Ethylenediamine, salt with phosphoric acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

Ames assay was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrationsviz.0,0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations: 0, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methodsi.e.Plate incorporation method and Pre-incubation method. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

In vitro mammalian chromosome aberration study:

In vitro mammalian chromosome aberration study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29th July 2016 “In Vitro Mammalian Chromosome Aberration Test. Blood samples were obtained by vein puncture using syringe from healthy donor (non smoker, non alcoholic) not receiving medication for at least 3 months and being in the range of 31-32 years age. Samples were collected in heparinized vials. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in RPMI₀medium and used at dose level of 0, 0.125, 0.25 or 0.5 mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system (1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0.25, 0.5 and 1 mg/mLof culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 °C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37°C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. Two slides was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation. 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. The test chemical is not mutagenic at the highest tested concentration of 0.5 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

In vitro mammalian cell gene mutation assay:

The test chemical did not induce gene mutation in Chinese hamster ovary (CHO) cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

This study was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other:

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 metabolic activation system

Test concentrations with justification for top dose:

0, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled Water
- Justification for choice of solvent/vehicle: The test chemical was solulble in Distilled Water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.

Statistics:

No data

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test).

Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.002 - 5 mg/plate based on the solubility and precipitation test.
In treated concentration 5 mg/plate (T8), there was no reduction in colony count as well as in background lawn in treated concentrations both in absence and in the presence of metabolic activation.
Based on the results of pre-experiment following doses were selected for the main study trials: 0.050, 0.158, 0.501, 1.582 and 5 mg/plate, both in the absence (-S9) as well as in the presence (+S9) of metabolic activation.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

Ames assay was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. T

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz. 0, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment.

Based on the pre-experiment results, the test item was tested with the following concentrations: 0, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

This in vitro assay was performed to assess the potential of the test chemical to induce structural / numerical chromosomal aberrations in one experiment (phase I). The induction of cytogenetic damage in human lymphocytes was assessed with and without metabolic activation. Due to the negative result in phase I, a second experiment (phase II) was performed.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human blood
- Suitability of cells: No data
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:Age: 31-32 years age
- Whether whole blood or separated lymphocytes were used if applicable: Separated lymphocytes were used
- Number of passages if applicable: No data
- Methods for maintenance in cell culture if applicable: No data
- Modal number of chromosomes: No data
- Normal (negative control) cell cycle time: No data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Blood cultures were set up in medium containing RPMI-1640, Fetal Bovine Serum, Phytohaemagglutinin, Heparin solution, Whole Blood and Antibiotic Solution
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically 'cleansed' against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 metabolic activation system

Test concentrations with justification for top dose:

0.00, 0.125, 0.25 or 0.5 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI0
- Justification for choice of solvent/vehicle: The test chemical was soluble in RPMI0

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

RPMI0

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks

DURATION
- Preincubation period: No data
- Exposure duration: Phase 1: 4 hrs (with and without metabolic activation system)
Phase 2: 4 hrs (with metabolic activation system) and 24 hrs (without metabolic activation system)
- Expression time: 16-21 hrs (with and without metabolic activation system- Phase I and II)
- Selection time (if incubation with a selection agent):No data
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa stain in phosphate buffer

NUMBER OF REPLICATIONS: No data

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cultures were incubated at 37 ± 2 °C for duration (exposure period) as mentioned. For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The labelled slides were dried over a slide warmer at 50°C and labelled. At least one slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant.

NUMBER OF CELLS EVALUATED: A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Mitotic index
- Any supplementary information relevant to cytotoxicity: To evaluate the toxicity of the test item a cytotoxicity assay was performed both in the presence and absence of metabolic activation system. 3 test concentrations (0.25, 0.5 and 1 mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with Negative control. The procedure for conducting cytotoxicity was the same as main experiment phase I up to the scoring of the mitotic index, except slide coding. Based on the results of cytotoxicity experiment following doses were selected for the main study: 0.125, 0.25 and 0.5 mg/mL culture media.


OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

A test item can be classified as clastogenic if:
 At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
 If the increase is dose-related
 Any of the results are outside the historical negative control range
A test item can be classified as non – clastogenic if:
 None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
 If there is no dose-related increase
 All results are within the historical negative control range
Statistical significance was confirmed by means of the non-parametric Mann Whitney Test. However, both biological and statistical significance should be considered together.

