DNA alkylation; SN2 >> Protein and/or DNA alkylation >> Dialkyl Alkylphosphonates
in vivo mutagenicity (Micronucleus) alerts by ISS -:- Alkyl (C5) or benzyl ester of sulphonic or phosphonic acid; H-acceptor-path3-H-acceptor
Aquatic toxicity classification by ECOSAR -:- Aliphatic Amines; Esters; Esters (phosphate)
DNA alerts for AMES, CA and MNT by OASIS -:- No alert found
in vitro mutagenicity (Ames test) alerts by ISS -:- Alkyl (C<5or benzyl ester of sulphonic or phosphonic acid
Empiric
Profile criteria -:- Target profiles
Lipinski Rule Oasis -:- Bioavailable
Organic functional groups -:- (nested) Dihydroxyl derivatives
Chemical elements -:- Group 14 - Carbon C; Group 15 - Nitrogen N; Group 15 - Phosphorus P; Group 16 - Oxygen O
Organic functional groups -:- Alcohol; Amine, tertiary; Phosphonate ester; Dihydroxyl derivatives; Aliphatic amine, tertiary; (Thio)Phosphonic acid derivatives
Organic functional groups, Norbert Haider (checkmol) -:- Hydroxy compound; Alcohol; Primary alcohol; Amine; Tertiary amine; Tertiary aliphatic amine; Phosphonic acid derivative; Phosphonic acid ester
Groups of elements -:- Non-Metals
Structure similarity -:- [90%,100%]
Organic functional groups (US EPA) -:- Miscellaneous sulfide (=S) or oxide (=O); Aliphatic Carbon [-CH3]; Aliphatic Carbon [-CH2-]; Aliphatic Carbon [CH]; Hydroxy, aliphatic attach [-OH]; Amino, aliphatic attach [-N<]; Phosphite, aliphatic attach [-O-P]; Phosphine oxide [O=P]
log Kow (calculated): -1.94

Analogue(s) selection (OECD principle 3 - Applicability domain)
Database(s) used: - Genotoxicity OASIS
Category boundaries (applicability domain):
- Active descriptor(s) range:
- log Kow: from -2.48 to 2.01 target chemical is in domain
- Response range:
- 58-161 E.coli E. Coli WP2 UvrA TA 102 W3102 E.coli WP2 PKM 101 E. Coli: from Negative to Negative
Profilers: - SN1 >> Iminium Ion Formation >> Aliphatic tertiary amines (DNA binding by OECD) (primary grouping) target chemical is in domain
- Organic functional groups (subcategorization) target chemical is in domain
Additional data pruning: Cannot calculate X descriptor(s) 21 value(s) from 12 chemical(s)
Manually eliminated data points: none

Principles of method if other than guideline:

- Software tool(s) used including version: OECD QSAR Toolbox
- Model(s) used: v4.5
- Model description: see field 'Justification for non-standard information'
- Justification of QSAR prediction: see field 'Justification for type of information'

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SMILES: CCOP(=O)(CN(CCO)CCO)OCC

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

E. coli WP2

Key result

Species / strain:

other: E. coli WP2 and S. thyphimurium TA 102

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Remarks:

Negative prediction for Ames mutagenicity via data exclusively from studies involving E.coli or TA 102 strains not covered by existing experimental Ames studies on the query compound.

Conclusions:

In this prediction the initial categorisation was defined by the identified formation of iminium ion alert, with subsequent subcategorisation via empirical profilers to derive a negative prediction for Ames mutagenicity via data exclusively from studies involving E.coli or TA 102 strains not covered by existing experimental Ames studies on the query compound.

Executive summary:

The E. coli WP2 and S. thyphimurium TA 102 strains have not been assessed for the query structure experimentally, however studies on other strains were shown to be negative.

In order to specifically assess activity towards E. coli and S. thyphimurium TA 102, it was determined that defining an endpoint in the OECD QSAR Toolbox with these strains specifically, cross referenced against any profiling alerts specific to Ames mutagenicity would allow for a robust prediction for the query structure.

The alert for S_N1 reactions leading to iminium ions was triggered from the general mechanistic “DNA Binding by OECD” profiler. The mechanism proposed involves P450 metabolism of Aliphatic tertiary amines leads to a reactive iminium species potentially leading to DNA adducts via an S_N1 mechanism.

Using the above the prediction was able to derive a conclusion of negative from the nearest neighbours to the test substance structure. In each case, these tertiary amines have results on either S. thyphimurium TA 102, or E coli WP2.  From the below it can be determined that for the specific strains the nearest neighbours containing tertiary amines, which are analogous with the specific moiety of the test substance, all results in these strains are negative, which is in concordance with the existing study results available for the target substance on other strains.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
OECD QSAR Toolbox

2. MODEL (incl. version number)
v4.5

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CCOP(=O)(CN(CCO)CCO)OCC

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
[Explain how the model fulfils the OECD principles for (Q)SAR model validation. Consider attaching the QMRF and/or QPRF or providing a link]
see TPRF information below

5. APPLICABILITY DOMAIN
[Explain how the substance falls within the applicability domain of the model]
see TPRF information below

6. ADEQUACY OF THE RESULT
see TPRF information below

Prediction summary
Predicted endpoint: Gene mutation; No effect specified; No species specified; No duration specified; No guideline specified
Predicted value: Negative
Unit/scale: Gene mutation I
Data gap filling method: Read-across analysis
Summary:
Prediction of potential for Ames mutagenicity in E.coli and TA 102 strains via RA to nearest neighbors to the query compound which also share the the same alerts within the endpoint specific profiler "in vitro mutagenicity (Ames test) alerts by ISS", supported by corresponding alerts from the "DNA binding by OASIS" profiler. In both cases alkylation of DNA nucleophiles is the proposed mechanism. The mechanism is linked in the endpoint specific profiler to organophosphorus pesticides with known effects.
The specific strains have not been assessed for the target compound, however studies on other strains were shown to be negative. therefore by subcategorizing for these organophosphorus compounds which have available data in the missing strains it is shown that the nearest compounds to the target are all negative, therefore lending weight to the existing argument from the study in the other strains that the compound is negative for Ames mutagenicity. it is noteworthy however that the prediction reported is out of parametric domain, as no compound with a lower partition coefficient than the target compound was available. However it was noted that when assessing the same profiling involving any strains a prediction within the parametric domain was possible, therefore for the purposes of this assessment of compounds with values in these specific strains, the prediction is still sound.
Predicted value: Negative
Predicted endpoint (OECD Principle 1 - Defined endpoint): Human Health Hazards -> Genetic Toxicity -> Gene mutation -> E. Coli WP2 UvrA TA 102
Prediction plot: (see Figure 1 below)

