N1,N2-dimethyl-N1-{2-[methyl(propan-2-yl)amino]ethyl}-N2-(propan-2-yl)ethane-1,2-diamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There is experimental data on the in vitro genetic toxicity in bacterial and mammalian cells for the target substance N2-dimethyl-N1-{2-[methyl(propan-2-yl)amino]ethyl}-N2-(propan-2-yl)ethane-1,2-diamine, CAS  1042950-30-0, available. An in vitro gene mutation study in bacteria with target substance N2-dimethyl-N1-{2-[methyl(propan-2-yl)amino]ethyl}-N2-(propan-2-yl)ethane-1,2-diamine, CAS 1042950-30-0 revealed negative results with and without metabolic activation. A chromosome aberration assay conducted with the target substance according to OECD TG 473 and in vivo micronucleus study (OECD 474), did not reveal mutagenic effects.

The potential to induce gene mutations in bacteria was assessed in an Ames test performed with the source substance Bis(2-dimethylaminoethyl)(methyl)amine (CAS No 3030-47-5). The study gave a negative result. In vitro cytogenicity and in vitro gene mutation studies in mammalian cells were performed with source substance Bis(2-dimethylaminoethyl)(methyl)amine (CAS No 3030-47-5). The results for all studies were consistently negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Aug. 20, 2012 to Febr.20, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The source and the target substances are aliphatic tertiary amines. Structurally, CAS 1042950-30-0 differs only in the presence of an additional (dimethylamino)propyl group linked to the central nitrogen from the source substance.
Tertiary amines can be generally represented by R-N(R´´)-R´; with elemental composition of only carbon, hydrogen and nitrogen, and where R is aliphatic hydrocarbon constituent of cyclic group.
All the amines, including tertiary, can form hydrogen bonds with water. Although tertiary amines lack a hydrogen atom attached to the nitrogen, and therefore can't form hydrogen bonds with themselves, however they can form hydrogen bonds with water molecules using the ion pair on the nitrogen. Solubility generally decreases as alkyl chains increase in length.
The source and target substances listed in Tables 1 and 2 of the read-across justification attached on this RSS, are registered as mono-constituent substances. The impurity profiles are well defined, and purity is generally high. Small changes in the impurity profile are unlikely to have significant influence on toxicological properties, which are dominated by irritation and corrosion - a feature of the target and source substances.
Thus, the chemical identity of the of the target and source substances is sufficiently clear for a meaningful comparison and assessment. 3.2 Common precursors and breakdown products
The considered substances (source and target substances) are aliphatic tertiary amines derived from ammonia [NH3] by substituting all the hydrogen atoms with alkyl groups (carbon and hydrogen containing molecular chains). Tertiary amines undergo metabolism via P-450 and MFOA, followed from C-oxidation and N-oxidation. For details see the attached documentation,

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

and OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Remarks:

Only minor deviation from study plan: The additional parameters were evaluated using the existing data: suspension growth (SG), cloning efficiency (CE), relative suspension growth (RSG) and relative total growth (RTG). No influence on the study results.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidin kinase gene (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
The cells used during the experimental studies were maintained in RPMI 1640 medium supplemented with 0.05% Pluronic® F68, 2 mM L-glutamine, 220 µg/mL sodium pyruvate, 100 µg/mL gentamycin, 2.5 µg/mL fungizone and fetal bovine serum (10% by volume), hereinafter referred to as growth medium.
Treatment medium was growth medium without sodium pyruvate, gentamycin and fungizone.
Cleansing medium used for reducing the spontaneous frequency of TK-/- mutants prior to experimental studies consists of growth medium supplemented with approximately 4.0 x 10-5 M thymidine, 1.2 x 10-4 M hypoxanthine, 3.3 x 10-5 M glycine and 7.2 x 10-7 M methotrexate.
Recovery medium is similar to cleansing medium, except that the methotrexate component is removed.
Selection medium is growth medium that contains 3 µg/mL of TFT (5-trifluorothymidine)

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: The indicator cell was the L5178Y mouse lymphoma cell line that is heterozygous at the TK locus (+/-). The particular clone (3.7.2C) used in this assay was isolated by American Type Culture Collection, 0801 University Blvd., Manassas, VA 20110-2209, USA.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (prepared from the livers of rats treated with Aroclor 1254) with cofactors

Test concentrations with justification for top dose:

Test concentrations for cytotoxicity testing:
39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/mL

Test concentrations for mutagenicity testing in the experiment without metabolic activation (3-hour or 24-hour exposure):
19.53, 39.06, 78.13, 156.3 and 312.5 µg/mL medium

Test concentrations for mutagenicity testing in the experiment with metabolic activation (3-hour exposure):
39.06, 78.13, 156.3, 312.5 and 625 µg/mL medium

Vehicle / solvent:

DMSO (dimethylsulfoxide)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Pre-incubation period: no
- Exposure duration:
3-hour or 24-hour exposure (without metabolic activation)
3-hour exposure (with metabolic activation)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-14 days

SELECTION AGENT (mutation assays): TFT (5-trifluorothymidine)

NUMBER OF REPLICATIONS: 2
without metabolic activation: 3h exposure and 24h exposure (2 experiments)
with metabolic activation: 3h exposure (2 experiments)

DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency

Evaluation criteria:

Evaluation criteria
The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment is a mutant frequency that is ≥ 2 times the concurrent background mutant frequency.
The observation of a mutant frequency that meets the minimum criterion for a single treated culture within a range of assayed concentrations is not sufficient evidence to evaluate a test item as a mutagen.

Assay acceptance criteria
An assay is considered acceptable for evaluation of the test results only if following criteria are satisfied.
a/ The mutant frequency in the negative control falls within the normal range (historical mean value).
b/ The plating efficiency of the negative control is ≥ 50%.
c/ At least one concentration of the positive control induces a clear increase in the mutant frequency (the mutant frequency of the positive control is ≥ 2 times the historical mean value of the negative control) with and without metabolic activation system..

