[nitrilotris(methylene)]trisphosphonic acid, ammonium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): Read across from ATMP-H: negative with and without activation in all strains tested (according/similar to OECD Test Guideline 471 and in compliance with GLP) (Monsanto, 1981).

Gene mutation (Bacterial reverse mutation assay / Ames test): Read across from ATMP-H: negative with and without activation in E. coli WP2 uvr A strain tested (according to OECD Test Guideline 471 and in compliance with GLP) (Envigo, 2018).

Cytogenicity in mammalian cells: Read across from ATMP-5Na negative in CHO cells (similar to OECD Test Guideline 473 and in compliance with GLP) (Covance Laboratories, 1998a)

Mutagenicity in mammalian cells: Read across from neutralised ATMP-H: negative with and without activation in L5178Y mouse lymphoma cells (similar to OECD Test Guideline 476 and in compliance with GLP) (SRI International, 1988).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-06-25 to 1998-08-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

up to 4400 µg active salt/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: none given in report

Untreated negative controls:

yes

Remarks:

growth medium

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without activation

Untreated negative controls:

yes

Remarks:

growth medium

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours (initial assay) 17.8 hours (confirmatory assay)
- Expression time (cells in growth medium): 16.8 hours (initial assay 2 hours (confirmatory assay)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (present for last 2 hours of incubation)
STAIN (for cytogenetic assays): 5% Giesma solution

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED:100 from each replicate culture where possible

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; other: assessment of percent confluence of cell monolayer and presence of mitotic or dead cells floating in medium.


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes


OTHER:

Evaluation criteria:

The test substance is considered positive for inducing chromosomal aberrations if there is significant dose-dependent increase (p<=0.01) in the number of cells with aberrations at one or more concentrations

Statistics:

Cochran-Armitage test for linear trend and Fisher's Exact test to compare percentage of cells with aberrations in treated cells with control results.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Non cytotoxic for 3h treatments. <500 µg/ml for continuous treatments without activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH was measured at 9.0 (culture medium pH 8.5)
- Precipitation: no precipitation was observed
- Other confounding effects:


RANGE-FINDING/SCREENING STUDIES: no precipitate observed in the absence of cells at a concentration of 4400 ug/ml

COMPARISON WITH HISTORICAL CONTROL DATA: control values were within historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: dead monolayers and approximately 85% reduction in monolayer confluence reported at 4400 µg/ml. Unhealthy monolayers and approximately 70% reduction reported at 3080 µg/ml. Unhealthy monolayers and approximately15% or 45% reduction in monolayer confluence at 2160 µg/ml. In absence of metabolic activation no cytotoxicity was seen with 3 hr treatment. With continuous treatment cytotoxicity was induced.  The top dose scored (250 µg/ml) had a relative mitotic index of 60%.  The higher dose (500 µg/ml) had a relative mitotic index of <10%. 

Table 1: Results of chromosome analysis Experiment 1 (3 h treatment, 20 incubation) without activation (total count from 2 cultures / 200 cells)

-		Untreated	Solvent* Control	Positive Control	Low dose 1510 µg/ml	Mid dose 2160 µg/ml	Mid dose 3080   µg/ml	High dose 4400 µg/ml
Cytotoxicity		-	-	-	no	no	no	no
		Percentage from 200 cells
	Percentage of cells with aberrations	3.0	1	52	3.5	0	3.0	0
Mitotic index (%)		14.8	20.8	NR	23.1	23.5	24.0	17.8
Polyploidy (%)		2.5	2.5	3.0	2.5	3.0	2.1	2.0
Endo reduplication (%)		2	1.5	1.0	2.0	1.5	0.5	2.5

 *Solvent control with water

NR not reported

 

 

Table 2: Results of chromosome analysis Experiment 2a (17.8 h treatment, 20 h incubation) without activation

-		Untreated	Solvent* Control	Positive Control	Low dose 31.3 µg/ml	Mid dose 62.6µg/ml	Mid dose 125 µg/ml	High dose 250 µg/ml
Cytotoxicity		-	-	-	no	no	no	no
		Percentage from 200 cells
	Percentage of cells with aberrations	0	0	12.8	1.5	0	1.0	1.5
Mitotic index (%)		6.5	7.0	NR	4.0	6.9	4.0	4.2
Polyploidy (%)		9.5	0.5	2.5	1.5	0	0.5	0.5
Endo reduplication (%)		0	0	0	1.5	0	0	0