Calculation approach (OECD principle 2 - Unambiguous algorithm):
takes the highest mode value from the 4 nearest neighbours Active descriptor: log Kow (calculated) Data usage: Maximal value* *When multiple values are available for the same chemical, their maximal value is taken in prediction calculations
Uncertainty of the prediction (OECD principle 4 - Uncertainty of the prediction):
The prediction is based on 4 values, 4 of them (100%) equal to predicted value Prediction confidence is measured by the p-value: 0.0123
Mechanistic interpretation: manually editable field Prediction of potential for Ames mutagenicity in E.coli and TA 102 strains via RA to nearest neighbors to the query compound which also share the the same alerts within the endpoint specific profiler "in vitro mutagenicity (Ames test) alerts by ISS", supported by corresponding alerts from the "DNA binding by OASIS" profiler. In the general mechanistic profiler a mechanism is proposed whereby the alkyl organophosphorus compound undergoes an SN2 nucleophilic attack from DNA bound Guanine at the N7 position, which ultimately forms a7-alkylguanine leaving group. This is supported by the equivalent alert in the endpoint specific profiler, which identifies a second mechanism, but states the major contributor to genotoxicity of organophosphorus compounds is Guanine N7-alkylation.
Adequacy of the prediction: manually editable field The E.coli and TA 102 strains have not been assessed for the target compound, however studies on other strains were shown to be negative. Therefore by categorizing for these organophosphorus compounds as discussed above and identifying those which have available data in the missing strains, it is shown that the nearest compounds to the target are all negative, therefore lending weight to the existing argument from the study in the other strains that the compound is negative for Ames mutagenicity. It is noteworthy however that the prediction reported is out of parametric domain, as no compound with a lower partition coefficient than the target compound was available. However it was noted that when assessing the same profiling involving any strains a prediction within the parametric domain was possible, therefore the profiling hypothesis could be considered within domain, and so for the purposes of this assessment of compounds with values in these specific strains, the prediction is considered sound.
Target profiles (OECD principle 5 - Chemical and biological mechanisms)
Profiles used for grouping/subcategorization
Profile criteria -:- Target profiles
Alkyl (C<5) or benzyl ester of sulphonic or phosphonic acid (in vitro mutagenicity (Ames test) alerts by ISS) (primary grouping) -:- Alkyl (C<5) or benzyl ester of sulphonic or phosphonic acid
DNA binding by OASIS (subcategorization) -:- SN2; SN2 >> Alkylation; SN2 >> Alkylation >> Alkylphosphates, Alkylthiophosphates and Alkylphosphonates
Chemical elements (subcategorization) -:- Group 14 - Carbon C; Group 15 - Nitrogen N; Group 15 - Phosphorus P; Group 16 - Oxygen O
Predefined
Profile criteria -:- Target profiles
US-EPA New Chemical Categories -:- Aliphatic Amines
Substance type -:- Discrete chemical; Organic; Mono constituent (predefined)
OECD HPV Chemical Categories -:- Tertiary Amines
General Mechanistic
Profile criteria -:- Target profiles
Protein binding by OECD -:- SN2; SN2 >> SN2 reaction at sp3 carbon atom; SN2 >> SN2 reaction at sp3 carbon atom >> Phosphonates
Protein binding by OASIS -:- SN2; SN2 >> Nucleophilic substitution at sp3 carbon atom; SN2 >> Nucleophilic substitution at sp3 carbon atom >> (Thio)Phosphonate esters
DNA binding by OASIS -:- SN2; SN2 >> Alkylation; SN2 >> Alkylation >> Alkylphosphates, Alkylthiophosphates and Alkylphosphonates
DNA binding by OECD -:- SN1; SN1 >> Iminium Ion Formation; SN1 >> Iminium Ion Formation >> Aliphatic tertiary amines
Endpoint Specific
Profile criteria -:- Target profiles
Protein binding alerts for Chromosomal aberration by OASIS -:- SN2; SN2 >> Protein and/or DNA alkylation; SN2 >> Protein and/or DNA alkylation >> Dialkyl Alkylphosphonates
in vivo mutagenicity (Micronucleus) alerts by ISS -:- Alkyl (C<5) or benzyl ester of sulphonic or phosphonic acid; H-acceptor-path3-H-acceptor
Aquatic toxicity classification by ECOSAR -:- Aliphatic Amines; Esters; Esters (phosphate)
DNA alerts for AMES, CA and MNT by OASIS -:- No alert found
in vitro mutagenicity (Ames test) alerts by ISS -:- Alkyl (C<5) or benzyl ester of sulphonic or phosphonic acid
Empiric
Profile criteria -:- Target profiles
Lipinski Rule Oasis -:- Bioavailable
Organic functional groups (nested) -:- Dihydroxyl derivatives
Chemical elements -:- Group 14 - Carbon C; Group 15 - Nitrogen N; Group 15 - Phosphorus P; Group 16 - Oxygen O
Organic functional groups -:- Alcohol; Amine, tertiary; Phosphonate ester; Dihydroxyl derivatives; Aliphatic amine, tertiary; (Thio)Phosphonic acid derivatives
Organic functional groups -:- Norbert Haider (checkmol) Hydroxy compound; Alcohol; Primary alcohol; Amine; Tertiary amine; Tertiary aliphatic amine; Phosphonic acid derivative; Phosphonic acid ester
Groups of elements -:- Non-Metals
Structure similarity -:- [90%,100%]
Organic functional groups (US EPA) -:- Miscellaneous sulfide (=S) or oxide (=O); Aliphatic Carbon [-CH3]; Aliphatic Carbon [-CH2-]; Aliphatic Carbon [CH]; Hydroxy, aliphatic attach [-OH]; Amino, aliphatic attach [-N<]; Phosphite, aliphatic attach [-O-P]; Phosphine oxide [O=P]
Log Know (calculated): -1.94

Analogue(s) selection (OECD principle 3 - Applicability domain)
Database(s) used: - Bacterial mutagenicity ISSSTY - Genotoxicity OASIS
Category boundaries (applicability domain): - Active descriptor(s) range: - log Kow: from -0.602 to 3.82 target chemical is out of domain –
Response range: - E. Coli WP2 UvrA TA 102 W3102 E.coli WP2 PKM 101 E. Coli: from Negative to Negative
Profilers: - Alkyl (C<5) or benzyl ester of sulphonic or phosphonic acid (in vitro mutagenicity (Ames test) alerts by ISS) (primary grouping) target chemical is in domain
- DNA binding by OASIS (subcategorization) target chemical is in domain
- Chemical elements (subcategorization) target chemical is in domain
Additional data pruning: none
Manually eliminated data points: none

Principles of method if other than guideline:

- Software tool(s) used including version: OECD QSAR Toolbox
- Model(s) used: v4.5
- Model description: see field 'Justification for non-standard information',
- Justification of QSAR prediction: see field 'Justification for type of information'

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SMILES: CCOP(=O)(CN(CCO)CCO)OCC

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

E. coli WP2

Key result

Species / strain:

other: E. coli WP2 and S. thyphimurium TA 102

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Remarks:

Negative prediction for Ames mutagenicity via data exclusively from studies involving E.coli or TA 102 strains not covered by existing experimental Ames studies on the query compound.

Conclusions:

Ames mutagenicity via data exclusively from studies involving E.coli or TA 102 strains and inclusive of all required strains was predicted to be negative by the SAR.

Executive summary:

The E. coli WP2 and S. thyphimurium TA 102 strains have not been assessed for the query structure experimentally, however studies on other strains were shown to be negative.

In order to specifically assess activity towards E. coli and S. thyphimurium TA 102, it was determined that defining an endpoint in the OECD QSAR Toolbox with these strains specifically, cross referenced against any profiling alerts specific to Ames mutagenicity would allow for a robust prediction for the query structure.

 

The endpoint specific profiler "in vitro mutagenicity (Ames test) alerts by ISS", supported by corresponding alerts from the "DNA binding by OASIS" profiler suggested the query structure may elicit methylation of N7 of guanine fragment in DNA, producing 7-methylguanine. The potential interaction with Guanine is of note because the GC pair is investigated in the already studied strains, therefore this effect would have already been picked up in the existing studies were it to occur, suggesting that this alerting mechanism does not occur in the query compound, further supporting the predicted negative result.

The prediction was performed using the alerts discussed above first using the endpoint specific alert and then subcategorised with the general mechanistic alert also. This lead to a conclusion of negative for mutagenicity from the nearest neighbours (determined by log K_OW) to the query structure. In each case, these organophosphates have results on either S. thyphimurium TA 102 and/or E. coli WP2.

 

Based on the identified activities in profilers relevant to the Ames mutagenicity endpoint as discussed above and identifying those compounds which have available data in the missing strains, it is shown that the nearest neighbours to the test substance are all negative, therefore lending weight of evidence to the existing argument from the Ames study in the other strains that the test substance has not been tested on are also negative for Ames mutagenicity test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

S. thyphimurium TA 102 or E. coli missing. Tested up to 2500 µg/plate without justification.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

4, 20, 100, 500 and 2500 µg/plate (main experiment and repetition with strain TA 98)
31.3, 62.5, 125, 250 and 500 µg/plate (repetition with strain TA 1535)

Vehicle / solvent:

- Solvents used: DMSO (for test substance and Trypaflavin) and deionized water (for Endoxan)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Trypaflavin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

DETERMINATION OF CYTOTOXICITY
- Method: degree of survival of treated cultures

Evaluation criteria:

The test substance is considered mutagenic if a biologically relevant and dose-dependent increase in the number of revertants exceeding twice the colony count of the corresponding solvent control is observed. This increase has to be reproducible in an independent second experiment.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: none reported

STUDY RESULTS: cf. "Any other information on results incl. tables"

HISTORICAL CONTROL DATA: not reported

Table 1: Ames test with and without metabolic activation

Metabolic Activation	Test Group	Dose Level (per plate)	Relative Revertant Colony Counts
			TA 1535	TA 1537	TA 98	TA 100
Without Activation	DMSO		1.00	1.00	1.00	1.00
	Test material	4 µg	2.46	0.46	1.64	0.72
		20 µg	1.77	0.96	1.62	0.87
		100 µg	1.04	1.00	1.64	1.35
		500 µg	1.00	1.16	1.45	0.91
		2500 µg	1.25	0.56	2.23	0.65
	Cyclophosphamid	150 µg	2.19*	-	-	1.30
	Trypaflavin	200 µg	-	***	0.41	-
With Activation	DMSO		1.00	1.00	1.00	1.00
	Test material	4 µg	2.00	1.33	1.17	0.83
		20 µg	1.92	1.40	2.25	0.92
		100 µg	2.01	1.07	2.61	0.83
		500 µg	2.11	1.11	2.09	1.10
		2500 µg	1.05	0.89	2.45	0.99
	Cyclophosphamid	150 µg	12.75*	-	-	2.79*
	Trypaflavin	200 µg	-	20.56*	35.61*	-

 

  -: not tested

    *: mutagenic effect

 ***: not determinable due to background growth

Table 2: Repetition of Ames test for Salmonella strains TA 98 and TA 1535

Metabolic Activation	Test Group	Dose Level (per plate)	Relative Revertant Colony Counts
			TA 1535	TA 98
Without Activation	DMSO		1.00	1.00
	Cyclophosphamid	150 µg	2.11*	-
	Trypaflavin	200 µg	-	***
With activation	DMSO		1.00	1.00
	Test material	4 µg	-	0.91
		20 µg	-	0.92
		31.3 µg	0.44	-
		62.5 µg	0.93	-
		100 µg	-	0.77
		125 µg	0.81	-
		250 µg	0.98	-
		500 µg	1.15	0.85
		2500 µg	-	0.69
	Cyclophosphamid	150 µg	10.63*	-
	Trypaflavin	200 µg	-	20.75*

Conclusions:

No genotoxicity was observed in in S. tythimurium TA 1535, 1537, 98 and 100 in the absence and presence of S9 at concentrations up to 2500 µg/plate.