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was noted at the top concentration of 312.5 µg/mL (without metabolic activation, 3-hour and 24-hour exposure), and at the top concentration of 625 µg/mL (with metabolic activation, 3-hour exposure).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary experiment
In the preliminary experiment without and with metabolic activation (24-hour or 3-hour exposure) cytotoxicity (decreased survival) was noted starting at concentrations of 312.5 or 625 µg/mL, respectively.
Hence, in the main study following concentration ranges were used:
a/ concentration range of 19.53 to 312.5 µg/mL (without metabolic activation, 3-hour or 24-hour exposure),
b/ concentration range of 39.06 to 625 µg/mL (with metabolic activation, 3-hour exposure).

Main study
1/Cytotoxicity:
Cytotoxicity (decreased survival) was noted
at the top concentration of 312.5 µg/mL (without metabolic activation, 3-hour and 24-hour exposure),
at the top concentration of 625 µg/mL (with metabolic activation, 3-hour exposure),

2/ Positive controls:
a/ Methylmethanesulfonate (at 10 or 15 µL/mL) – without metabolic activation
It caused pronounced increases in the mutation frequency ranging from 1589.27 to 2712.47 per 1E+06 clonable cells.
b/ 3-Methylcholanthrene (at 2.5 or 4.0 µg/mL) – with metabolic activation
It caused pronounced increases in the mutation frequency ranging from 898.28 to 3466.39 per 1E+06 clonable cells.

3/ Negative controls
a/ without metabolic activation
The values of mutation frequencies of the negative controls ranged from 50.18 to 82.96 per 1E+06 clonable cells.
b/ with metabolic activation
The values of mutation frequencies of the negative controls ranged from 89.03 to 101.24 per 1E+06 clonable cells.

4/ Test sample – Pentamethyldiethylentriamine
a/ without metabolic activation
The mutation frequencies of the cultures treated with test substance ranged from 50.18 to 71.30 per 1E+06 clonable cells (3 hours exposure) and from 62.65 to 90.30 per 1E+06 clonable cells (24 hours exposure).
b/ with metabolic activation
The mutation frequencies of the cultures treated with test substance ranged from 85.50 to 101.52 per 1E+06 clonable cells (3 hours exposure, first assay) and from 58.14 to 102.97 per 1E+06 clonable cells (3 hours exposure, second assay).

These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 1E+06 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

Ratio of small to large mutant colonies
In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.43 to 1.50 for test substance treated cells and from 0.42 to 1.36 for the negative controls.

Remarks on result:

other: strain/cell type: TK +/-

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative without and with metabolic activation

Under the present test conditions, Pentamethyldiethylentriamine, tested up to cytotoxic concentrations, in the absence and presence of metabolic activation in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, Pentamethyldiethylentriamine also did not exhibit clastogenic potential at the concentration-range investigated.
According to the evaluation criteria for this assay, these findings indicate that Pentamethyldiethylentriamine, tested up to cytotoxic concentrations in the absence and presence of metabolic activation, did neither induce mutations nor have any chromosomal aberration potential.

Executive summary:

The test substance, Pentamethyldiethylentriamine, was tested for in-vitro mutagenicity in the Mouse Lymphoma Assay according to (EC) No. 440/2008 B.17 and OECD Guideline No.476: In Vitro Mammalian Cell Gene Mutation Test.

 

The substance was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; the experiment with S9 mix was carried out in two independent assays.

The vehicle DMSO (dimethylsulfoxide) served as the negative control.

 

In the preliminary experiment without and with metabolic activation (24-hour or 3-hour exposure) cytotoxicity (decreased survival) was noted starting at concentrations of 312.5 or 625 µg/mL, respectively.

 

Hence, in the main study the concentration range of 19.53 to 312.5 µg/mL was used in the experiments without metabolic activation with a 3-hour or 24-hour exposure time and a concentration-range of 39.06 to 625 µg/mL was used in the experiments with metabolic activation (3-hour exposure).

 

Methylmethanesulfonate (at 10 or 15 µL/mL) was employed as a positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene (at 2.5 or 4.0 µg/mL) in the presence of exogenous metabolic activation.

 

In the main study, cytotoxicity (decreased survival) was noted in the absence of metabolic activation (3-hour and 24-hor exposure) at the top concentration of 312.5 µg/mL and in the experiments with metabolic activation (3-hour exposure) at the top concentration of 625 µg/mL.

 

The values of mutation frequencies of the negative controls ranged from 50.18 to 82.96 per 1E+06 clonable cells in the experiments without metabolic activation and from 89.03 to 101.24 per 1E+06 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data-range.

 

The mutation frequencies of the cultures treated with Pentamethyldiethylentriamine ranged from 50.18 to 71.30 per 1E+06 clonable cells (3 hours exposure) and from 62.65 to 90.30 per 1E+06 clonable cells (24 hours exposure) in the experiments without metabolic activation and from 85.50 to 101.52 per 1E+06 clonable cells (3 hours exposure, first assay) and from 58.14 to 102.97 per 1E+06 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 1E+06 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

 

In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.43 to 1.50 for Pentamethyldiethylentriamine treated cells and from 0.42 to 1.36 for the negative controls.

 

The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3-MC) caused pronounced increases in the mutation frequency ranging from 1589.27 to 2712.47 per 1E+06 clonable cells in the case of MMS and ranging from 898.28 to 3466.39 per 1E+06 clonable cells in the case of 3-MC.

 

In addition, the colony size ratio was moderately shifted towards an increase in small colonies, ranging from 1.71 to 2.58 in the case of MMS.

 

Results

Under the present test conditions, Pentamethyldiethylentriamine, tested up to cytotoxic concentrations, in the absence and presence of metabolic activation in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.