If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Statistics:

Statistical significance at the p < 0.05 was evaluated by means of the non-parametric Mann-Whitney test

Species / strain:

lymphocytes: Human perpheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No Significant change in pH was observed at 0 h and 4 h when compared with negative controls.
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: There was slight precipitation observed at 1 mg/mL concentration.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item a cytotoxicity assay was performed both in the presence and absence of metabolic activation system. 3 test concentrations (0.25, 0.5 and 1 mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with Negative control. Cytotoxicity was observed in all treated concentration of 1 (T3) mg/mL both in the absence and in the presence of metabolic activation (1%).

In the absence of S9 mix, the mean mitotic index observed was 10.08 (NC), 8.44 (T1), 6.38 (T2), 4.46 (T3) and 8.35 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.09 (NC), 8.64 (T1), 6.49 (T2), 4.84 (T3) and 8.65 (PC).

In the cytotoxicity experiment, the highest test concentration 1 (T3) mg/ mL of treated culture media showed more than 50% reduction the mitotic index when compared to the respective Negative control both in the presence or absence of metabolic activation confirms the cytotoxicity effect. Hence this concentration was not selected for the main study.

Hence, 0.5 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation.

Hence the concentrations selected for the main study are 0.125, 0.25 or 0.5 mg/mL. The main study was performed in two independent phases

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: Please refer table remarks section

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Cytotoxicity Experiment     

Before conducting the chromosomal aberration study,Ethylenediamine, salt with phosphoric acid (CAS No. 14852-17-6)was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.25 (T1), 0.5 (T2) and 1.0 (T3) mg/mL of culture media. Cytotoxicity was observed in all treated concentration of 1 (T3) mg/mL both in the absence and in the presence of metabolic activation (1%).

In the absence of S9 mix, the mean mitotic index observed was 10.08 (NC), 8.44 (T1), 6.38 (T2),  4.46 (T3) and 8.35 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.09 (NC), 8.64 (T1), 6.49 (T2), 4.84 (T3) and 8.65 (PC).

In the cytotoxicity experiment, the highest test concentration 1 (T3) mg/ mLof treated culture mediashowed more than 50% reduction the mitotic index when compared to the respective Negative control both in the presence or absence of metabolic activation confirms the cytotoxicity effect. Hence this concentration was not selected for the main study.

Hence, 0.5 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation.

The main study was performed in twoindependentphases;

PHASE I         

In the experiment, the cultures were exposed to Ethylenediamine, salt with phosphoric acid (CAS No. 14852-17-6) for a short period of time (4 h) both in the absence and in the presence of metabolic activation system (1%).The mean percentage of aberrant cells was 0.333 (NC), 0.333 (T1), 0.667 (T2), 0.667 (T3) and 11.000 (PC) in the absence of metabolic activation and 0.333 (NC),     0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.000 (PC)in the presence of metabolic activation at the concentration of 0.0 (NC), 0.125 (T1), 0.25 (T2) and 0.5 (T3) mg/mL and positive controls, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of30 µg/mL in the presence of metabolic activation (1%) causedsignificant increase in percent aberrant cells.Even though the analysis did not reveal any statistical significance, the increase was biologically significant.

During thetreatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index observed at the tested concentrations.The observed mean mitotic indexin the absence of metabolic activation were 9.95, 8.80, 7.90, 7.04 and 8.35 andin the presence ofmetabolic activation were 10.00, 8.94, 8.15, 7.44 and 8.53 for0.0 (NC), 0.125 (T1), 0.25 (T2) and 0.5 (T3) mg/mLand 30 µg/mL(PC)concentrations respectively.

 

PHASE II          

The phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the Phase II, test item concentrations used were   0.0 (NC), 0.125 (T1), 0.25 (T2) and 0.5 (T3) mg/mL and 30 µg/mL (PC) culture both in absence and presence of metabolic activation (2%). The duration of exposure to the test item in presence of metabolic activation system was 4 hours and in absence of metabolic activation the duration of exposure was 24 hours. The mean percent aberrant cells were 0.333 (NC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.333 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (T1),     0.333 (T2), 0.333 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.0 (NC), 0.125 (T1), 0.25 (T2) and 0.5 (T3) mg/mL of culture and positive control, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of30 µg/mL in the presence of metabolic activation (2%) causedsignificant increase in percent aberrant cells.Though the analysis did not reveal any statistical significance, the increase was biologically significant.