Executive summary:

In a reverse gene mutation assay in bacteria conducted similarly to OECD TG 471, strains TA 1535, TA 1537, TA 98 andTA 100 of S. typhimurium were exposed to the test item diluted in dimethylsulfoxide at concentrations ranging from 4 to 2500 µg/plate in the presence or absence of mammalian metabolic activation using the plate incorporation method.  

 

There was no evidence of cytotoxicity and no increase of the number of revertant colonies was observed for any of the four tester strains. The positive controls, Cyclophosphamide and Trypaflavin induced an appropriate response. 

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Apr - 18 May 1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

2015

Deviations:

no

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian cell gene mutation test

Specific details on test material used for the study:

- Date received: 26 Mar 1981
- Color: amber
- Stability: 2 years
- Storage conditions: room temperature (~20°C), ambient humidity, in the dark

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: 3.7.2 clone of Fischer L5178Y cells
- Maintenance: Frozen stocks are maintained in liquid nitrogen; Laboratory cultures are maintained at a density less or equal to 1 x 10E6 cells/mL.

MEDIA USED
- Type and identity of media:
Growth medium: RPMI 1640 supplemented with 10% (v/v) horse serum, 2 mM glutamine, 22 mg/mL sodium
pyruvate, 50 mg/mL pluronic solution and penicillin-streptomycin
Treatment medium: growth media with reduced serum (5%)
Cloning medium: growth medium w/o pluronic solution and with noble agar (final concentration: 0.35%)
Selective medium: cloning medium with 4 µg/mL TFT
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

other: TK+/-

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 mix

Test concentrations with justification for top dose:

Experiment I
Without S9 mix: 31.3, 62.5, 125, 250, and 500 µL/L
With S9 mix: 250, 500, and 1000 µL/L

Experiment II
Without S9 mix: 200, 250, 300, 350, and 400 µL/L
With S9 mix: 1000, 1100, and 1200 µL/L

The highest chosen dose reduced the growth rate by 50-90% based on the results of the preliminary cytotoxicity test (for experiment I) and on the results of experiment I (for experiment II).

Vehicle / solvent:

- Solvent used: water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density: 6 x 10E5 cells/mL at the beginning of treatment; 3 x 10E6 cells cloned in soft agar for TFT resistant cells, 600 cells cloned in non-selective medium for viable counts.

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days after the end of the treatment, cells were plated for determination of the cloning efficiency and the mutation frequency on agar plates containing TFT selective medium. The agar plates were incubated for 10-12 days.

SELECTION AGENT: Trifluorothymidine (TFT)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth

Evaluation criteria:

The substance is considered mutagenic if it produces a dose-dependent increase in mutation frequency over three doses to a level at least 2.5 times the solvent control. If this increase is observed for the highest dose in a reproducible manner, than the substance is considered mutagenic even without an observed dose response.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

at ≥ 0.3 µL/mL without activation, at ≥ 1.1 µL/mL with activation

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

solvent control

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: None reported

RANGE-FINDING/SCREENING STUDIES:
- Doses were selected based on the results of a range-finding assay from a range producing 10-100% relative suspension growth.

STUDY RESULTS: see Tables below

HISTORICAL CONTROL DATA: not reported

Table 1: Experiment I - with and without metabolic activation

		w/o S9 mix		with S9 mix
Observed parameter		Relative cloning efficiency	Mutant frequency x 10E6	Relative cloning efficiency	Mutant frequency x 10E6
Negative control		76	63	161	108
		88	67	75	152
Solvent control		100	54	100	129
		100	70	100	130
Positive control (EMS 0.5 µL/mL w/o S9 mix, DMN 0.3 µL/mL with S9 mix)		99	522	19	1058
Test material	0.0313	147	36	-	-
(µL/mL)	0.0625	98	60	-	-
	0.125	115	55	-	-
	0.250	93	79	135	119
	0.500	74	241	166	161
	1.000	-	-	67	300

Table 2: Experiment II - with and without metabolic activation

		w/o S9 mix		with S9 mix
Observed parameter		Relative cloning efficiency	Mutant frequency x 10E6	Relative cloning efficiency	Mutant frequency x 10E6
Negative control		57	92	87	123
		83	54	140	117
Solvent control		100	55	100	114
		100	61	100	158
Positive control (EMS 0.5 µL/mL w/o S9 mix, DMN 0.3 µL/mL with S9 mix)		62	651	19	863
Test material	0.20	103	66	-	-
(µL/mL)	0.25	96	107	-	-
	0.30	55	201	-	-
	0.35	81	203	-	-
	0.40	50	297	-	-
	1.00	-	-	80	261
	1.10	-	-	45	348
	1.20	-	-	48	380

-: not tested

EMS: ethylmethylsulfonate

DMN: dimethylnitrosamine

Conclusions:

In this study, there was a concentration related positive response of induced mutant colonies over background.

Executive summary:

In a mammalian cell gene mutation assay performed similarly to the OECD TG 490, L5178Y TK+/- mouse lymphoma cells cultured in vitro were exposed to the test item diluted in water in the presence and absence of Rat S9 mammalian metabolic activation.  

The following concentrations were used:

 

Experiment I

Without S9 mix: 31.3, 62.5, 125, 250, and 500 µL/L

With S9 mix: 250, 500, and 1000 µL/L

 

Experiment II

Without S9 mix: 200, 250, 300, 350, and 400 µL/L

With S9 mix: 1000, 1100, and 1200 µL/L

 

The highest chosen dose reduced the growth rate by 50-90% based on the results of the preliminary cytotoxicity test (for experiment I) and on the results of experiment I (for experiment II).

 

 

An increase in mutation frequency was seen at concentrations of 0.25 µL/mL or greater, reaching significant levels at 0.30 µL/mL. In the presence of an activation system, the test item is less toxic and a significant increase in mutation frequency was seen at concentrations greater or equal to 1.10 µL/mL.

 

The positive controls did induce the appropriate response.  

 

In this study, there was a concentration related positive response of induced mutant colonies over background.

 

This study is classified as acceptable.

 

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Apr 1981 - 22 Sept 1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1983

Deviations:

no

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Date received: 26 Mar 1981
- Color: amber
- Stability: 2 years
- Storage conditions: room temperature (~20°C), ambient humidity, in the dark

Target gene:

Not applicable

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: 3.7.2 clone of Fischer L5178Y cells
- Maintenance: Frozen stocks are maintained in liquid nitrogen; Laboratory cultures are maintained at a density less or equal to 1 x 10^6 cells/mL.

MEDIA USED
- Type and identity of media:
Growth medium: RPMI 1640 supplemented with 10% (v/v) horse serum, 2 mM glutamine, 22 mg/mL sodium pyruvate, 50 mg/mL pluronic solution and penicillin-streptomycin
Treatment medium: growth media with reduced serum (5%)
Cloning medium: growth medium w/o pluronic solution and with noble agar (final concentration: 0.35%)
Selective medium: cloning medium with 4 μg/mL TFT

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

other: TK+/-

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 mix

Test concentrations with justification for top dose:

Experiment I
Without S9 mix: 31.3, 62.5, 125, 250, and 500 μL/L
With S9 mix: 250, 500, 1000, 1500, and 2000 μL/L

The highest chosen dose reduced the growth rate by 0-90% based on the results of the range-finding assay-

Vehicle / solvent:

- Solvent used: water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 3 x 10E5 cells/mL

DURATION
- Exposure duration: 4 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN (for cytogenetic assays): Giemsa (for abberations) and Hoechst stain (for sister chromatid exchange)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- After treatment, cells are resuspended in growth medium and incubated with agitation for 21 h. After addition of 0.1 µg/mL colcemid for additional 3 h, cells are harvested, exposed to hypotonic medium and fixed in Carboy's fixative. The cell suspension is distributed on slides and stained for the intended use.

NUMBER OF CELLS EVALUATED: 50

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 50

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (of 500 randomly chosen cells)

Evaluation criteria:

A substance is considered positive if it differs significantly at the 0.01 level by a Student's t-test (one-tailed for aberrations and two-tailed for sister chromatid exchange).

Statistics:

Student's t-test, p < 0.01 and p < 0.05

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

with and without activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 1.5 µL/mL with activation

Vehicle controls validity:

valid

Remarks:

solvent control

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE FINDING STUDIES:
- Doses were selected based on the results of a range-finding assay from a range producing 10-100% relative suspension growth.

Table 1: Results of chromosomal aberration test with and without metabolic activation

		w/o S9 mix				with S9 mix
Observed parameter		Cell count	Cells with aberrations (%)		Mitotic index (%)	Cell count	Cells with aberrations (%)		Mitotic index (%)
			Struct.	Numer.			Struct.	Numer.
Negative control		50	4	6	4.4	50	0	0	8.6
		50	2	0	6.3	50	4	2	9.6
Solvent control		50	0	0	5.8	50	4	4	10.2
		50	2	0	7.0	50	0	0	6.8
Positive control (EMS 0.5 µL/mL w/o S9 mix, DMN 0.2 µL/mL with S9 mix)		50	12**	8**	5.5	50	16**	10**	6.8
Test material	0.0313	50	2	2	5.5	-	-	-	-
(µL/mL)	0.0625	50	0	2	7.0	-	-	-	-
	0.1250	50	4	2	5.6	-	-	-	-
	0.2500	50	4	4	6.8	50	10*	0	9.4
	0.5000	50	24**	12**	8.8	50	6	8	8.4
	1.000	-	-	-	-	50	4	6	9.6
	1.500	-	-	-	-	50	32**	38**	6.8
	2.000	-	-	-	-	50	72**	34**	4.5

-: not tested

*: significantly greater than solvent control (p <0.05, Student's t-test)

**: significantly greater than solvent control (p <0.01, Student's t-test)

EMS: ethylmethylsulfonate

DMN: dimethylnitrosamine

Conclusions:

There was a concentration related positive response of chromosome aberration induced over background.