In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, Pentamethyldiethylentriamine also did not exhibit clastogenic potential at the concentration-range investigated.

According to the evaluation criteria for this assay, these findings indicate that Pentamethyldiethylentriamine, tested up to cytotoxic concentrations in the absence and presence of metabolic activation, did neither induce mutations nor have any chromosomal aberration potential.

 

 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

From Aug. 20, 2012 to Febr.20, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

he source and the target substances are aliphatic tertiary amines. Structurally, CAS 1042950-30-0 differs only in the presence of an additional (dimethylamino)propyl group linked to the central nitrogen from the source substance.
Tertiary amines can be generally represented by R-N(R´´)-R´; with elemental composition of only carbon, hydrogen and nitrogen, and where R is aliphatic hydrocarbon constituent of cyclic group.
All the amines, including tertiary, can form hydrogen bonds with water. Although tertiary amines lack a hydrogen atom attached to the nitrogen, and therefore can't form hydrogen bonds with themselves, however they can form hydrogen bonds with water molecules using the ion pair on the nitrogen. Solubility generally decreases as alkyl chains increase in length.
The source and target substances listed in Tables 1 and 2 of the read-across justification attached on this RSS, are registered as mono-constituent substances. The impurity profiles are well defined, and purity is generally high. Small changes in the impurity profile are unlikely to have significant influence on toxicological properties, which are dominated by irritation and corrosion - a feature of the target and source substances.
Thus, the chemical identity of the of the target and source substances is sufficiently clear for a meaningful comparison and assessment. 3.2 Common precursors and breakdown products
The considered substances (source and target substances) are aliphatic tertiary amines derived from ammonia [NH3] by substituting all the hydrogen atoms with alkyl groups (carbon and hydrogen containing molecular chains). Tertiary amines undergo metabolism via P-450 and MFOA, followed from C-oxidation and N-oxidation. For details see the attached documentation

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

and OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Remarks:

Only minor deviation from study plan: The additional parameters were evaluated using the existing data: suspension growth (SG), cloning efficiency (CE), relative suspension growth (RSG) and relative total growth (RTG). No influence on the study results.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidin kinase gene (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
The cells used during the experimental studies were maintained in RPMI 1640 medium supplemented with 0.05% Pluronic® F68, 2 mM L-glutamine, 220 µg/mL sodium pyruvate, 100 µg/mL gentamycin, 2.5 µg/mL fungizone and fetal bovine serum (10% by volume), hereinafter referred to as growth medium.
Treatment medium was growth medium without sodium pyruvate, gentamycin and fungizone.
Cleansing medium used for reducing the spontaneous frequency of TK-/- mutants prior to experimental studies consists of growth medium supplemented with approximately 4.0 x 10-5 M thymidine, 1.2 x 10-4 M hypoxanthine, 3.3 x 10-5 M glycine and 7.2 x 10-7 M methotrexate.
Recovery medium is similar to cleansing medium, except that the methotrexate component is removed.
Selection medium is growth medium that contains 3 µg/mL of TFT (5-trifluorothymidine)

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: The indicator cell was the L5178Y mouse lymphoma cell line that is heterozygous at the TK locus (+/-). The particular clone (3.7.2C) used in this assay was isolated by American Type Culture Collection, 0801 University Blvd., Manassas, VA 20110-2209, USA.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (prepared from the livers of rats treated with Aroclor 1254) with cofactors

Test concentrations with justification for top dose:

Test concentrations for cytotoxicity testing:
39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/mL

Test concentrations for mutagenicity testing in the experiment without metabolic activation (3-hour or 24-hour exposure):
19.53, 39.06, 78.13, 156.3 and 312.5 µg/mL medium

Test concentrations for mutagenicity testing in the experiment with metabolic activation (3-hour exposure):
39.06, 78.13, 156.3, 312.5 and 625 µg/mL medium

Vehicle / solvent:

DMSO (dimethylsulfoxide)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Pre-incubation period: no
- Exposure duration:
3-hour or 24-hour exposure (without metabolic activation)
3-hour exposure (with metabolic activation)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-14 days

SELECTION AGENT (mutation assays): TFT (5-trifluorothymidine)

NUMBER OF REPLICATIONS: 2
without metabolic activation: 3h exposure and 24h exposure (2 experiments)
with metabolic activation: 3h exposure (2 experiments)

DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency

Evaluation criteria:

Evaluation criteria
The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment is a mutant frequency that is ≥ 2 times the concurrent background mutant frequency.
The observation of a mutant frequency that meets the minimum criterion for a single treated culture within a range of assayed concentrations is not sufficient evidence to evaluate a test item as a mutagen.

Assay acceptance criteria
An assay is considered acceptable for evaluation of the test results only if following criteria are satisfied.
a/ The mutant frequency in the negative control falls within the normal range (historical mean value).
b/ The plating efficiency of the negative control is ≥ 50%.
c/ At least one concentration of the positive control induces a clear increase in the mutant frequency (the mutant frequency of the positive control is ≥ 2 times the historical mean value of the negative control) with and without metabolic activation system..

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was noted at the top concentration of 312.5 µg/mL (without metabolic activation, 3-hour and 24-hour exposure), and at the top concentration of 625 µg/mL (with metabolic activation, 3-hour exposure).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary experiment
In the preliminary experiment without and with metabolic activation (24-hour or 3-hour exposure) cytotoxicity (decreased survival) was noted starting at concentrations of 312.5 or 625 µg/mL, respectively.
Hence, in the main study following concentration ranges were used:
a/ concentration range of 19.53 to 312.5 µg/mL (without metabolic activation, 3-hour or 24-hour exposure),
b/ concentration range of 39.06 to 625 µg/mL (with metabolic activation, 3-hour exposure).