The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment.

Treatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index was observed at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 9.94, 9.04, 8.10, 6.85 and 8.34 andin the presence ofmetabolic activation were 10.07, 9.19, 8.46, 7.60 and 8.50 for0.0 (NC), 0.125 (T1), 0.25 (T2) and 0.5 (T3)and30 µg/mL(PC)concentrations respectively.

Note: NC: Negative control; T1: Test concentration1; T2: Test concentration 2; T3: Test concentration 3; PC: Positive Control.

Conclusions:

The test chemical is not mutagenic at the highest tested concentration of 0.5 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

In vitro mammalian chromosome aberration study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29th July 2016 “In Vitro Mammalian Chromosome Aberration Test.

 

Blood samples were obtained by vein puncture using syringe from healthy donor (non smoker, non alcoholic) not receiving medication for at least 3 months and being in the range of 31-32 years age. Samples were collected in heparinized vials. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in RPMI₀medium and used at dose level of 0, 0.125, 0.25 or 0.5 mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system (1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0.25, 0.5 and 1 mg/mLof culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 °C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37°C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. Two slides was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation. 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis.

 

The test chemical is not mutagenic at the highest tested concentration of 0.5 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE for the target CAS is summarized based on data from various test chemicals

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HGPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

9

Details on mammalian cell type (if applicable):

- Type and identity of media: F12-D5 medium and incubated at 37°C in a 5% CO2 atmosphere.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

10

Details on mammalian cell type (if applicable):

- Type and identity of media: F12D5 medium
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Fresh S9-1iver homogenate was isolated from Aroclor 1254-induced rats

Test concentrations with justification for top dose:

9. 0, 0.125, 0.25, 0.5, 1, 2, 4 or 8 µL/mL
10. Upto 1 µL/mL

Vehicle / solvent:

9. - Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The test chemical was soluble in Distilled water

10. No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: dimethylnitrosamine

Remarks:

9

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

10

Details on test system and experimental conditions:

9. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 5 hrs
- Expression time (cells in growth medium): 18-24 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: The colony-forming potential of 100-200 treated cells was used as the measure of treatment-induced cytotoxicity. The colony-forming ability was determined by the viable fraction of the plated cells used to correct the mutant frequency for the individual treated cultures and to detect variations in the growth ability of the cells.

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

10. METHOD OF APPLICATION: in medium
Cells at the start of the experiment: 1000000 cells

DURATION
- Preincubation period: No data
- Exposure duration: 5 hrs
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

9. The cell line was observed for gene mutation at the locus specified
10. A test result was considered a positive effect of the test chemical whenever the frequency of mutants corrected for colony-forming ability was statistically different from the concurrent control value at a minimum of 2 consecutive doses and/or there was evidence of a dose-related effect of treatment.

Statistics:

9. Statistical analysis of the mutation data for this assay has been described by Slesinski et al.
10. Data were analyzed for significant differences from the concurrent solvent control values by transformation of the mutant frequency values using the procedure of Box and Cox (1964) and statistical comparison to the solvent control values with either Student's or Cochrans's t test. Variances of historical control data were used for statistical comparisons for concurrent controls with zero mutants.