*Solvent control with water

NR not reported

 

Table 3: Results of chromosome analysis Experiment 1, (3 h treatment, 20 h incubation) with activation

-		Untreated	Solvent* Control	Positive Control	Low dose 1510 µg/ml	Mid dose 2160 µg/ml	Mid dose 3080 µg/ml	High dose 4400 µg/ml
Cytotoxicity		-	-	-	no	no	no	no
		Percentage from 200 cells
	Percentage of cells with aberrations	1.5	1.0	60.0	1.0	8.0	1.5	1.0
Mitotic index (%)		16.0	19.9	NR	21.9	15.7	16.9	11.7
Polyploidy (%)		3.0	2.5	2.5	2.0	2.0	2.0	1.0
Endo reduplication (%)		1.5	0	1.5	1.0	6.0	0	0

*Solvent control with water

NR not reported

 

Table 4: Results of chromosome analysis Experiment 2, (3 h treatment, 20 h incubation) with activation

-		Untreated	Solvent* Control	Positive Control	Low dose 1510 µg/ml	Mid dose 2160 µg/ml	Mid dose 3080 µg/ml	High dose 4400 µg/ml
Cytotoxicity		-	-	-	no	no	no	no
		Percentage from 200 cells
	Percentage of cells with aberrations	1.5	2.0	52	1.0	1.5	0.5	0.5
Mitotic index (%)		13.1	14.1	NR	12.8	13.8	13.3	13.3
Polyploidy (%)		2.0	1.5	2.0	2.0	3.0	2.0	2.5
Endo reduplication (%)		2.0	1.5	0.5	2.5	2.9	1.0	2.5

*Solvent control with water

NR not reported

Conclusions:

ATMP-5Na has been tested for clastogenicity in vitro in Chinese hamster Ovary (CHO) cells in a study conducted according to OECD Test Guideline 473 and in compliance with GLP (Covance Laboratories, 1998a). The test substance did not induce chromosome aberrations in vitro when tested up to cytotoxic concentration with or without metabolic activation. Positive, negative and solvent controls gave the expected results. It is concluded that ATMP-5Na does not cause chromosomal aberrations under the conditions of the test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 November 2017 to 23 November 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only one test strain of E. coli was used to detect mutations via cross-linking.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

Only one test strain of E. coli was used to detect mutations via cross-linking.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: On the day of the experiment, the test item ATMP-H was dissolved in deionized water. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable for this period unless specified otherwise by the Sponsor.
- Preliminary purification step (if any): The dose selection was adjusted to the purity of 33%.
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

To evaluate the toxicity of the test item a pre-experiment was performed. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test). In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. Since no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration.
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Remarks:

no treatment

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

ACTIVATION: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10 % (v/v) in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: 48 hours at 37°C in the dark

SELECTION AGENT (mutation assays): selective agar

NUMBER OF REPLICATIONS: triplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: number of revertants
- Any supplementary information relevant to cytotoxicity: not specified

Rationale for test conditions:

To evaluate the toxicity of the test item a pre-experiment was performed. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test). In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. Since no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration.

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 the colony count of the corresponding 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 and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not specified
- Effects of osmolality: not specified
- Evaporation from medium: not specified
- Water solubility: not specified
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES: In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: within the range of positive historical control data
- Negative (solvent/vehicle) historical control data: within the range of negative historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: number of revertants

Table 1: Summary of experiment 1

Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
Without Activation		WP2 uvrA
Deionised water		48 ± 8
Untreated		48 ± 4
ATMP-H	3 µg	43 ± 5
	10 µg	48 ± 8
	33 µg	44 ± 8
	100 µg	48 ± 10
	333 µg	43 ± 9
	1000 µg	47 ± 11
	2500 µg	40 ± 7
	5000 µg	36 ± 5
MMS	2.0 µL	953 ± 87
With Activation
Deionised water		55 ± 3
Untreated		56 ± 11
ATMP-H	3 µg	51 ± 8
	10 µg	57 ± 8
	33 µg	51 ± 13
	100 µg	49 ± 15
	333 µg	51 ± 7
	1000 µg	55 ± 7
	2500 µg	39 ± 9
	5000 µg	47 ± 13
2-AA	10.0 µg	534 ± 112