Main study
1/Cytotoxicity:
Cytotoxicity (decreased survival) was noted
at the top concentration of 312.5 µg/mL (without metabolic activation, 3-hour and 24-hour exposure),
at the top concentration of 625 µg/mL (with metabolic activation, 3-hour exposure),

2/ Positive controls:
a/ Methylmethanesulfonate (at 10 or 15 µL/mL) – without metabolic activation
It caused pronounced increases in the mutation frequency ranging from 1589.27 to 2712.47 per 1E+06 clonable cells.
b/ 3-Methylcholanthrene (at 2.5 or 4.0 µg/mL) – with metabolic activation
It caused pronounced increases in the mutation frequency ranging from 898.28 to 3466.39 per 1E+06 clonable cells.

3/ Negative controls
a/ without metabolic activation
The values of mutation frequencies of the negative controls ranged from 50.18 to 82.96 per 1E+06 clonable cells.
b/ with metabolic activation
The values of mutation frequencies of the negative controls ranged from 89.03 to 101.24 per 1E+06 clonable cells.

4/ Test sample – Pentamethyldiethylentriamine
a/ without metabolic activation
The mutation frequencies of the cultures treated with test substance ranged from 50.18 to 71.30 per 1E+06 clonable cells (3 hours exposure) and from 62.65 to 90.30 per 1E+06 clonable cells (24 hours exposure).
b/ with metabolic activation
The mutation frequencies of the cultures treated with test substance ranged from 85.50 to 101.52 per 1E+06 clonable cells (3 hours exposure, first assay) and from 58.14 to 102.97 per 1E+06 clonable cells (3 hours exposure, second assay).

These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 1E+06 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

Ratio of small to large mutant colonies
In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.43 to 1.50 for test substance treated cells and from 0.42 to 1.36 for the negative controls.

Remarks on result:

other: strain/cell type: TK +/-

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative without and with metabolic activation

Under the present test conditions, Pentamethyldiethylentriamine, tested up to cytotoxic concentrations, in the absence and presence of metabolic activation in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, Pentamethyldiethylentriamine also did not exhibit clastogenic potential at the concentration-range investigated.
According to the evaluation criteria for this assay, these findings indicate that Pentamethyldiethylentriamine, tested up to cytotoxic concentrations in the absence and presence of metabolic activation, did neither induce mutations nor have any chromosomal aberration potential.

Executive summary:

The test substance, Pentamethyldiethylentriamine, was tested for in-vitro mutagenicity in the Mouse Lymphoma Assay according to (EC) No. 440/2008 B.17 and OECD Guideline No.476: In Vitro Mammalian Cell Gene Mutation Test.

 

The substance was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; the experiment with S9 mix was carried out in two independent assays.

The vehicle DMSO (dimethylsulfoxide) served as the negative control.

 

In the preliminary experiment without and with metabolic activation (24-hour or 3-hour exposure) cytotoxicity (decreased survival) was noted starting at concentrations of 312.5 or 625 µg/mL, respectively.

 

Hence, in the main study the concentration range of 19.53 to 312.5 µg/mL was used in the experiments without metabolic activation with a 3-hour or 24-hour exposure time and a concentration-range of 39.06 to 625 µg/mL was used in the experiments with metabolic activation (3-hour exposure).

 

Methylmethanesulfonate (at 10 or 15 µL/mL) was employed as a positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene (at 2.5 or 4.0 µg/mL) in the presence of exogenous metabolic activation.

 

In the main study, cytotoxicity (decreased survival) was noted in the absence of metabolic activation (3-hour and 24-hor exposure) at the top concentration of 312.5 µg/mL and in the experiments with metabolic activation (3-hour exposure) at the top concentration of 625 µg/mL.

 

The values of mutation frequencies of the negative controls ranged from 50.18 to 82.96 per 1E+06 clonable cells in the experiments without metabolic activation and from 89.03 to 101.24 per 1E+06 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data-range.

 

The mutation frequencies of the cultures treated with Pentamethyldiethylentriamine ranged from 50.18 to 71.30 per 1E+06 clonable cells (3 hours exposure) and from 62.65 to 90.30 per 1E+06 clonable cells (24 hours exposure) in the experiments without metabolic activation and from 85.50 to 101.52 per 1E+06 clonable cells (3 hours exposure, first assay) and from 58.14 to 102.97 per 1E+06 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 1E+06 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

 

In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.43 to 1.50 for Pentamethyldiethylentriamine treated cells and from 0.42 to 1.36 for the negative controls.

 

The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3-MC) caused pronounced increases in the mutation frequency ranging from 1589.27 to 2712.47 per 1E+06 clonable cells in the case of MMS and ranging from 898.28 to 3466.39 per 1E+06 clonable cells in the case of 3-MC.

 

In addition, the colony size ratio was moderately shifted towards an increase in small colonies, ranging from 1.71 to 2.58 in the case of MMS.

 

Results

Under the present test conditions, Pentamethyldiethylentriamine, tested up to cytotoxic concentrations, in the absence and presence of metabolic activation in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.

In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, Pentamethyldiethylentriamine also did not exhibit clastogenic potential at the concentration-range investigated.

According to the evaluation criteria for this assay, these findings indicate that Pentamethyldiethylentriamine, tested up to cytotoxic concentrations in the absence and presence of metabolic activation, did neither induce mutations nor have any chromosomal aberration potential.