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

9

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

10

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce gene mutation in Chinese hamster ovary (CHO) cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed usingChinese hamster ovary (CHO) cell linein the presence and absence of S9 metabolic activation system. The test chemical was dissolved in water and used at dose level of 0, 0.125, 0.25, 0.5, 1, 2, 4 or 8µl/mL. The methods described for determining colony-forming ability and the frequency of mutants resistant to 6-thioguanine were followed. Approximately 20-24 h prior to the mutation test, 5 × 10⁵cells were inoculated into each of two 25-cm²culture flasks containing F12-D5 medium, and incubated at 37°C in a 5% CO₂atmosphere. On the day of testing, appropriate concentrations of the test agent were added to duplicate cultures of cells, and cultures were treated for 5 h at 37°C. The cells were allowed a period of 18-24 h of recovery from treatment before chemical-induced cytotoxicity was determined. Treatment of cells in the presence of an S9 metabolic activation system was performed identically, with the exception that F12 medium without serum was used. The colony-forming potential of 100-200 treated cells was used as themeasure of treatment-induced cytotoxicity. At 2-3-day intervals after treatment, cells were subcultured. After a period of at least 7 days to allow expression of the mutant phenotype, cells were dissociated with 0.075% trypsin, counted and plated. The colony-forming ability determined by the viable fraction of the plated cells was used to correct the mutant frequency for the individual treated cultures and to detect variations in the growth ability of the cells. Statistical analysis of the mutation data for this assay has been described by Slesinski et al. Based on the observations made,the test chemical did not induce gene mutation inChinese hamster ovary (CHO) cell linein the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another study, In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed as per the HGPRT assay usingChinese hamster ovary cellsin the presence and absence of S9 metabolic activation system. The test chemical was used up to a dose level of 1.0µL/mL. Because the test chemical is highly alkaline and significantly altered the pH of the medium, cultures exposed to high EDA concentrations were equilibrated with a 10 CO2:90% air mixture during exposure to EDA to attempt to buffer the alkalinity, and thus test the highest possible concentrations. Cells were treated with the control and test material for 5 hours both with and without metabolic activation. The surviving fraction was determined after an expression period of at least 7 days following subculture at 2-3 day intervals in F12D5 medium. For each experiment, mutants were selected by plating a total of 1000000 cells in 5-100 mm culture dishes with F12D5 medium supplemented with 2 ug/ml (12 uM) thioguanine. Colonies were stained and counted either manually or with an Artek 880 colony counter. None of the mutant frequencies for test chemical treated cells were statistically different from concurrent controls and the frequency of mutants for all doses was within the historical range of variation observed in the spontaneous mutation frequency for this test by us and others. The relatively high doses up to 0.1% by volume (1.0 ul/ml) tested in this assay were attained only by equilibrating cultures with 10% CO₂in air to attempt to neutralize the alkaline effects of the test chemical upon the medium. Although this CO₂equilibration procedure resulted in greater variability in the survival determinations in the separate experiments, very steep dose-survival effects were noted consistently in all experiments with the test chemical within the 2-fold range of concentrations between 0.5 and 1.0 ul/ml (0.05 and 0.10% by volume). Variability evident in the survival and plating efficiency values was likely caused by small variations in the CO₂equilibration and by growth variations typical of CHO cells in this test system. The lack of mutagenic effects of the test chemical in the repeated tests indicated that these variations were not of sufficient magnitude to affect the sensitivity of the test. Based on the observations made, thetest chemical did not induce gene mutation inChinese hamster ovary cellsin the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available, the test chemical did not induce gene mutation in Chinese hamster ovary (CHO) cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test chemical was tested for mutagenicity in this sperm abnormality assay by Bruce and Heddle. In brief, the assay was used as an in vivo method to study mutagenicity because it has been shown that mice exposed to mutagenic agents exhibit significantly increased numbers of abnormally shaped sperm cells. The abnormal sperm morphology is likely caused by small deletions, point mutations or both. Three male mice per dose level were exposed to the test chemical via intraperitoneal injection at 0 (vehicle), 125, 250 and 500 mg/kg bw/day for 5 consecutive days. The highest dose level was close to the pre-determined LD50 value. Mice treated with benzo(a)pyrene served as positive controls. All mice were sacrificed 30 days after the last injection. Sperm samples were then prepared accordingly to calculate the number of abnormally shaped sperm cells. A total of 333 sperm cells from each animal were examined under high magnification. The results were plotted as percent abnormal sperm after subtraction of the frequency found in the control group. Threshold for biological significance was breached if the treated group exceeded the control group by 1% (10/1000). The test chemical was considered active when positive results were obtained consistently, i.e., when dose response curves could be constructed, or the results could be repeated. No significant increase in the proportion of abnormal sperm cells could be observed following treatment with the test chemical. In contrast, the positive control group showed a distinct increase in the proportion of abnormal sperm cells at 125 mg/kg bw/day (lowest dose tested). Based on the presented data, the test chemical is non-mutagenic in mouse sperm cells following intraperitoneal injections at doses up to 500 mg/kg bw.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: gene mutation

Remarks:

Sperm abnormality assay

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data provided is from a peer-reviwed publication.