Executive summary:

In a mammalian cell cytogenetics assay (Chromosome aberration test), L5178Y cell cultures were exposed to the test item in water with and/or without Rat S9 metabolic activation.

The following concentrations were used:

Without S9 mix: 31.3, 62.5, 125, 250, and 500 μL/L

With S9 mix: 250, 500, 1000, 1500, and 2000 μL/L

 

The highest chosen dose reduced the growth rate by 0-90% based on the results of the range-finding assay.

 

In this assay, a significant increase in structural aberrations was seen at the highest concentration (0.5 µL/mL). In the presence of an activated system, there was a significant increase at 1.5 and 2 µL/mL.

In both the activation and nonactivation portions, there was also a significant increase in numerical aberrations at these concentrations.

 

Positive controls induced the appropriate response.  

 

There was a concentration related positive response of chromosome aberration induced over background.

 

This study is classified as acceptable.

 

 

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

• Micronucleus test (Combined OECD 474+489, 2022, rel. 1, key): negative up to the TG maximum recommended dose.


• Comet assay (Combined OECD 474+489, 2022, rel. 1, key): negative in liver and glandular stomach up to TG maximum recommended dose; equivocal in duodenum up to TG maximum recommended dose.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 December 2021 to 27 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to the guideline study, as a combined OECD 474+489 micronucleus and comet study. All validity criteria were met under both guidelines. The study was conducted under GLP.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Official Journal of the European Union No. L142, amendment to EC No. 440/2008, 14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Amber glass bottle, placed in refrigeration (2-8°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Test material concentrations were stirred and treated with ultra-sonic waves to obtain a homogeneous suspension.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Not stated
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Not stated
- Reactivity of the test material with the incubation material used (e.g. plastic ware): Not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): N/A
- Preliminary purification step (if any): N/A
- Final concentration of a dissolved solid, stock liquid or gel: 500, 1000, 2000 mg/kg/day
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: N/A

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han

Details on species / strain selection:

Crl: WI(Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Sex; DRF phase had 3 males and 3 females per group. Main study phase; control and 2000 mg/kg/day had 8 males per group (5 main study animals and 3 TK animals). At 500 or 1000 there were 5 males per group (main study only). Positive control EMS and CP there were 3 males per group (main study only).
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 170.4 - 185.3 (based on group means). Body weights of the rats at the start of the treatment were within 20% of the sex mean.
- Assigned to test groups randomly: Yes.
- Fasting period before study: Nofood
- Housing: Up to 5 animals of the same sex and same dose group were housed together. Housed in polycarbonate cages containing sterilized sawdust as bedding material equipped with water bottles.
- Diet: SM R/M-Z pellets (Sponsor: SSNIFF® Spezialdiäten GmbH, Soest, Germany) available ad libitum. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: Municipal tap water, available ad libitum in water bottles.
- Acclimation period: at least 5 days before commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 - 24 °C
- Humidity: 40 - 70 %:
- Air changes: Ten or more air changes per hour:
- Photoperiod: 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 17 Dec 2021 To: 27 Jan 2022

Route of administration:

oral: gavage

Vehicle:

Corn Oil (Supplier: Fagron, Capelle a/d Ijssel, The Netherlands)
- Justification for choice of solvent/vehicle: Was selected as shown to be suitable in previous in vivo studies using the test material and a solubility test
- Concentration of test material in vehicle: 50 - 200 mg/mL (based on dose level of 500 - 2000 mg/kg/day and dose volume of 10 mL/kg).
- Amount of vehicle (if gavage or dermal): Dose Volume of 10 mL/kg.

Details on exposure:

The test material was suspended in corn oil. Adjustment was made for specific gravity of the vehicle (0.92). In the main study an adjustment was made for specific density of the test material (1.161 g/mL). Test material concentrations were stirred and treated with ultra-sonic waves to obtain a homogeneous suspension. This resulted in yellow translucent suspensions for all formulations. Test material concentrations were dosed within 3 hours after preparation.

Duration of treatment / exposure:

3 consecutive days

Frequency of treatment:

Vehicle and the test material formulations were dosed daily
CP was dosed once daily.
EMS was dosed twice daily.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Negative control group

Dose / conc.:

500 mg/kg bw/day

Remarks:

Test. material

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Test material

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Test material

Dose / conc.:

200 mg/kg bw/day

Remarks:

Postive control - EMS (Comet)

Dose / conc.:

19 mg/kg bw/day

Remarks:

Positive control - CP (micronucleus)

No. of animals per sex per dose:

DRF phase had 3 males and 3 females per group. Main study phase; control and 2000 mg/kg/day had 8 males per group (5 main study animals and 3 TK animals). At 500 or 1000 there were 5 males per group (main study only). Positive control EMS and CP there were 3 males per group (main study only).

Positive control(s):

Alkaline Comet test: Ethyl Methanesulfonate (EMS; CAS no. 62-50-0, Sigma Aldrich, Steinheim, Germany):
-Justification for choice of solvent/vehicle: Based on guideline.
- Concentration of test material in vehicle: 20 mg/mL (based on dose level of 200 mg/kg/day and dose volume of 10 mL/kg) dissolved in physiological saline.
- Amount of vehicle: Dose Volume of 10 mL/kg.

Micronucleus test: Cyclophosphamide (CP; CAS No. 6055-19-2; Sigma Aldrich Chemie GmbH, Steinheim, Germany)
-Justification for choice of solvent/vehicle: Based on guideline.
- Concentration of test material in vehicle: 1.9 mg/mL (based on dose level of 19 mg/kg/day and dose volume of 10 mL/kg) dissolved in physiological saline.
- Amount of vehicle: Dose Volume of 10 mL/kg.

EMS positive control dose twice daily for 3 days via oral gavage.
CP positive control once daily for 3 days via oral gavage.

The positive controls were selected based on the guidelines.

Tissues and cell types examined:

Liver, glandular stomach and duodenum cell. Based on the visual scoring of at least 150 cells per tissue per animal.

Details of tissue and slide preparation:

Liver
The isolation method was based on the publication of Hu et al (2002). A portion of
0.6-0.7 gram from the liver was removed and minced thoroughly on aluminum foil in ice. The minced liver tissue was added to 10 mL of collagenase (20 Units/mL; Sigma Aldrich, Zwijndrecht, The Netherlands) dissolved in HBSS (Ca2+- and Mg2+-free) and incubated in a shaking water bath at 37 °C for 20 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40 g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359 g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+- and Mg2+-free) and kept on ice.

Isolation of glandular stomach cells
This isolation method for glandular stomach is based on the JACVAM Comet validation study Uno et al (2015). The stomach was cut open and washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca++, Mg++ free, Life Technologies, Breda, the Netherlands). The fore-stomach was removed and discarded. The glandular stomach was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA (Merck, Darmstadt, Germany)). The glandular stomach was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days. Therefore, this layer contains a high number of apoptotic cells which disturb the interpretation in the Comet assay. Moreover, since the lifetime of these cells is very short it is unlikely that these cells play a role in carcinogenesis (Uno et al (2015)). The glandular stomach was then rinsed with mincing buffer incomplete (without DMSO) and transferred to a petri-dish containing 10 mL mincing buffer. The glandular stomach was then scraped multiple times with a cell scraper and the cells were collected in the mincing buffer present in the petri-dish. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution, pH 7.5 (DMSO (Merck) was added immediately before use). The cell suspension was filtered through a 100 µm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.

Isolation of duodenum
This isolation method for duodenum is based on the JACVAM Comet validation study, Uno et al (2015).
The duodenum was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA without DMSO). The duodenum was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The duodeunum was cut open and the surface epithelia of the glandular epithelia were gently scraped 3-4 times with a cell scraper to remove apoptotic cells in the upper cell layer. This layer was discarded. The duodenum was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The duodenum was then scraped multiple times with a cell scraper and the cells are collected in the mincing buffer present in the petri-dish.
The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca++, Mg++ free, and phenol red free if available), pH 7.5 (DMSO was added immediately before use). The cell suspension was filtered through a 100 µm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.

Preparation of Comet Slides
To the cell suspension, melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 µL was layered on a pre-coated Comet slide (Trevigen) in duplicate. Three slides per tissue were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 45 to 47 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area.

Lysis, Electrophoresis and Staining of the Slides
The cells on the slides were immersed overnight (approximately 16 h) in pre-chilled lysis solution (Trevigen) in the refrigerator (set to maintain 4°C). After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4) in order to remove residual detergent and salts prior to the alkali unwinding step. The slides were then placed in freshly prepared alkaline solution for 20 and 30 minutes, for stomach/duodenum and liver, respectively, at room temperature in the dark to allow DNA unwinding. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 – 1 Volt/cm. The electrophoresis was performed for 20 to 30 minutes under constant cooling (actual temperature 4.0 - 4.5°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in absolut ethanol (99.6%, Merck) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.