 

 

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

in vitro chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 10, 2021 - December 23, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

Adopted 29 July 2016

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Batch No.: UW20226411
Purity: 98%
Expiry Date: 14 February 2022
Storage conditions: Room Temperature

Species / strain / cell type:

lymphocytes: human lymphocyte

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Lymphocyte cultures, provided by Biopredic International (France).
- Suitability of cells:
- Normal cell cycle time (negative control):

For lymphocytes:
- Sex, age and number of blood donors: Main Assay I: 2 Female donors, 31 and 34 years old. Main Assay II: 2 Female donors, 22 and 28 years old.
- Whether whole blood or separated lymphocytes were used: Whole blood
- Whether blood from different donors were pooled or not: yes
- Mitogen used for lymphocytes: Colcemid

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Treatment medium in the presence of S9 metabolism:
Test item or control solution: 0.05mL
S9 mix: 1.00mL
Culture medium (without PHA: 3.95mL
Treatment medium in the absence of S9 metabolism:
Test item or control solution: 0.05mL
Culture medium (without PHA): 4.95mL

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: One batch of S9 tissue fraction, provided by Trinova Biochem GmbH; Species: Rat; Strain: Sprague Dawley; Tissue: Liver; Inducing Agents: Phenobarbital – 5,6-Benzoflavone (Producer MOLTOX,Molecular Toxicology, Inc.; Batch Number 4318)
- method of preparation of S9 mix: The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each10 mL): S9 tissue fraction (1.0 mL); NADP (100 mM; 0.4mL); G-6-P (100 mM; 0.5mL); MgCl2 (100 mM; 0.2 mL); MgCl2 (100 mM, 5.0 mL); Distilled Water (2.9 mL)
- concentration or volume of S9 mix and S9 in the final culture medium: S9 mix 1.00mL in treatment medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Tests for ALKOXYRESORIJFIN-O-DRAI.KYLASE ACTIVITIES Assays for ethoxyresomfin-O-deethylase (EROD), pentoxy-, benzyl- and methoxyresorufin-O-dealkylases (PROD, BROD, & PROD, MROD) were conducted using a modification of the methods of Burke, et al., Biochem Pham 34:3337, 1985. Fold-inductions were calculated as the ratio of the sam le vs. uninduced specific activities (SA s). Control SA's (pmoles/min/ mg protein) were 130.2, 84, 41.1, & 34 for BROD, EROD, MROD and PROD, respectively. TEST FOR THE PRESENCE OF ADVENTITIOUS AGENTS and PROMUTAGEN ACTIVATION were conducted.

Test concentrations with justification for top dose:

Dose levels of 2.00, 1.33, 0.889, 0.593, 0.395, 0.263, 0.176, 0.117 and 0.0780 μL/mL were
selected for the short time exposure ofMain Assay 1. For the continuous treatment in the
absence of S9 metabolism, the additional dose level of 0.0520 μL/mL was also employed.
In the short treatment series, the cytotoxicity observed at higher concentrations was not adequate to select dose levels for the scoring of chromosomal aberrations, hence treatments were repeated (Main Assay 2) using more closely spaced concentrations as follows: 2.00, 1.74, 1.51, 1.32, 1.14, 0.994, 0.865, 0.752 and 0.654 μL/mL.

Vehicle / solvent:

Sterile water for injection

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Cyclophosphamide (short-term treatment, -+S9,18.0 and 23.0 μg/mL ); Mitomycin-C (long-term treatment;0.300 and 0.450 μg/mL )

Details on test system and experimental conditions:

Preparation of the test item: Solutions of the test item, as received, were prepared immediately before use in sterile water for injection. Solutions were prepared on a volume/volume basis with correction for the displacement due to the volume of the test item. All test item solutions were used within 30 minutes from the initial preparation.

Rationale for test conditions:

Principles of the method
The in vitro chromosome aberration test detects structural changes in the chromosomes of human lymphocytes after in vitro treatment with the test item. Lymphocytes in whole blood cultures are stimulated to divide by exposure to phytohaemagglutinin (PHA). After approximately 48 hours, cells are treated with the test item or control solutions. Since cultured lymphocytes have little ability to metabolise indirect mutagens to reactive derivatives, the assay is performedboth in the absence and presence of an "S9" metabolising system.
The cells are exposed to the test item for a short treatment time with and without metabolic activation and for a long treatment time until harvest without metabolic activation The spindle poison colcemid is used to arrest the cells in metaphase and post treatment mitosis are harvested for analysis at a time corresponding to about 1.5 cell cycle lengths after the beginning of treatment (24 hours) to allow detection of chemicals which may cause cell cycle delays.

Evaluation criteria:

Criteria for outcome

In this assay, the test item is considered as clearly positive if the following criteria are met:

– At least one dose level shows a statistically significant increase in aberration-bearing cells (excluding gaps), compared with the concurrent negative control.

– Any of the results is outside the distribution of the historical negative control data.

– The increase is dose-related when evaluated with an appropriate trend test (Cochran- Armitage).

The test item is considered clearly negative if the following criteria are met:

– None of the dose levels shows a statistically significant increase in aberration-bearing cells (excluding gaps).

– There is no concentration related increase, when evaluated with an appropriate trend test.

– All the results are inside the distribution of the historical control data.

Statistics:

For the statistical analysis, Fisher’s Exact Test was used to compare the number of cells
bearing aberrations (assumed to be Poisson distributed) in control and treated cultures.
Bonferroni’s corrections were applied for multiple comparisons. The analysis was performed
using sets of data either including or excluding gaps.Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduction of the mitotic index

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Solubility test: A preliminary solubility trial was performed using sterile water for injection. This solvent was selected since it is compatible with the survival of the cells and the S9 metabolic activity. The test item was found to be miscible in water at the concentration of 200 μL/mL. An
aliquot of this stock solution added to culture medium in the ratio 1:100, gave a clear solution. Based on these results, the final concentration of 2.00 μL/mL in sterile water for injection was selected as the highest dose level to be used in the cytogenetic assay (the upper limit to be tested as indicated in the Study Protocol). Following treatment with the test item, no precipitate or opacity visible by eye were noticed at the beginning or by the end of treatment, in the absence or presence of S9 metabolism at any dose level, in any experiment.