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

A significant increase in the proportion of abnormally shaped sperm cells has been observed in mice between four to eleven weeks after exposure to mutagenic agents (Proc. Natl. Acad. Sci. U.S.A. 1975; 72: 4425-4429). The abnormal sperm morphology is likely caused by small deletions, point mutations or both according to data from previous studies

GLP compliance:

not specified

Type of assay:

other: Sperm abnormality assay

Species:

mouse

Strain:

other: Hybrid mice (C57BL/6 X C3H/He)

Sex:

male

Details on test animals or test system and environmental conditions:

The rats were housed in suspension, wire bottom cages, in air conditioned rooms with automated light and dark cycles, and were allowed food (Purina Chow) and water ad lib. They were not treated with antibiotics or insecticides.

Route of administration:

intraperitoneal

Vehicle:

Water

Details on exposure:

The highest dose was selected by chosing a convenient value close to the LD50, which was initially established in the study.

Duration of treatment / exposure:

Five consecutive days

Frequency of treatment:

Once daily

Post exposure period:

30 days

Remarks:

0 (vehicle), 125, 250 and 500 mg/kg bw/day

No. of animals per sex per dose:

3 to 4.

Control animals:

yes, concurrent vehicle

Positive control(s):

Benzo(a)pyrene

Tissues and cell types examined:

Sperm cells

Details of tissue and slide preparation:

Sperm suspensions were prepared by mincing the cauda epididymis in 2 ml phosphate buffered physiological saline, pipetting the resulting suspension and filtering it through an 80 um stainless steel mesh to remove tissue fragments. A fraction of each suspension was mixed 10:l with 1% eosin-Y in water. Thirty min later smears were made, allowed to dry in air, and mounted under a coverslip with Permount. Three hundred and thirty-three sperm were examined at high magnification from each animal for a total of approx 1000 per treated group.

Evaluation criteria:

The results were plotted as percent abnormal sperm after subtraction of the frequency found in the control group. Threshold for biological significance was breached if the treated group exceeded the control group by 1% (10/1000). The test chemical was considered active when positive results were obtained consistently, i.e., when dose response curves could be constructed, or the results could be repeated.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the presented data, the test chemical is non-mutagenic in mouse sperm cells following intraperitoneal injections at doses up to 500 mg/kg bw.

Executive summary:

The test chemical was tested for mutagenicity in this sperm abnormality assay by Bruce and Heddle. In brief, the assay was used as an in vivo method to study mutagenicity because it has been shown that mice exposed to mutagenic agents exhibit significantly increased numbers of abnormally shaped sperm cells. The abnormal sperm morphology is likely caused by small deletions, point mutations or both. Three male mice per dose level were exposed to the test chemical via intraperitoneal injection at 0 (vehicle), 125, 250 and 500 mg/kg bw/day for 5 consecutive days. The highest dose level was close to the pre-determined LD50 value. Mice treated with benzo(a)pyrene served as positive controls. All mice were sacrificed 30 days after the last injection. Sperm samples were then prepared accordingly to calculate the number of abnormally shaped sperm cells. A total of 333 sperm cells from each animal were examined under high magnification. The results were plotted as percent abnormal sperm after subtraction of the frequency found in the control group. Threshold for biological significance was breached if the treated group exceeded the control group by 1% (10/1000). The test chemical was considered active when positive results were obtained consistently, i.e., when dose response curves could be constructed, or the results could be repeated. No significant increase in the proportion of abnormal sperm cells could be observed following treatment with the test chemical. In contrast, the positive control group showed a distinct increase in the proportion of abnormal sperm cells at 125 mg/kg bw/day (lowest dose tested). Based on the presented data, the test chemical is non-mutagenic in mouse sperm cells following intraperitoneal injections at doses up to 500 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Data available for the target chemical and its various test chemicals was reviewed to determine its mutagenic nature. The studies are as mentioned below:

Ames assay:

Ames assay was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrationsviz.0,0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations: 0, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methodsi.e.Plate incorporation method and Pre-incubation method. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

In vitro mammalian chromosome aberration study:

In vitro mammalian chromosome aberration study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29th July 2016 “In Vitro Mammalian Chromosome Aberration Test.

 

Blood samples were obtained by vein puncture using syringe from healthy donor (non smoker, non alcoholic) not receiving medication for at least 3 months and being in the range of 31-32 years age. Samples were collected in heparinized vials. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in RPMI₀medium and used at dose level of 0, 0.125, 0.25 or 0.5 mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system (1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0.25, 0.5 and 1 mg/mLof culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 °C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37°C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. Two slides was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation. 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis.