Table 2: Summary of experiment 2

Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ± SD)
Without Activation			WP2 uvrA
Deionised water			32 ± 5
Untreated			34 ± 4
ATMP-H	33 µg		36 ± 5
	100 µg		35 ± 5
	333 µg		32 ± 6
	1000 µg		31 ± 2
	2500 µg		33 ± 4
	5000 µg		19 ± 2
MMS	2.0 µL		945 ± 40
With Activation
Deionised water			42 ± 2
Untreated			49 ± 12
ATMP-H	33 µg		34 ± 3
	100 µg		43 ± 2
	333 µg		37 ± 4
	1000 µg		36 ± 4
	2500 µg		35 ± 5
	5000 µg		28 ± 3
2-AA	10.0 µg		495 ± 16

MMS: methyl methane sulfonate

2-AA: 2-aminoanthracene

Conclusions:

ATMP-H has been tested in a valid bacterial reverse mutation assay, according to OECD Test Guideline 471 and in compliance with GLP, using E. coli WP2 uvr A. No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to limit concentration. Appropriate positive, negative and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The restrictions were that only four strains of bacteria were used.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

insufficient range of strains to meet requirements of current guideline

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: Salmonella typhimurium TA98, 100, 1535, 1537

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced mouse and rat liver S9

Test concentrations with justification for top dose:

0.01, 0.04, 0.2, 1, 3,10ul Dequest 2000/plate and 25ul Dequest 2000 Dequest/plate for spot test. Expressed as ATMP: 0.0065, 0.026, 0.13, 0.65, 1.95, 6.5ul/plate for plate incorporation; up to 9ul for spot test

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: none given

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 and TA 100 without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium nitrite

Remarks:

TA 1535 without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA 98 and TA 100 with activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 1535 and TA 1537 with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); as impregnation on paper disk


DURATION
- Preincubation period: none
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

NUMBER OF REPLICATIONS: single applications (spot test), triplicate plates (plate incorporation)

DETERMINATION OF CYTOTOXICITY
- Method: other: reduction in microbial lawn

Evaluation criteria:

A positive response is indicated if three of more treatments are significantly greater than the solvent control with a significant dose-response.

Statistics:

Analysis of log10 revertants per plate used Bartlett's test for homogeneity of variance and comparison of treatments with controls used within-levels pooled variance and a one-sided t-test. Where values were found to be significant, a Grubb's test was applied and significance of dose-response was evaluated by a t-test.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1-3 μL/plate for plate incorporation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1-3 μL/plate for plate incorporation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1-3 μL/plate for plate incorporation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1-3 μL/plate for plate incorporation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the main study toxicity was seen as follows: +S9; 10ul, 3ul (TA98, TA1535); 10ul, 3ul, 1ul (TA100, TA1537) -S9: 10ul, 3ul (all strains)

Remarks on result:

other: No mutagenic potential

No increase in revertants in any strain/metabolic activation condition. Full details of results presented in report.  Positive and negative values acceptable.

Conclusions:

ATMP-H has been tested for mutagenicity in vitro in Salmonella typhimurium TA98, 100, 1535, 1537 in a study conducted according to a protocol similar to OECD Test Guideline 471 and in compliance with GLP. ATMP-H did not increase the number of revertants when tested up to cytotoxic concentration with or without metabolic activation. The vehicle and positive controls gave the expected results. ATMP-H was concluded to be negative for mutagenicity in bacteria under the conditions of the test.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

: not tested in the absence of metabolic activation

Principles of method if other than guideline:

Method: Clive et al

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

0-1.2 µl Dequest 2000/ml (calculated by previous reviewer as 0-0.78 µl active acid/ml). These concentrations were positive in the previous experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no information

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 2 days


SELECTION AGENT (mutation assays): 5ug/ml trifluorothymidine

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 6x10E06 (1st experiment); 9x10E06 (2nd experiment)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG)

Evaluation criteria:

Positive result: Dose-related increase in number of mutant colonies and mutant frequency at one or more concentrations of greater than or equal to twice control levels with an RTG of greater than 10%.