Osmolality and pH results: Following treatment with the test item, no remarkable variation of pH or osmolality was observed at any dose level, in the absence or presence of S9 metabolism. The results are available in the Attachment under Overall Results.

Selection of dose levels for scoring: For all experiments the mitotic index (MI) was scored for each of the treatment series

Main Assay: following the short termtreatment in the absence of S9 metabolism, marked toxicity was observed at the highest dose level where the mitotic index was reduced to 11% of the concurrent negative control. At the next lower concentration of 1.33 μL/mL, test item treatments induced a mild toxicity reducing the mitotic index to 68% of the concurrent negative control. No remarkable toxicity was observed over the remaining dose range. In the presence of S9 metabolism the mitotic index was reduced to 38% and 57% of the concurrent negative control at the two highest dose levels, respectively. No remarkable toxicity was observed over the remaining dose range. Following the continuous treatment in the absence of S9, dose related toxic effects were observed reducing the relative mitotic index below 10% at the three highest dose levels. At
the intermediate concentration of 0.263 μL/mL a moderate reduction in mitotic index (MI
= 53%), judged suitable for scoring, was observed. As the toxicity levels observed for the short treatment series, were not considered adequate for the scoring of chromosomal aberrations, an additional experiment (Main Assay 2) was performed using a narrowed concentration range. A less pronounced toxic effect was noticed and no relevant reduction in mitotic index was observed at any dose level in the presence of S9 metabolism. In its absence, test item treatment at the highest dose level yielded moderate toxicity with a relative mitotic index of 50% over the concurrent negative.

Assay results: In this table the number and types of aberrations are presented, together with the total number of aberrations (chromatid type and chromosome type), including and excluding
gaps. The total number of aberrant metaphases, including and excluding gaps, is also shown.
Following treatment with the test item, no remarkable increase in the incidence of cells bearing aberrations, excluding gaps, over the concurrent negative control value, was observed in any treatment series. No relevant increase in numerical aberrations (polyploid and endoreduplicated cells) was observed at any dose level and treatment condition. Marked increases in the frequency of cells bearing aberrations (including and excluding gaps) were seen in the cultures treated with the positive control items,Mitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system.

Evaluation: The incidence of cells with structural aberrations (excluding gaps) in vehicle control cultures fell within the normal range based on historical control data (confidence interval: mean value ± 2 standard deviations). Adequate number of cells (300 at each test point) and test item concentrations were analysed. The positive control itemsMitomycin-C and Cyclophosphamide induced statistically significantincreases in the incidence of cells bearing structural aberration, compared with the concurrent negative control and the responses were compatible with historical control ranges. The study was accepted as valid. Test item treatments did not induce statistically significant increases in the incidence of cellsbearing aberrations and no statistically significant dose-effect relationship was indicated. All the results were inside the distribution of the historical data for negative controls with the exception of the result obtained at the intermediate dose level (1.32 μL/mL) in the presence of S9, that slightly exceeded the upper confidence limit of historical control data. However, the observed value fell within the confidence limits for the short treatment series in the absence of S9 metabolism, hence it was considered a chance event without any biological relevance. On the basis of these results and in accordance with the criteria for outcome of the study, the test item was not considered to induce chromosomal aberrations in human lymphocytes.

Remarks on result:

other: no mutagenetic results

Conclusions:

On the basis of these results, it is concluded that the test item does not induce chromosome aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254, prepared according to MARON and AMES (1983) was obtained from Trinova Biochem. S9 was collected from male rats.

Test concentrations with justification for top dose:

Test cncentrations: 31.6, 100, 316, 1000, 3160 and 5000 µg of the test item per plate in the plate corporation and preincubation test, without or with metabolic activation.
Plates 3 per concentration and experiment
Experiments 2 independent experiments, each with and without metabolic activation

The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. No signs of cytotoxicity and no test item precipitation was noted.
Hence, 5000 µg of the test item per plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test for the experiments without and with metabolic activation, respectively.

Vehicle / solvent:

water

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

1st independent experiment - Plate Incorporation Method
Sterile top agar containing 0.6 % agar and 0.5% NaCl was molten on the day of the test. 10 mL of a sterile solution of 0.5 mM L-histidine HCl /0.5 mM Biotin were added to 100 mL of molten agar. 2 mL of this top agar were distributed into culture tubes held at 45 °C in a heating block. 0.1 mL of Salmonella cell suspension (containing approximately 108 viable cells in the late exponential or early stationary phase), 0.1 mL of test item solution (or 0.1 mL vehicle or 0.1 mL positive control) and 0.5 mL of S9 mix were added to these culture tubes. In the assay without metabolic activation, the S9 mix was substituted with 0.5 mL phosphate buffer mentioned above.
The test components were mixed by vortexing the soft agar for 3 sec at low speed and then poured onto a coded 27.5 mL minimal glucose agar plate (Minimal Glucose Agar medium E). To achieve a uniform distribution of the top agar on the surface of the plate, the uncovered plate was quickly tilted and rotated and then placed on a level surface with the cover on and finally allowed to harden.
Immediately, the plates were inverted and placed in a dark 37 °C incubator for 48 h and could be stored after incubation for up to 24 hours at 4 °C. The revertant colonies on the test plates and on the control plates were counted with a colony counter , and the presence of the background lawn on all plates was confirmed. A lawn that was thin compared with the lawn on the negative control plate was evidence of bacterial toxicity.
Routine examination of the background lawn of bacterial growth resulting from the trace of histidine added to the top agar could be an aid in determining the presence of toxic effects. If massive cell death has occurred, the background lawn on the test plates would have been sparse compared with control plates.
In this case more histidine would be available to the individual surviving bacteria and they would undergo more cell divisions, consequently appearing as small colonies which could be mistaken for revertants if the absence of a normal background lawn is not noted.