Evaluation criteria:

To prevent bias, slides were randomly coded (per tissue) before examination of the Comets. An adhesive label with study identification number and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty Comets (50 comets of each replicate LMAgarose circle) were examined per sample.
The following criteria for scoring of Comets were used:
• Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.
In addition, the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal in the repeat experiment. The occurrence of hedgehogs was scored in all treatment groups and the control.

The in vivo comet is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The positive control EMS should produce at least a statistically significant increase in the percentage Tail Intensity compared to the vehicle treated animals. The response should be compatible with the data in the historical control database.
c) Adequate numbers of cells and doses have been analysed
d) The highest test dose is the MTD or 2000 mg/kg/day
ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis.

Statistics:

ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data .
A test material is considered positive in the Comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
When all criteria are met, the test chemical is considered to induce DNA strand breakage in the tissues examined within the test system.
A test material is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
When all criteria are met, the test chemical is considered not to induce DNA strand breakage in the tissues examined within the test system.
As the Multiple Sequentially-rejective U-test and Welch t test show that there are statistically significant differences between one or more of the test material groups and the vehicle control group a Linear regression (p < 0.05) was performed to test whether there is a significant trend in the induction.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF THE DOSE FORMULATION ANALYSIS:

Accuracy:
A small response at the retention time of the test material was observed in the chromatograms of the vehicle. It was considered not to derive from the vehicle since a similar response was obtained in the analytical blanks.

The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85%-115%).

Homogeneity:
The dose formulation samples were homogeneous (i.e. RSD ≤ 10%).The mean number of micronucleated polychromatic erythrocytes scored in test material treated groups were compared with the corresponding solvent control group.


RESULTS OF RANGE-FINDING STUDY

- Dose range: 2000 mg/kg bw administered to 3 male and 3 female animals for a total of 3 consecutive days
- Solubility: A solubility test was performed based on visual assessment.
- Clinical signs of toxicity in test animals: None
- Evidence of cytotoxicity in tissue analysed: None
- Rationale for exposure: Maximum according to test guideline
- Harvest times: N/A
- High dose with and without activation: N/A
- Other: N/A

RESULTS OF DEFINITIVE STUDY

- Tail Intenisity (Comet Assay)
A statistical significant increase (p<0.05) in the mean Tail Intensity (%) was observed in duodenum cells of test material treated male animals compared to the vehicle treated animals. The high and intermediate dose groups showed tail intensity increases compared to vehicle-treated animals and in addition a dose related response was observed (significant trend analysis (P<0.0001)). All the results obtained were within the 95% confidence interval of historical control database of the negative controls.

A statistically significant increase (p<0.05) in the mean Tail Intensity (%) was observed in glandular stomach cells of test item treated male animals compared to the vehicle treated animals for the low dose group (500 mg/kg body weight); the intermediate and high dose groups did not show a statistical increase. However, no dose related response was observed (confirmed by a trend analysis) and these values were within the historical control database of the negative controls.

No increase in the mean Tail Intensity (%) was observed in liver cells of test material treated male animals compared to the vehicle treated animals.


Ethyl Methanesulfonate, the positive control material, induced a significant increase and in the mean Tail Intensity in the male animals liver, duodenum and glandular stomach cells. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.

- Appropriateness of dose levels and route:
Dose level and route of exposure were selected according to test guideline and guided by a DRF study

Results Summary Tables

Overview Tail Intensity in Liver Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	2.09	0.35
Test Material 500 mg/kg	1.78	0.28
Test Material 1000 mg/kg	1.89	0.78
Test Material 2000 mg/kg	1.56	0.54
EMS 200 mg/kg	78.04	3.03

Overview Tail Intensity in Duodenum Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	3.90	1.84
Test Material 500 mg/kg	4.81	0.67
Test Material 1000 mg/kg	6.48	1.31
Test Material 2000 mg/kg	9.43	2.27
EMS 200 mg/kg	50.79	3.47

Overview Tail Intensity in Glandular Stomach Cells of Male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	5.23	0.97
Test Material 500 mg/kg	6.73	1.91
Test Material 1000 mg/kg	5.72	1.04
Test Material 2000 mg/kg	6.22	1.38
EMS 200 mg/kg	52.67	7.18

 

Conclusions:

The Comet assay undertaken on cells of male rats sampled 3-4 hours post dosing at up to 2000 mg/kg with the test item (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report as found to be valid. The test material is not genotoxic in the Comet assay in liver and glandular stomach cells. The assay was equivocal in duodenum cells as there was a significant dose-response shown however within historical control range of the vehicle control. No morphologic alterations were found which indicate that there was no cytotoxicity present.

Executive summary:

Study Objective

The objective of this study was to obtain information on the potential genotoxicity of the test material when administered to rats at the maximum recommended dose in accordance with current regulatory guidelines, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow and by measuring the increase in DNA strand breaks in liver, duodenum and glandular stomach.

 

Study Design

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic materials. Moreover, historical control background data has been generated with this strain.

 

The study procedures described in this report were based on the most recent OECD (29 July 2016) and EC (14 February 2017) guidelines.

 

The test material was a brownish-orange, slightly viscous liquid. The test material was suspended in corn oil.

 

Based on the results of the dose-range finding study, test concentrations of 2000 mg/kg/day for male animals was selected as maximum dose for the main test (the highest dose required in the current guideline). Since there were no substantial differences in toxicity between sexes only males were used in the main study.

 

A small response at the retention time of the test material was observed in the chromatograms of the The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85%-115%). The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%).

 

In the main study male animals were dosed three times by oral gavage with vehicle or with 500, 1000 and 2000 mg test material per kg body weight for three consecutive days. A positive control group for the comet assay was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight and a positive control group for the micronucleus assay was dosed once by oral gavage with 19 mg cyclophosphamide (CP) per kg body weight. In total 6 treatment groups were used, each consisting of 5 animals, with exception of the positive control and TK animals (3 animals per group).

 

In addition, blood for bioanalysis of the test material in plasma was collected from TK animals for the 2000 mg/kg group (highest dose group) and from TK animals for the vehicle control group.

 

In those animals used for bioanalysis (toxicokinetic animals), blood was sampled 0.5, 1, 2, 4 and 6 h after the second dose of either vehicle or the highest concentration of the test material. Vehicle dosed animals showed levels below the lower limit of quantification in the plasma. All test material dosed animals showed increased levels of the test material in the plasma, confirming systemic exposure.

 

Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia tissues were isolated.  Single cell suspensions from liver, glandular stomach and duodenum were made followed by Comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed. Bone marrow smears were prepared for micronucleus analysis.

 

Study Results (Comet Assay)

A statistical significant increase (p<0.05) in the mean Tail Intensity (%) was observed in duodenum cells of test material treated male animals compared to the vehicle treated animals. The high and intermediate dose groups showed tail intensity increases compared to vehicle-treated animals and in addition a dose related response was observed (significant trend analysis (P<0.0001)). All the results obtained were within the 95% confidence interval of historical control database of the negative controls.

 

A statistically significant increase (p<0.05) in the mean Tail Intensity (%) was observed in glandular stomach cells of test item treated male animals compared to the vehicle treated animals for the low dose group (500 mg/kg body weight); the intermediate and high dose groups did not show a statistical increase. However, no dose related response was observed (confirmed by a trend analysis) and these values were within the historical control database of the negative controls.

 

No increase in the mean Tail Intensity (%) was observed in liver cells of test material treated male animals compared to the vehicle treated animals.

 

Conclusion

The Comet assay undertaken on cells of male rats sampled 3-4 hours post dosing at up to 2000 mg/kg with the test item (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report as found to be valid. The test material is not genotoxic in the Comet assay in liver and glandular stomach cells. The assay was equivocal in duodenum cells as there was a significant dose-response shown however within historical control range of the vehicle control. No morphologic alterations were found which indicate that there was no cytotoxicity present.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 December 2021 to 27 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to the guideline study, as a combined OECD 474+489 micronucleus and comet study. All validity criteria were met under both guidelines. The study was conducted under GLP.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Official Journal of the European Union No. L142, amendment to EC No. 440/2008, 14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Amber glass bottle, placed in refrigeration (2-8°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Test material concentrations were stirred and treated with ultra-sonic waves to obtain a homogeneous suspension.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Not stated
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Not stated
- Reactivity of the test material with the incubation material used (e.g. plastic ware): Not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): N/A
- Preliminary purification step (if any): N/A
- Final concentration of a dissolved solid, stock liquid or gel: 500, 1000, 2000 mg/kg/day
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: N/A

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han

Details on species / strain selection:

Crl: WI(Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Sex: DRF phase had 3 males and 3 females per group. Main study phase; control and 2000 mg/kg/day had 8 males per group (5 main study animals and 3 TK animals). At 500 or 1000 there were 5 males per group (main study only). Positive control EMS and CP there were 3 males per group (main study only).
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 170.4 - 185.3 (based on group means). Body weights of the rats at the start of the treatment were within 20% of the sex mean.
- Assigned to test groups randomly: Yes.
- Fasting period before study: Nofood
- Housing: Up to 5 animals of the same sex and same dose group were housed together. Housed in polycarbonate cages containing sterilized sawdust as bedding material equipped with water bottles.
- Diet: SM R/M-Z pellets (Sponsor: SSNIFF® Spezialdiäten GmbH, Soest, Germany) available ad libitum. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: Municipal tap water, available ad libitum in water bottles.
- Acclimation period: at least 5 days before commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 - 24 °C
- Humidity: 40 - 70 %:
- Air changes: Ten or more air changes per hour:
- Photoperiod: 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 17 Dec 2021 To: 27 Jan 2022

Route of administration:

oral: gavage

Vehicle:

Corn Oil (Supplier: Fagron, Capelle a/d Ijssel, The Netherlands)
- Justification for choice of solvent/vehicle: Was selected as shown to be suitable in previous in vivo studies using the test material and a solubility test
- Concentration of test material in vehicle: 50 - 200 mg/mL (based on dose level of 500 - 2000 mg/kg/day and dose volume of 10 mL/kg).
- Amount of vehicle (if gavage or dermal): Dose Volume of 10 mL/kg.

Details on exposure:

The test material was suspended in corn oil. Adjustment was made for specific gravity of the vehicle (0.92). In the main study an adjustment was made for specific density of the test material (1.161 g/mL). Test material concentrations were stirred and treated with ultra-sonic waves to obtain a homogeneous suspension. This resulted in yellow translucent suspensions for all formulations. Test material concentrations were dosed within 3 hours after preparation.

Duration of treatment / exposure:

3 consecutive days

Frequency of treatment:

Vehicle and the test material formulations were dosed daily
CP was dosed once daily.
EMS was dosed twice daily.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Negative control group

Dose / conc.:

500 mg/kg bw/day

Remarks:

Test. material

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Test material

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Test material

Dose / conc.:

200 mg/kg bw/day

Remarks:

Postive control - EMS (Comet)

Dose / conc.:

19 mg/kg bw/day

Remarks:

Positive control - CP (micronucleus)

No. of animals per sex per dose:

DRF phase had 3 males and 3 females per group. Main study phase; control and 2000 mg/kg/day had 8 males per group (5 main study animals and 3 TK animals). At 500 or 1000 there were 5 males per group (main study only). Positive control EMS and CP there were 3 males per group (main study only).

Positive control(s):

Alkaline Comet test: Ethyl Methanesulfonate (EMS; CAS no. 62-50-0, Sigma Aldrich, Steinheim, Germany):
-Justification for choice of solvent/vehicle: Based on guideline.
- Concentration of test material in vehicle: 20 mg/mL (based on dose level of 200 mg/kg/day and dose volume of 10 mL/kg) dissolved in physiological saline.
- Amount of vehicle: Dose Volume of 10 mL/kg.

Micronucleus test: Cyclophosphamide (CP; CAS No. 6055-19-2; Sigma Aldrich Chemie GmbH, Steinheim, Germany)
-Justification for choice of solvent/vehicle: Based on guideline.
- Concentration of test material in vehicle: 1.9 mg/mL (based on dose level of 19 mg/kg/day and dose volume of 10 mL/kg) dissolved in physiological saline.
- Amount of vehicle: Dose Volume of 10 mL/kg.

EMS positive control dose twice daily for 3 days via oral gavage.
CP positive control once daily for 3 days via oral gavage.

The positive controls were selected based on the guidelines.

Tissues and cell types examined:

Bone Marrow. The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%).

Details of tissue and slide preparation:

Bone marrow was sampled 24 hours after second dosing. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 4 mL of fetal calf serum. The cell suspension was collected and centrifuged at 216 g for 5 min.

The supernatant was removed with a Pasteur pipette. Approximately 500 µl serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. At least two slides were prepared per animal.
The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer (Hematek 3000, Siemens Healthcare, Den Haag, The Netherlands). This staining is based on Giemsa. The dry slides were automatically mounted with a coverslip with an automated coverslipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

Evaluation criteria:

To prevent bias, all slides were randomly coded before examination. An adhesive label with study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated. Parts on the slides that contained mast cells that might interfere with the scoring of micronucleated polychromatic erythrocytes were not used for scoring.

Acceptability Criteria of the Micronucleus Test
A micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control material induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes.
d) The highest test dose is the MTD or 2000 mg/kg/day
ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis.

Statistics:

ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data.
A test material is considered positive in the micronucleus test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided,
p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
Positive results in the micronucleus test indicate that a test chemical induces micronuclei, which are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species.
A test material is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
Negative results indicate that, under the test conditions, the test chemical does not produce micronuclei in immature erythrocytes of the test species.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF THE DOSE FORMULATION ANALYSIS:

Accuracy:
A small response at the retention time of the test material was observed in the chromatograms of the vehicle. It was considered not to derive from the vehicle since a similar response was obtained in the analytical blanks.

The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85%-115%).

Homogeneity:
The dose formulation samples were homogeneous (i.e. RSD ≤ 10%).The mean number of micronucleated polychromatic erythrocytes scored in test material treated groups were compared with the corresponding solvent control group.


RESULTS OF RANGE-FINDING STUDY

- Dose range: 2000 mg/kg bw administered to 3 male and 3 female animals for a total of 3 consecutive days
- Solubility: A solubility test was performed based on visual assessment.
- Clinical signs of toxicity in test animals: None
- Evidence of cytotoxicity in tissue analysed: None
- Rationale for exposure: Maximum according to test guideline
- Harvest times: N/A
- High dose with and without activation: N/A
- Other: N/A

RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei (for Micronucleus assay):
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of test material treated animals compared to the vehicle treated animals.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the within the 95% control limits of the distribution of the historical negative control database.

Cyclophosphamide, the positive control material, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.

- Ratio of PCE/NCE (for Micronucleus assay):
The animals of the groups, which were treated with test material showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test material on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

- Appropriateness of dose levels and route:
Dose level and route of exposure were selected according to test guideline and guided by a DRF study

Results Summary Table

 

 

Mean Number of Micronucleated Polychromatic Erythrocytes and Ratio of Polychromatic/Normochromatic Erythrocytes

Group	Treatment	Number of Animals	Dose (mg/kg body weight)	Number of micronucleated polychromatic erythrocytes (mean ± S.D.) ^(1)			Ratio polychromatic/ normochromatic erythrocytes (mean ± S.D.) ^(2)
	MALES
1	Vehicle Control	5	0	4.4	±	2.3	0.96	±	0.02
2	Test Material	5	500	5.4	±	2.3	0.87	±	0.15
3	Test Material	5	1000	2.8	±	1.5	0.94	±	0.08
4	Test Material	5	2000	5.8	±	2.3	0.92	±	0.04
6	CP	3	19	68.7	±	13.7^(3)	0.37	±	0.05^(3)

Vehicle control = Corn oil

CP = Cyclophosphamide.

(1)  At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.

(2)  The ratio was determined from at least the first 500 erythrocytes counted.

(3)  Significantly different from corresponding control group (Students t test, P < 0.001).

Conclusions:

The test item was not found to be clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described.

Executive summary:

Study Objective

The objective of this study was to obtain information on the potential genotoxicity of the test material when administered to rats at the maximum recommended dose in accordance with current regulatory guidelines, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow and by measuring the increase in DNA strand breaks in liver, duodenum and glandular stomach.

 

Study Design

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic materials. Moreover, historical control background data has been generated with this strain.

 

The study procedures described in this report were based on the most recent OECD (29 July 2016) and EC (14 February 2017) guidelines.

 

The test material was a brownish-orange, slightly viscous liquid. The test material was suspended in corn oil.

 

Based on the results of the dose-range finding study, test concentrations of 2000 mg/kg/day for male animals was selected as maximum dose for the main test (the highest dose required in the current guideline). Since there were no substantial differences in toxicity between sexes only males were used in the main study.

 

A small response at the retention time of the test material was observed in the chromatograms of the The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85%-115%). The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%).

 

In the main study male animals were dosed three times by oral gavage with vehicle or with 500, 1000 and 2000 mg test material per kg body weight for three consecutive days. A positive control group for the comet assay was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight and a positive control group for the micronucleus assay was dosed once by oral gavage with 19 mg cyclophosphamide (CP) per kg body weight. In total 6 treatment groups were used, each consisting of 5 animals, with exception of the positive control and TK animals (3 animals per group).

 

In addition, blood for bioanalysis of the test material in plasma was collected from TK animals for the 2000 mg/kg group (highest dose group) and from TK animals for the vehicle control group.

 

In those animals used for bioanalysis (toxicokinetic animals), blood was sampled 0.5, 1, 2, 4 and 6 h after the second dose of either vehicle or the highest concentration of the test material. Vehicle dosed animals showed levels below the lower limit of quantification in the plasma. All test material dosed animals showed increased levels of the test material in the plasma, confirming systemic exposure.

 

Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia tissues were isolated.  Single cell suspensions from liver, glandular stomach and duodenum were made followed by Comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed. Bone marrow smears were prepared for micronucleus analysis.

 

Study Results (Micronucleus)

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test material compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the distribution of the historical negative control database. Cyclophosphamide, the positive control material, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.

The groups that were treated with the test material showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test material on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

 

Conclusions

In conclusion, the test material is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Summary of genetic toxicity data

 

A summary of all available data is included within table 7.6.1/1.