 

The test chemical is not mutagenic at the highest tested concentration of 0.5 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

In vitro mammalian cell gene mutation assay:

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed usingChinese hamster ovary (CHO) cell linein the presence and absence of S9 metabolic activation system. The test chemical was dissolved in water and used at dose level of 0, 0.125, 0.25, 0.5, 1, 2, 4 or 8µl/mL. The methods described for determining colony-forming ability and the frequency of mutants resistant to 6-thioguanine were followed. Approximately 20-24 h prior to the mutation test, 5 × 10⁵cells were inoculated into each of two 25-cm²culture flasks containing F12-D5 medium, and incubated at 37°C in a 5% CO₂atmosphere. On the day of testing, appropriate concentrations of the test agent were added to duplicate cultures of cells, and cultures were treated for 5 h at 37°C. The cells were allowed a period of 18-24 h of recovery from treatment before chemical-induced cytotoxicity was determined. Treatment of cells in the presence of an S9 metabolic activation system was performed identically, with the exception that F12 medium without serum was used. The colony-forming potential of 100-200 treated cells was used as themeasure of treatment-induced cytotoxicity. At 2-3-day intervals after treatment, cells were subcultured. After a period of at least 7 days to allow expression of the mutant phenotype, cells were dissociated with 0.075% trypsin, counted and plated. The colony-forming ability determined by the viable fraction of the plated cells was used to correct the mutant frequency for the individual treated cultures and to detect variations in the growth ability of the cells. Statistical analysis of the mutation data for this assay has been described by Slesinski et al. Based on the observations made,the test chemical did not induce gene mutation inChinese hamster ovary (CHO) cell linein the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another study, In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed as per the HGPRT assay using Chinese hamster ovary cellsin the presence and absence of S9 metabolic activation system. The test chemical was used up to a dose level of 1.0µL/mL. Because the test chemical is highly alkaline and significantly altered the pH of the medium, cultures exposed to high EDA concentrations were equilibrated with a 10 CO2:90% air mixture during exposure to EDA to attempt to buffer the alkalinity, and thus test the highest possible concentrations. Cells were treated with the control and test material for 5 hours both with and without metabolic activation. The surviving fraction was determined after an expression period of at least 7 days following subculture at 2-3 day intervals in F12D5 medium. For each experiment, mutants were selected by plating a total of 1000000 cells in 5-100 mm culture dishes with F12D5 medium supplemented with 2 ug/ml (12 uM) thioguanine. Colonies were stained and counted either manually or with an Artek 880 colony counter. None of the mutant frequencies for test chemical treated cells were statistically different from concurrent controls and the frequency of mutants for all doses was within the historical range of variation observed in the spontaneous mutation frequency for this test by us and others. The relatively high doses up to 0.1% by volume (1.0 ul/ml) tested in this assay were attained only by equilibrating cultures with 10% CO₂in air to attempt to neutralize the alkaline effects of the test chemical upon the medium. Although this CO₂equilibration procedure resulted in greater variability in the survival determinations in the separate experiments, very steep dose-survival effects were noted consistently in all experiments with the test chemical within the 2-fold range of concentrations between 0.5 and 1.0 ul/ml (0.05 and 0.10% by volume). Variability evident in the survival and plating efficiency values was likely caused by small variations in the CO₂equilibration and by growth variations typical of CHO cells in this test system. The lack of mutagenic effects of the test chemical in the repeated tests indicated that these variations were not of sufficient magnitude to affect the sensitivity of the test. Based on the observations made, the test chemical did not induce gene mutation inChinese hamster ovary cellsin the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available from the target chemical and its various read across chemicals, the test chemical does not exhibit gene mutation in vitro. Hence it is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

The test chemical is regarded to be classified as Not Classified for Germ cell mutagenicity. The decision is based on data from studies equivalent or similar to OECD Test Guidelines in which the test chemical or read-across chemicals have tested negative for mutagenicity in bacteria, for mutagenicity in Chinese hamster ovary cells, and for clastogenic effects in human peripheral lymphocytes. The decision is further supported by data from a sperm abnormality assay in which a read-across chemical showed no mutagenic potential in mice following intraperitoneal injections at doses up to 500 mg/kg bw/day.