2nd independent experiment - Preincubation Method
The test item was preincubated with the test strain (containing approximately 108 viable cells in the late exponential or early stationary phase) and sterile buffer (0.5 mL) or the metabolic activation system (0.5 mL) for 20 minutes at 37 °C prior to mixing with the overlay agar and pouring onto the surface of a minimal agar plate. 0.1 mL of the test item solution (or 0.1 mL vehicle or 0.1 mL positive control), 0.1 mL of bacteria, and 0.5 mL of S9 mix or sterile buffer, were mixed with 2 mL of overlay agar. Tubes were aerated during preincubation by using a shaker. The remaining steps were the same as described for the plate incorporation method.

Rationale for test conditions:

The test item was completely dissolved in water for injection shortly before use. The vehicle water for injection was employed as the negative control. Fresh preparations of the test item were used for the treatment in all experimental parts.
Prior to the main test two preliminary cytotoxicity tests (plate incorporation test, without and with metabolic activation) were carried out in test strain TA100.
Cytotoxicity is evidenced by a reduction in the number of spontaneous revertants by at least 50%, a clearing or diminution of the background lawn or by the degree of survival of the treated cultures. Insolubility could have been assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye. The recommended maximum test concentration for soluble non-cytotoxic test items is 5 mg/plate or 5 µL/plate. For non-cytotoxic test items that are not soluble at 5 mg/plate or 5 µL/plate, one or more concentrations tested should be insoluble in the final treatment mixture. Test items that are cytotoxic already below 5 mg/plate are tested up to a cytotoxic concentration. Interference of precipitates with the scoring should be avoided.
In the main study 6 different concentrations of the test item were tested, with half-log intervals between plates (31.6, 100, 316, 1000, 3160 and 5000 µg PU-2019-872 per plate in the plate corporation and preincubation test, without or with metabolic activation).

Evaluation criteria:

Bacteria colonies were counted employing the Biosys Biocount 5000 γ system. Print outs of the colony counts were filed with the raw data. Occurrence of test item precipitation would have been documented after visual inspection of the cultures with the unaided eye. Cytotoxicity is defined as reduction in the number of colonies by more than 50% compared to the solvent control and/or a scarce background lawn.
Acceptance Criteria:
The results of the negative and positive control cultures should be within the range of the historical data generated by LPT.
The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
TA98: 20 - 60
TA100: 100 - 200
TA102: 240 - 320
TA1535: 10 - 35
TA1537: 3 - 20
A test item is considered to show a positive response if
- the number of revertants is significantly increased (p  0.05, U-test according to MANN and WHITNEY, see section ‎6, reference ‎3) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient (section ‎6, reference ‎3) may be applied.
Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of Salmonella typhimurium.
A test item for which the results do not meet the above-mentioned criteria is considered as non-mutagenic in the AMES test.

Where concurrent negative or positive control data fall outside the range, they may be acceptable and considered for the inclusion into the historical control distribution as long as these data are not extreme outliers.

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:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with metabolic activation/Preincubation test: 5000 µg/plate with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3160 and 5000 µg/plate without metabolic activation/ Preincubation test: at 3160 µg/plate without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

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:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Preincubation test: at 3160 and 5000 µg/plate without metabolic activation and at 5000 µg/plate with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test item tested up to a cytotoxic concentration of 5000 µg/plate, without and with metabolic activation, respectively, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test.

Six concentrations ranging from 31.6 to 5000 µg of the test item/plate were employed in the plate incorporation test and in the preincubation test for the experiments without and with metabolic activation, respectively.

Cytotoxicity
Cytotoxicity in form of reduction of the number of revertants by more than 50% was noted in the plate incorporation test at concentrations of 3160 and 5000 µg of the test item/plate in test strain TA98 without metabolic activation and at a concentration of 5000 µg of the test item/plate in test strain TA1537 with metabolic activation. In the preincubation test, cytotoxicity in form of reduction of the number of revertants by more than 50% was noted for the following concentration(s) and test strains: at 3160 µg of the test item/plate in test strain TA98 without metabolic activation, at 3160 and 5000 µg of the test item//plate in test strain TA102 without metabolic activation, at 5000 µg of the test item/plate in test strain TA102 with metabolic activation and at 5000 µg of the test item/plate in test strain TA1537 without metabolic activation.
Mutagenicity
No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to a concentration of 5000 µg/plate, in the plate incorporation test and in the preincubation test for the experiments without and with metabolic activation, respectively.
The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

In conclusion, under the present test conditions, the test item tested up to a cytotoxic concentration of 5000 µg/plate, without and with metabolic activation, respectively, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test.

Executive summary:

The test item tested up to a cytotoxic concentration of 5000 µg/plate, without and with metabolic activation, respectively, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 21, 2021 - March 3, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(Adopted July 2016)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Batch No.: UW20226411
Purity: 98%
Expiry Date: 14 February 2022
Storage conditions: Room Temperature

Species:

rat

Strain:

CD-1

Remarks:

[Hsd: ICR(CD-1)]

Details on species / strain selection:

The specieswere those stated in the regulations, giving a valid model for the assessment of in vivo genotoxicity.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy - Age at study initiation:
- Weight at study initiation: 181.0 -199.3 g.
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Housing: Animals were housed up to 5 animals/cage in polisulphone H-temp solid bottomed cages with nesting material provided into suitable bedding bags
- Diet (e.g. ad libitum): commercially available laboratory rodent diet ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ±15
- Photoperiod (hrs dark / hrs light): 12

Route of administration:

oral: gavage

Vehicle:

The vehicle used in this study was sterile water of injectable grade (batch nos. 20B0703 and
20C3004 obtained from Baxter, Italy).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Fresh suspensions of the test item were prepared for each day’s work in the vehicle. Concentrations of solutions were calculated and expressed in terms of test item as received. All dose levels in this Report are expressed to three significant figures.
For the Main Assay, dose levels were selected on the basis of a preliminary toxicity experiment,
where two groups of two male and two female animals each were dosed on two consecutive days at 600 and 300mg/kg body weight/day. The maximum dose level of 600mg/kg body weight/day was selected on the basis of information received from the Sponsor (Safety Data Sheet).
Animals were inspected approximately 1 hour after dosing, 3 hours after dosing and at the end of working day daily throughout the study for signs of reaction to treatment. Besides, they were weighed daily starting from Day 1 of treatment.
Animals were sacrificed approximately twenty-four hours after dosing and bone marrow smear slides were prepared . Scoring was performed on slides prepared from the femurs of animals from the high dose group.