Statistics:

No statistical analysis of results was carried out.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Remarks:

Concentrations based on data from previous experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: test solution was neutralised to eliminate pH effects. Slight drop in pH seen at highest concentration but approximate fall in 0.08pH units only
- Precipitation: white precipitate reported at concentrations greater than 0.61µl/ml

RANGE-FINDING/SCREENING STUDIES: not conducted as this is a follow-up experiment

COMPARISON WITH HISTORICAL CONTROL DATA: no data

In the first experiment all treated cultures had higher RTGs than the control culture and a dose-related increase in mutant frequency was seen (see Table 1). The experiment was discounted because of the low control growth.

Table 1: Results of Mammalian Mutagenicity assay 1 with tester strain L5178Y in presence of metabolic activation

Concentration µl/ml	Mutant frequency x10E-06	RTG %
0*	23	100
0.49	22	195
0.51	33	134
0.77	48	149
0.96	59	133
1.2	58	121

In the second experiment no increases in mutant frequency and no cytotoxicity were seen. It had been proposed that a selective advantage of mutants in the presence of test substance was the reason for the increase in revertants observed in the previous experiment. No effects on levels of spontaneous mutants were seen when cells were plated immediately after treatment. This finding does not support the proposed reason for the previously observed positive result.

Table 2: Results of Mammalian Mutagenicity assay 2 with tester strain L5178Y in presence of metabolic activation

Concentration µl/ml	Mutant frequency x10E-06	RTG %
0*	101	100
0.61	91	100
0.77	112	74
0.96	98	92
1.2	100	88
1.5	112	103
Positive control	659	46

Conclusions:

A neutralized solution of ATMP acid was tested for mutagenicity to mammalian cells under GLP. No increase in the number of revertants was observed in the presence of metabolic activation when tested up to the solubility limit. The test results indicate that the positive findings in the previous experiment with un-neutralised ATMP acid were an artefact of pH. It is concluded that ATMP acid is not mutagenic to mammalian cells under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus assay oral gavage study in mouse: Read across from ATMP-5Na Negative (OECD Test Guideline 474 and in compliance with GLP) (Covance Laboratories, 1998b).

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

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:

12/96 final draft

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Crl: CD-1 (ICR) BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Labs Raleigh NC
- Age at study initiation: approx 8 weeks
- Weight at study initiation: 30.6 - 33.9 g (males); 23.6-28.4 g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: 5 animals per polycarbonate cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Amount of vehicle (if gavage or dermal): 10 ml/kg

Duration of treatment / exposure:

2 days

Frequency of treatment:

24, 48h

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Range finding experiments: 3 male and 3 female per dose; 6 males per dose in main experiment.

Control animals:

yes, concurrent vehicle

Positive control(s):

- cyclophosphamide;
- Justification for choice of positive control(s): none given
- Route of administration: oral gavage
- Doses / concentrations: 80 mg/kg

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on two range-finding studies.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 500 mg/kg bw, 1000 mg/kg bw and positive control harvested at 24 hours; 2000 mg/kg bw and vehicle control harvested at 24 and 48 hours.


DETAILS OF SLIDE PREPARATION: Giesma stain

METHOD OF ANALYSIS: scored for micronuclei and PCE NCE ratio

Evaluation criteria:

Criteria for positive: Statistically significant increase in micronucleated PCEs or at least one dose level and a statistically significant dose-related response.

Statistics:

ANOVA followed by Dunnett's t-test when ANOVA positive

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

up to limit concentration

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

No clinical toxicity and no cytotoxicity to the bone marrow was observed in any treated animals. No increase in the frequency of micronucleated erythrocytes occurred in any test substance dose.

Table 1: Mean Results of in vivo micronucleus test in mouse bone marrow

-	Solvent Control (water)		Positive Control (Cyclophosphamide)	Low dose 500 mg/kg	Mid dose 1000 mg/kg	High dose 2000 mg/kg
Sampling time (h)	24	48	24	24	48	24	48
Number of cells analysed	2000	2000	2000	2000	2000	2000	2000
Micronucleated cells per animal %	0.07	0.07	2.73	0.04	0.09	0.02	0.06
Ratio PCE/NCE	0.52	0.47	0.50	0.59	0.44	0.41	0.60

Conclusions:

ATMP-5Na has been tested for clastogenicity in vivo in a reliable study conducted according to OECD Test Guideline 474 and in compliance with GLP. No increase in the frequency of micronucleated erythrocytes was observed. It is concluded that ATMP-5Na is negative for the induction of micronuclei under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Reliable information is available on the cytogenicity (in vitro and in vivo) of ATMP-5Na. This information is directly applicable to the registered substance, ATMP-xNH4 as genetic toxicity will not be affected by which sodium salt is tested. For endpoints where reliable studies were not available for ATMP-xNH4, key studies for ATMP-H (CAS 6419-19-8) were read across to ATMP-xNH4. See attachment to IUCLID Section 13 for justification of read-across.

 

ATMP-H has been tested for mutagenicity in vitro in Salmonella typhimurium TA98, 100, 1535, 1537 in a study conducted according to a protocol similar to OECD Test Guideline 471 and in compliance with GLP (Monsanto, 1981). ATMP-H did not increase the number of revertants when tested up to cytotoxic concentration with or without metabolic activation. The vehicle and positive controls gave the expected results. ATMP-H was concluded to be negative for mutagenicity in bacteria under the conditions of the test.

 

ATMP-H has been tested in a valid bacterial reverse mutation assay, according to OECD Test Guideline 471 and in compliance with GLP, using E. coli WP2 uvr A (Envigo, 2018). No increase in the number of revertants was observed in any test strain, with or without metabolic activation when tested up to limit concentration. Appropriate positive, negative and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

 

ATMP-5Na has been tested for clastogenicity in vitro in Chinese hamster Ovary (CHO) cells in a study conducted according to OECD Test Guideline 473 and in compliance with GLP (Covance Laboratories, 1998a). The test substance did not induce chromosome aberrations in vitro when tested up to cytotoxic concentration with or without metabolic activation. Positive, negative and solvent controls gave the expected results. It is concluded that ATMP-5Na does not cause chromosomal aberrations under the conditions of the test.

 

A neutralised solution of ATMP-H has been tested for mutagenicity to mammalian cells in mouse lymphoma L5178Y cells in a study conducted according to a protocol similar to OECD Test Guideline 476 and in compliance with GLP (SRI International, 1988). No increase in the number of revertants was observed in the presence of metabolic activation when tested up to the solubility limit. The test results indicate that the positive findings in the previous experiment with unneutralised ATMP acid were an artefact of pH (SRI International, 1982). It is concluded that ATMP-H is not mutagenic to mammalian cells under the conditions of this test.

In a supporting bacterial mutagenicity study, conducted in a similar manner to OECD Test Guideline 471 and pre-GLP, ATMP-H (Dequest 2000) was tested for mutagenicity to bacteria. No mutagenicity was observed (reliability 4) (Monsanto, 1977).

In a supporting bacterial mutagenicity study, not conducted according to any guideline and pre-GLP, ATMP sodium salt was tested for reverse mutation in two strains of Salmonella typhimurium. No increase in the number of revertants was seen. It is concluded that the test substance does not induce mutations in the strains tested, under the conditions of the test (reliability 4) (Manley, 1981).

 

ATMP-5Na has been tested for clastogenicity in vivo in a reliable study conducted according to OECD Test Guideline 474 and in compliance with GLP. No increase in the frequency of micronucleated erythrocytes was observed. It is concluded that ATMP-5Na is negative for the induction of micronuclei under the conditions of this test (Covance Laboratories, 1998b).

Justification for classification or non-classification

Based on the available data for ATMP-5Na and ATMP-H, no classification for genetic toxicity is required for ATMP-xNH4 according to Regulation (EC) No 1272/2008.