 

Table 7.6.1/1 : Summary of genotoxicity data:

 

Test n°	Test Guideline / Reliability	Focus	Strains / cells tested	Metabolic activation	Test concentration	Outcome
1 (Herbold, 1980)	Ames Test (OECD 471, no GLP claimed) WoE, rel.2	Gene mutation	S. typhimurium TA 1535, TA 1537, TA 98, TA 100	-S9 +S9	Tested up to 2,500 µg/plate	-S9: not mutagenic + S9: not mutagenic
2 (BFS, 2022)	Ames test: (QSAR: Alkylphosphate mechanism) WoE, rel.2	Alkylphosphate reaction with Guanine	E. coli and/or S. typhimurium TA 102 strains	-S9 +S9	N/A	Not mutagenic
3 (BFS, 2022)	Ames test: (QSAR: Iminium ion mechanism) WoE, rel.2	Generation of reactive iminium species via P450 metabolism of Aliphatic tertiary amines	E. coli and/or S. typhimurium TA 102 strains	-S9 +S9	N/A	Not mutagenic
4 (Majeska & Matheson, 1982)	In vitromammalian chromosome aberration test (equiv. OECD 473, no GLP claimed,) Key, rel. 1	Clastogenicity	Fischer L5178Y mouse lymphoma cells	-S9 +S9	Tested up to 500 µg/ml without S9 2000 µg/ml with S9 (up to cytotoxicity limit)	-S9: mutagenic + S9: mutagenic
5 (Majeska & Matheson) 1981)	In vitro gene mutation (TK+/-) assay (equiv. OECD 490, no GLP claimed) Key, rel. 1	Gene mutation	L5178Y TK+/- mouse lymphoma cells	-S9 +S9	Tested up to 500 µL/L without S9 1000 µL/L with S9 (up to cytotoxicity limit)	-S9: mutagenic + S9: mutagenic
6 (Jacobs, 2022)	In vivo micronucleus test (OECD 474, GLP) Key, rel.1	Clastogenicity / Aneugenicity	Male rats (Wistar Han) Bone Marrow	N/A	Up to 2000 mg/kg bw/day (recommended limit concentration)	Not clastogenic
7 (Jacobs, 2022)	In vivo Comet assay (OECD 489, GLP) Key, rel.1	Gene mutation	Male rats (Wistar Han) Liver Glandular Stomach Duodenum	N/A	Up to 2000 mg/kg bw/day (recommended limit concentration)	Liver: not genotoxic Glandular stomach: not genotoxic Duodeum: Equivocal for genotoxicity*

* see commentary for further evaluation

 

Gene mutation test in bacteria (Test n° 1-3)

 

A bacterial reverse mutation assay (Ames test) was performed with the substance (Test n°1). This study was used to conclude on the potential of the substance to induce gene mutation in bacteria. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains with any dose of test material, either in the presence or absence of metabolic activation. The test indicates that the substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. 

 

As E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 strains was not evaluated in this study, it was not possible to conclude on the cross-linking potential of the substance.  According to the OECD TG 471 guideline certain oxidising mutagens, cross-linking agents and hydrazines may be detected specifically by the two strains mentioned above. Diethyl bis(2-hydroxyethyl)aminomethylphosphonate has no cross-linking or oxidizing properties, nor is it a hydrazine derivative. To support the absence of mutagenic potential within the Ames test, a (Q)SAR assessment was performed using the OECD Toolbox v4.5, focussing on specific mechanisms of toxicity (Test n° 2 & 3). Test n° 2 assessed formation of Alkylphosphates, and n° 3 the formation of Iminium Ion from Aliphatic Tertiary Amines.

 

Via categorization (for organophosphorus and tertiary amine compounds) based on the substance’s identified activities in profilers relevant to the Ames mutagenicity endpoint, and identification of those compounds which have available data in the missing strains, it was shown that the nearest neighbours to the test substance are all negative. As such, it was concluded that no evidence of mutagenicity was identified for the substance in the model predictions.

 

Based on weight-of-evidence, the substance is therefore considered as non-mutagenic according to the Ames test. 

 

Chromosome aberration Assay (Test n° 4)

 

In a mammalian cell cytogenetics assay, equivalent to an in vitro chromosome aberration test (OECD 473), mouse lymphoma L5178Y cells were exposed for 4 hours to the test substance in water at concentrations of up to 500 µL/L without metabolic activation, and up to 2,000 µL/L with metabolic activation (induced rat liver S9). Doses were selected following a range-finding study. 50 cells per concentration were analysed.

 

A statistically significant increase in the number of structural aberrations was observed after treatment at the highest concentration (0.5 µL/mL) without metabolic activation, and at 1.5 and 2 µL/mL with metabolic activation. At the same concentrations, there was also a significant increase in numerical aberrations.. All acceptability criteria were met.

 

The test item is considered to be clastogenic in this in vitro chromosome aberration test.

 

Gene mutation assay in mammalian cells (Test n° 5)

 

In a mammalian cell gene mutation assay performed similarly to the OECD TG 490, L5178Y TK+/- mouse lymphoma cells cultured in vitro were exposed to the test item diluted in water in the presence and absence of Rat S9 mammalian metabolic activation. Cells were exposed for 4h at 5 concentrations up to 500 µL/mL (-S9), and up to 1,000 µL/mL (+S9) in two independent experiments. The highest chosen dose reduced the growth rate by 50-90% based on the results of the preliminary cytotoxicity test (for experiment I) and on the results of experiment I (for experiment II).

 

Substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments, with evidence of a concentration-related positive response of induced mutant colonies over background. The increase in mutation frequency was seen at concentrations of 0.25 µL/mL or greater, reaching significance at 0.30 µL/mL. In the presence of an activation system, the test item is less cytotoxic and a significant increase in mutation frequency was seen at concentrations greater or equal to 1.10 µL/mL. All acceptability criteria were met.

 

The test item is considered to be mutagenic in this in vitro gene mutation test.

 

Micronucleus test in vivo (Test n° 6)

 

Based on the final decision on the testing proposal (communication number TPE-D-2114546045-54-01/F), an in vivo mammalian alkaline comet assay combined with in vivo mammalian erythrocyte micronucleus test (test methods: OECD 489 & 474) was performed.

 

In the mammalian in vivo micronucleus test (OECD 474), the potential cytogenicity of the test material was investigated when administered to rats by measuring the (increase in the) number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow.

 

Based on the results of the dose-range finding study, 2000 mg/kg bw/day was selected as maximum dose for the main test (the highest dose required in the current guideline). Male animals were used, as no sex difference was identified in response to the test material during the dose-range finding study. In the main study male animals were dosed three times by oral gavage with vehicle or with 500, 1000 and 2000 mg/kg bw for three consecutive days. A positive control group was dosed once by oral gavage with 19 mg cyclophosphamide (CP) per kg body weight. In total 6 study groups were used (including a positive control for the Comet assay); each consisted of 5 animals, with exception of the positive control and TK animals (3 animals per group).

 

In addition, blood for bioanalysis of the test material in plasma was collected from TK animals for the 2000 mg/kg bw group (highest dose group) and from TK animals for the vehicle control group. In those animals used for bioanalysis (toxicokinetic animals), blood was sampled 0.5, 1, 2, 4 and 6 h after the second dose of either vehicle or the highest concentration of the test material. Vehicle dosed animals showed levels below the lower limit of quantification in the plasma. All test material dosed animals showed increased levels of the test material in the plasma, confirming systemic exposure.

 

Approximately 3-4 hours after the last dose the animals were sacrificed, and tissues were isolated. Bone marrow smears were prepared for micronucleus analysis.

 

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test material compared to the vehicle treated animals. The groups that were treated with the test material showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test material on erythropoiesis. All acceptability criteria were met.

 

The test item is considered to be not clastogenic in this in vivo Micromucleus test.

 

Gene mutation assay in vivo (Test n° 7)

 

Based on the final decision on the testing proposal (communication number TPE-D-2114546045-54-01/F), an in vivo mammalian alkaline comet assay (test method: OECD 489) combined with in vivo mammalian erythrocyte micronucleus test (test methods: OECD 489 & 474) was performed.

 

In the mammalian in vivo alkaline comet gene mutation assay (OECD 489), the potential genotoxicity of the test material was elucidated when administered to rats by measuring the increase in DNA strand breaks in liver, duodenum and glandular stomach.

 

Based on the results of the dose-range finding study, 2000 mg/kg bw/day was selected as maximum dose for the main test (the highest dose required in the current guideline). Male animals were used, as no sex difference was identified in response to the test material during the dose-range finding study. In the main study male animals were dosed three times by oral gavage with vehicle or with 500, 1000 and 2000 mg/kg bw for three consecutive days. A positive control group for the comet assay was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight. In total 6 groups (including a positive control for the MNT assay) were used; each consisting of 5 animals, with exception of the positive control and TK animals (3 animals per group). In addition, blood for bioanalysis of the test material in plasma was collected from TK animals for the 2000 mg/kg group (highest dose group) and from TK animals for the vehicle control group.

 

In addition, blood for bioanalysis of the test material in plasma was collected from TK animals for the 2000 mg/kg bw group (highest dose group) and from TK animals for the vehicle control group. In those animals used for bioanalysis (toxicokinetic animals), blood was sampled 0.5, 1, 2, 4 and 6 h after the second dose of either vehicle or the highest concentration of the test material. Vehicle dosed animals showed levels below the lower limit of quantification in the plasma. All test material dosed animals showed increased levels of the test material in the plasma, confirming systemic exposure.

 

Approximately 3-4 hours after the last dose the animals were sacrificed, and tissues were isolated.  Single cell suspensions from liver, glandular stomach and duodenum were made followed by Comet slide preparation. The slides were analyzed and the Tail Intensity (%) was assessed.

 

All criteria for an acceptable assay were met. No increase in the mean Tail Intensity (%) was observed in liver cells of test material treated male animals compared to the vehicle treated animals. The test material is not genotoxic in the Comet assay in liver and cells.

 

A statistically significant increase in the mean Tail Intensity (%) was observed in glandular stomach cells of test item treated male animals compared to the vehicle treated animals for the low dose group (the intermediate and high dose groups did not show a statistical increase). No dose related response was observed (confirmed by a trend analysis) and values were within the historical control database of the negative controls. The test material is not genotoxic in the Comet assay in glandular stomach cells.

 

A significant increase in the mean Tail Intensity (%) was observed in duodenum cells of test material treated male animals compared to the vehicle treated animals. The high and intermediate dose groups showed tail intensity increases compared to vehicle-treated animals and in addition a dose related response was observed (significant trend analysis (P<0.0001)). There were no morphologic alterations in the duodenum following three administrations of the test item at doses of up to 2000 mg/kg bw/day, ruling out confounding tissue site toxicity. The assay was concluded to be equivocal in duodenum cells as there was dose-response shown, however this was within historical control range of the vehicle control.

 

Weight-of-Evidence Evaluation

 

Seven genetic toxicity studies are available of sufficient quality and reliability to assess the respective endpoints: two in silico QSAR studies, three in vitro studies and two in vivo studies (table 7.6.1/2).

 

Table 7.6.1/2: summary of study outcomes according to Annex and key mechanism

 

	Gene Mutation	Cytogenicity
Annex VII  (in silico / in vitro):	Ames (OECD 471, 1980, rel. 2): negative Ames (QSAR, 2022, rel. 2, WoE): negative Ames (QSAR, 2022, rel. 2, WoE): negative
Annex VIII (in vitro):	Gene mutation (equiv. OECD 490, 1981, rel.1, key): positive	Chromosome aberration (equiv. OECD 490, 1982, rel.1, key): positive
Annex IX:/X (in vivo):	Genotoxicity (OECD 489, 2022, rel. 1, key): Stomach = negative Liver = negative Duodenum = equivocal	Cytogenicity (OECD 474, 2022, rel. 1, key): negative
Outcome:	No immediately conclusive outcome, WoE and expert judgement required to reach conclusion	No concern for cytogenicity (clastogen or aneugen)

 

Cytogenicity:

 

A mammalian cell cytogenetic assay in vitro, equivalent to mammalian cell chromosome aberration test (OECD 473), reported an increase in incidence of chromosome aberrations, triggering the requirement for further investigation in vivo, in accordance with the ECHA Integrated Testing Strategy for mutagenicity (ECHA R7a, Figure R.7.7-1). Subsequently, an in vivo mammalian alkaline comet assay combined with in vivo mammalian erythrocyte micronucleus test (test method: OECD 489 & 474) was performed based on the final decision on the testing proposal (communication number TPE-D-2114546045-54-01/F). Under the experimental conditions described, the substance was not found to be clastogenic or aneugenic in the bone marrow micronucleus test of male rats up to a dose of 2000 mg/kg bw (the maximum recommended dose in accordance with current regulatory guidelines). No indication of systemic genotoxic activity of the parent compound or a metabolite was reported in the in vivo genotoxicity test. 

 

Conclusion: There is no concern for cytogenicity for Diethyl bis(2-hydroxyethyl)aminomethylphosphonate.

 

Mutagenicity:

 

In a mammalian cell gene mutation assay performed similarly to the OECD TG 490, an increase in gene mutations was reported following exposure of L5178Y TK+/- mouse lymphoma cells to the test item. This triggered the requirement for further investigation in vivo, in accordance with the ECHA Integrated Testing Strategy for mutagenicity (ECHA R7a, Figure R7.7-1). Subsequently, an in vivomammalian alkaline comet assay combined with in vivo mammalian erythrocyte micronucleus test (test method: OECD 489 & 474) was performed based on the final decision on the testing proposal (communication number TPE-D-2114546045-54-01/F).

 

No indication of systemic genotoxic activity of the parent compound or a metabolite was reported in the in vivo genotoxicity test according to OECD 489. Under the experimental conditions described, the test material is not genotoxic in the comet assay in liver and glandular stomach cells. The assay was equivocal in duodenum cells as there as a significant dose-response shown however this was within historical control range of the vehicle control. Further explanation on the detail of the results for this tissue are outlined below.

 

Converse to the other organs evaluated, for duodenum, the vehicle control % tail intensity was very close to the lower range of the HCD. Furthermore, the fairly high standard deviations of the mean tail intensity scores reported within the study indicated that there was an overlap between dosed, control and negative HCD groups. Review of the tail % scores for individual animals in all groups confirms this generalised pattern of high variability, for example, within the control group % intensity scores from 1.70 – 7.69 were recorded. Table 7.6.1/3 outlines the % tail intensities reported for Duodenum, and table 7.6.1/4 the HCD for the study period.

 

Table 7.6.1/3: Duodenum mean tail intensity (%)

 

	Tail Intensity (%)	S.D.
Vehicle Control	3.90	1.84
Test Material 500 mg/kg	4.81	0.67
Test Material 1000 mg/kg	6.48	1.31
Test Material 2000 mg/kg	9.43	2.27
EMS 200 mg/kg	50.79	3.47

Table 7.6.1/4: Historical Control Data for Comet (experiments performed at test site November 2018 - November 2021).

	Duodenum Tail Intensity (%) Males and Females
Mean	6.7
Standard Deviation	3.2
Number of observations	26
Lower control limit (95% control limits)	0.4
Upper control limit (95% control limits)	13.1

In order to establish an outcome for this endpoint and subsequent self-classification according to CLP, further consideration to the interpretation of alkaline Comet assays must be given. Interpretation of comet assay results is conventionally based on four parameters¹:

1. whether or not at least one test dose is statistically significant versus the concurrent control.


2. whether or not there is a statistically significant positive trend analysis reflecting a dose response.

iii. where data points fall with respect to laboratory historical control data.

1. demonstration of exposure of the examined tissue(s) to the test agent.

Using the above criteria, the assay is clearly negative for all criteria for liver. For glandular stomach, the assay is positive for I; however there is no positive trend in the dose response (ii), and the values were within the range of the lab’s historical control database (iv), therefore the outcome was concluded to be negative. For duodenum, the assay is positive for i, ii and iv. However, the wide standard deviations reported both within the study and the HCD led to lack of clarity over iii and consequent assignment of an equivocal outcome for this organ.

 

Within a review of the performance and data interpretation of the in vivo comet assay reported by van der Leede et. al.², discussion was offered on how to determine between Comet assay outcomes which are equivocal, and those which are inconclusive. Importantly, the authors considered that where there exists a single experiment within which the response is neither considered negative nor positive, it is more fitting to consider the response inconclusive than equivocal (which they reserved for instances where multiple independent experiments were available). Based on this, it is argued that the OECD 489 Comet assay is negative for liver and glandular stomach, and inconclusive for duodenum.

 

Conclusion: By appraisal of the weight-of-evidence and application of expert judgement, it is concluded that based on the negative result of the systemic organ liver and the local organ glandular stomach and the equivocal result observed with the local organ duodenum, there is no concern for systemic in vivo genotoxicity for Diethyl bis(2-hydroxyethyl)aminomethylphosphonate.

 

Relevant evidence from the wider dataset:

Within the wider available dataset for the test item, no evidence could be found to further support germ cell mutagenicity. The sub-chronic 90-day study (equiv. OECD 408, rel.2, key) identified no changes in reproductive organs. Similarly, although a reproductive and developmental toxicity screening study (OECD 421, 1995, rel 1, key) identified histopathological findings within the male reproductive organs (i.e. degeneration of germinal epithelium in the testes and interstitial infiltration with mixed cells in the epididymides), these were considered incidental and non-treatment related, and were not related to any effects on reproductive function.

 

Conclusion:

Following weight-of-evidence analysis and application of expert judgement, it is concluded that no additional self-classification is proposed regarding mutagenicity according to the CLP and to the GHS.

 

References:

1. Maronpot RR, Hobbs CA, Hayashi SM. Role of pathology peer review in interpretation of the comet assay. J Toxicol Pathol. 2018 Jul;31(3):155-161. doi: 10.1293/tox.2018-0019. Epub 2018 May 11. PMID: 30093784; PMCID: PMC6077155.


2. Bas-jan van der Leede, Ann Doherty, Melanie Guérard, Jonathan Howe, Mike O’Donovan, Ulla Plappert-Helbig, Véronique Thybaud,Performance and data interpretation of the in vivo comet assay in pharmaceutical industry: EFPIA survey results, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 2014, Volumes 775–776, Pages 81-88, https://doi.org/10.1016/j.mrgentox.2014.09.008.

Justification for classification or non-classification

Harmonised classification

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP). 

 

Self Classification

Based on information available no additional self-classification is proposed regarding mutagenicity according to the CLP and to the GHS.