Duration of treatment / exposure:

around 23-24 hours for administration with test item; 24 hours for administration with positive control. An additional group of 3 satellite rats was dosed once with test item at 600mg/kg to performthe blood samplings for proof of exposure, pre-dose, 1 and 2 hours after dosing.

Frequency of treatment:

24 h interval between 2 administrations

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

five animals per sex and dose

Control animals:

yes, concurrent vehicle

Positive control(s):

The vehicle used in this study was sterile water of injectable grade (batch nos. 20B0703 and
20C3004 obtained from Baxter, Italy).

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

Animals were sacrificed at the appropriate sampling time as indicated in the experimental scheme. One femur of each animal was rapidly dissected out and cleaned of surrounding tissue. In order to extract the bone marrow, the bone was cut at the proximal end and irrigated with foetal calf serum using a syringe. The suspension of cells was aspirated and this procedure wasrepeated several times. The suspension thus obtained was centrifuged at 1000 rpm for 5 minutes and the supernatant completely removed. The cells of the sediment were then resuspended and transferred onto clean microscope slides as smear preparations.These slides were air-dried and then fixed with methanol for 10 minutes. Subsequently slides were stained with haematoxylin and eosin solutions. Three slides were made from each animal.The slides were randomly coded by a person not involved in the subsequent microscope scoring. The slides were examined under low power and one or two slides from each animal were selected according to staining and quality of smears. Immature polychromatic erythrocytes (PCE) stain a pink-purple colour (since they retain basic ribosomal material for approximately 24 h after enucleation) and can be distinguished from the pink normochromatic
erythrocytes (NCE). The polychromatic cells are also slightly larger and have more diffuse boundaries. Erythrocytes lack nuclei, making micronuclei obvious when present. The criteria of Schmid (1976) will be used to score micronuclei.At least four thousand PCEs per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time, the numbers of normal and micronucleated normochromatic erythrocytes (NCEs) were also recorded.

Evaluation criteria:

Acceptance criteria: The incidence of micronucleated PCEs of the vehicle control group falls within the historical negative control range. The positive control item induces a significant increase in the frequency of micronucleated PCEs and the response falls within the historical positive control range.The appropriate number of doses and cells has been analysed.

Criteria for outcome of assay: The test item is considered to induce micronuclei if: At least one of the treatment groups exhibits a statistically significant increase (p<0.05) in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control. The increase is dose-related when evaluated with an appropriate trend test. Any of these results are outside the distribution of the historical negative control data (95% control limits).
Where increases in the incidence of micronucleated PCEs are observed, which are statistically
significant, but fall within the range of negative control values of this laboratory, then these data are used to demonstrate that these increases do not have biological significance

Statistics:

After completion of microscopic analysis, the data for each slide were decoded and the results analysed as described below. Only counts obtained from polychromatic cells were subjected to statistical analysis. Using the original observations (and not the micronucleus frequencies per 1000 cells), a modified χ2 calculation was employed to compare treated and control groups. The degree of heterogeneity within each group was first calculated and where this was significant, it was considered in the comparison between groups. In this case the variance ratios (F) were calculated from the between-groups and within group χ2 values. In addition, a test for a lineartrend (Snedecor and Cochran) was performed in order to evaluate dose effect relationship.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: two male and two female animals each were dosed on two consecutive days at 600 and 300mg/kg body weight/day
- Clinical signs of toxicity in test animals: body weight loss (300 mg/kg bw); body weight loss, semiclosed yes, hypoactivity
- Evidence of cytotoxicity in tissue analysed: no

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Following treatment with the test item, no increase in the incidence of micronucleated PCEs over the concurrent vehicle control was observed in any group. Increases in the frequency of micronucleated PCEs were observed in the positive control group, indicating the correct functioning of the test system.
- Appropriateness of dose levels and route: Preparations of test item were analysed with respect to concentration and homogeneity (high and low concentrations) by the Analytical Chemistry Department of the test institute-
- Bone Marrow cell Toxicity: The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no inhibitory effect on erythropoietic cell division was observed at any dose level. The results confirmed the identity of the test item and concentrations and homogeneity of all preparations were within the acceptance limits (85-115%; precision CV < 10%)
- Observations: Following treatment with the test item, hypoactivity, semiclosed eyes and body weight loss were observed in the animals from the high dose group. Body weight loss was also seen in the intermediate dose group. No signs were observed in any other treatment group.
- Statistical evaluation: The incidence of micronucleated PCEs in the vehicle control fell within the distribution of the historical negative control range. A statistically significant increase in the incidence of micronucleated PCEs over the control values was seen in the positive control group and the response was compatible with those generated in the historical control database. The appropriate number of doses and cells was analysed. The study was accepted as valid since the above mentioned criteria were met. Following treatment with test item, no statistically significant increase in the incidence of micronucleated PCEs over the control value was observed in any treatment group and no dose effect relationship was noted. Bone marrow exposure was indicated by bioanalytical results. On the basis of the above mentioned results and in accordance with the criteria for outcome of the study, the test item was not considered to induce micronuclei in the polychromatic erythrocytes of treated rats

Conclusions:

On the basis of the results obtained, it is concluded that test item, administered by oral gavage, does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The available data for genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification