[[(phosphonomethyl)imino]bis[ethylenenitrilobis(methylene)]]tetrakisphosphonic acid, ammonium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No genetic toxicity data are available for DTPMP-xNH4, however, information is available for DTPMP-H and sodium salts of DTPMP. The use of read-across data between members of the category is in accordance with the rationale outlined in the read-across justification included in Section 13.

Gene mutation (Bacterial reverse mutation assay / Ames test): DTPMP-7Na was negative with and without activation in Salmonella typhimurium TA98, TA100, TA 1535, TA 1538 and E. coli WP2 uvrA (OECD Test Guideline 471 and in compliance with GLP) (Japan Oilstuff Inspectors Corporation, 2001a).

Gene mutation (Bacterial reverse mutation assay / Ames test): DTPMP -H was negative with and without activation in Salmonella typhimurium TA98, TA100, TA 1535, TA 1538 and E. coli WP2 uvrA (OECD TG 471) (BMI Inc, 2003).

Mutagenicity in mammalian cells: DTPMP was negative in Chinese hamster ovary cells (similar to OECD TG 476) (Pharmakon Research International, 1984).

Mutagenicity in mammalian cells: read-across from DTPMP-xNa; negative in mouse lymphoma L5278Y cells (similar to OECD Test Guideline 476 and in compliance with GLP) (Central Toxicology Laboratory, 1997).

Mutagenicity in mammalian cells: DTPMP-H was negative in Chinese hamster ovary cells (similar to OECD TG 476) (Microbiological Associates, 1983).

Mutagenicity in mammalian cells: DTPMP-H was negative in L5178Y cells (similar to OECD TG 476) (SRI International, 1983).

Cytogenicity in mammalian cells: DTPMP-7Na was positive in Chinese hamster lung CHL/IU cells (OECD Test Guideline and in compliance with GLP 473) (Japan Oilstuff Inspectors Corporation, 2001b).

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:

15-Jun-2001 to 18-Sep-2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines on Industrial Chemicals

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

mammalian cell line, other: CHL/IU (originally derived from female Chinese hamster lung)

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's minimum essential 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 applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital- and 5,6-benzoflavone-induced male rat liver S9

Test concentrations with justification for top dose:

625-5000 µg mixed sodium salts/ml (pulse treatment)
150-3000 µg mixed sodium salts/ml (24-hour continuous treatment)
50-400 µg mixed sodium salts/ml (48-hour continuous treatment)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: saline
- Justification for choice of solvent/vehicle: solubility of test substance in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9, 0.1 µg/ml (pulse treatment), 0.03 µg/ml (continuous treatment)

Positive control substance:

mitomycin C

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with S9, 15 µg/ml (pulse treatment)

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: pulse treatment 6 hours; continuous treatment, 24 or 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): pulse treatment 24 hours; continuous treatment 24 or 48 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid, 0.1 µg/ml, 2 hours
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 100 cells /culture, 2 cultures/treatment

DETERMINATION OF CYTOTOXICITY
- Method: other: cell density after crystal violet staining

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: mitotic index

Evaluation criteria:

Negative: both the incidence of aberrant cells (excluding gaps) and that of numerical aberrations <5%
Inconclusive: either of these incidences is 5-10%
Positive: either of these incidences >10%

Statistics:

None

Key result

Species / strain:

mammalian cell line, other: CHL/IU

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

pulse treatment ( 6 and 24 hour exposure)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=2500 µg mixed sodium salts/ml (pulse treatment),

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mammalian cell line, other: CHL/IU

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

48 hour treatment

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=50 µg mixed sodium salts/ml (48-hour continuous treatment)

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: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: soluble in water
- Precipitation: no precipitation observed
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
- No, but cell survival was evaluated at 156-5000 µg mixed sodium salts/ml (pulse treatment, 24-hour continuous treatment and 48-hour continuous treatment) and the data used to determine the concentrations that were to be evaluated for chromosome aberrations

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cell survival:
- Pulse treatment -S9, µg mixed sodium salts/ml: 156 (109%), 313 (109%), 625 (97%), 1250 (96%), 2500 (76%), 5000 (58%)
- Pulse treatment +S9, µg mixed sodium salts/ml: 156 (98%), 313 (104%), 625 (103%), 1250 (108%), 2500 (102%), 5000 (77%)
- 24-hour continuous treatment -S9, µg mixed sodium salts/ml: 156 (115%), 313 (81%), 625 (47%), 1250 (37%), 2500 (19%), 5000 (0%)
- 48-hour continuous treatment -S9, µg mixed sodium salts/ml: 156 (50%), 313 (25%), 625 (7%), 1250 (5%), 2500 (1%), 5000 (0%)

Full details of the results from the 48 hours treatment (result reported as positive) are not presented in the study report. A graph of the results indicates that the incidence of cells with structural aberrations was 2.5% at 50 µg/ml; 5% at 100; 15% at 150; 37% at 200 and 49% at 300 µg/ml.

Table 1 Chromosome aberration test - treatment time 6 hours

Concentration µg/ml	+/- S9 mix	No. of cells analyzed	Chromatid breaks	Chromatid exchanges	No. of cells with aberrations (%)	No. of cells with gaps	Cell growth index (%)	Polyploid cells
Control	-	200	0	0	0	1	100	0
625	-	200	1	0	1	0	98	0
1250	-	200	3	1	4	0	91	0
2500	-	200	6	1	7	4	71	2
5000	-	200	9	1	9	3	41	0
Positive control	-	200	54	121	139	1	-	0
Control	+	200	0	0	1	0	100	0
625	+	200	0	2	2	1	91	0
1250	+	200	0	1	1	0	95	0
2500	+	200	3	3	4	0	99	0
5000	+	200	1	0	1	0	67	0
Positive control	+	200	7	42	46	1	-	0

 

Chromosome aberration test - treatment time 24 hours

Concentration µg/ml	+/- S9 mix	No. of cells analyzed	Chromatid breaks	Chromatid exchanges	No. of cells with aberrations (%)	No. of cells with gaps	Cell growth index (%)	Polyploid cells
Control	-	200	2	0	2	1	100	0
4.7	-	200	1	1	2	1	101	0
9.4	-	200	2	0	2	0	102	0
18.8	-	200	4	0	4	1	104	0
37.5	-	200	3	2	5	0	107	0
75	-	200	2	1	3	3	103	0
150	-	200	8	0	8	2	113	0
300	-	-	TOXIC
Positive control	-	200	30	30	58	0	-	0
Control	+	200	2	0	2	0	100	0
50	+	200	4	1	5	0	99	0
100	+	200	7	4	11	2	68	0
150	+	200	31	9	34	0	57	0
200	+	200	67	9	74	1	43	0
300	+	200	87	10	97	2	20	0
400	+	-	TOXIC
Positive control	+	200	47	56	86	0	-	0

 

Conclusions:

In a reliable in vitro chromosome aberration assay, conducted according to OECD guideline 473 and in compliance with GLP, a dose-related increase in the number of cells with aberrations was observed after 48 hours treatment.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

02-Jul-2001 to 26-Jul-2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(only 2-AA used as positive control +S9)

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines on Industrial Chemicals

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Target gene:

histidine (Salmonella typhimurium)
tryptophan (Escherichia coli)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital- and 5,6-benzoflavone-induced rat liver S9

Test concentrations with justification for top dose:

Test 1-1: 78-5000 µg mixed sodium salts/plate -S9, 156-5000 µg/plate +S9
Test 1-2: 20-5000 µg mixed sodium salts/plate -S9 for S typhimurium
Test 2: 20-5000 µg mixed sodium salts/plate -S9 for S typhimurium, 78-5000 µg mixed sodium salts/plate -S9 for E coli, 156-5000 µg mixed sodium salts/plate +S9
20-5000ug/plate -S9; 156-5000ug/plate +S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9; TA98, 0.1 µg/plate; TA100, 0.01 µg/plate

Positive control substance:

furylfuramide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9 ; TA1535, 0.5 µg/plate

Positive control substance:

sodium azide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9; TA1537, 80 µg/plate

Positive control substance:

9-aminoacridine

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9, WP2 uvrA, 2 µg/plate

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with S9; TA98, 0.5 µg/plate; TA100, 1 µg/plate; TA1535, TA1537, 2 µg/plate; WP2 uvrA, 10 µg/plate

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: other: growth inhibition

Evaluation criteria:

Posltive: mean number of revertant colonies more than double the negatlve control value and signlflcant concentration-dependent Increase

Statistics:

No statistical analysis performed

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 625 µg mixed sodium salts/plate -S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 625 µg mixed sodium salts/plate -S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 625 µg mixed sodium salts/plate -S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 625 µg mixed sodium salts/plate -S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=1250 µg mixed sodium salts/plate -S9

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: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: soluble in water
- Precipitation: no precipitation observed
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
- Concentrations of 5-5000 µg/plate
- No growth inhibition in any strain
- Bacteriostatics observed at 1250 µg/plate -S9 in all strains
- No precipitation

COMPARISON WITH HISTORICAL CONTROL DATA:
- Negative/solvent control: within the range of historical data
- Positive controls: within the range of historical data

All treated cultures had less than 2-fold increase in mutant frequency and no evidence of a dose-response.  All positive controls gave a greater than 2-fold increase in mutant numbers.

Table 1 Experiment 1 Mean number of revertants (average of 3 plates)

 

Concentration (µg/plate)	+/- S9 mix	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Control	-	102	10	23	14	7
78	-	102	8	21	16	9
156	-	88	7	18	16	6
313	-	71	5	18	8	1
625	-	0	0	7	0	0
1250	-	0	0	0	0	0
2500	-	0	0	0	0	0
5000	-	0	0	0	0	0
Positive control	-	528	422	631	559	194
Control	+	111	11	25	24	6
156	+	133	11	30	24	10
313	+	148	11	21	22	6
625	+	140	9	23	17	6
1250	+	122	6	21	11	3
2500	+	108	4	15	9	3
5000	+	98	5	9	8	2
Positive control	+	501	260	648	446	154
Mean number or revertants 16-19.07.2001

Table 2 Experiment 2 Mean number of revertants (average of 3 plates)

Concentration (µg/plate)	+/- S9 mix	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Control	-	104	11	-	16	7
20	-	101	6	-	16	8
39	-	94	8	-	12	8
78	-	106	8	-	14	8
156	-	97	9	-	14	5
313	-	81	6	-	14	4
1250	-	0	0	-	0	0
5000	-	0	0	-	0	0
Positive control	-	522	411	-	604	211

 

Table 3 Experiment 3 Mean number of revertants (average of 3 plates)

Concentration (µg/plate)	+/- S9 mix	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Control	-	99	8	20	21	7
20	-	105	10	-	18	7
39	-	107	11	-	19	6
78	-	110	10	22	19	6
156	-	103	11	14	17	7
313	-	86	8	16	15	4
625	-	-	-	13	-	-
1250	-	0	0	0	0	0
2500	-	-	-	0	-	-
5000	-	0	0	0	0	0
Positive control	-	510	419	655	601	183
Control	+	127	10	21	23	8
156	+	130	9	29	26	9
313	+	132	12	17	19	7
625	+	127	12	22	18	6
1250	+	114	8	15	14	7
2500	+	119	6	13	6	3
5000	+	103	6	12	3	2
Positive control	+	679	304	758	517	152

 

Conclusions:

In a reliable study, conducted according to OECD guideline 471, no genotoxicity was seen in a bacterial mutagenicity assay. The study was performed in compliance with GLP.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14-Jan-2003 to 17-Jan-2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

other: Standards for mutagenicity tests using microorganisms (Notification no. 77, 1988, and Notification no. 67, 1997); Amendment of the reporting form of the results of the mutagenicity tests using microorganisms (Notification no. 653, 1997)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(only duplicate plates used)

GLP compliance:

no

Remarks:

Japanese guideline notification no. 76

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital- and 5,6-benzoflavone-induced male rat liver S9

Test concentrations with justification for top dose:

10, 20, 39, 78, 156, 313, 625, 1250 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: test substance supplied in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

furylfuramide

Remarks:

-S9; TA100, WP2 uvrA, 0.01 µg/plate; TA98, 0.1 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

-S9; TA1535, 0.5 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191 (2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine.2HCl

Remarks:

-S9; TA1537, 1.0 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

+S9; TA100, TA98, TA1537, 5 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

+S9; TA1535, 2 µg/plate; WP2 uvrA, 10 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: duplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: other: growth inhibition

Evaluation criteria:

"If the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was judged positive."

Statistics:

Not done

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=313 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=313 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=313 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=313 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=625 µg/plate

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: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: test substance supplied as a solution in water
- Precipitation: no precipitate seen
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
- Concentrations tested: 1.2, 4.9, 20, 78, 313, 1250, 5000 µg/plate without S9
- Growth inhibition in all S. typhimurium TA strains at 313 µg/plate and above and in WP2 uvrA at 1250 µg/plate and above
- No precipitate
- Highest concentrations selected for the main test: 313 µg/plate for all S. typhimurium TA strains and 1250 µg/plate for WP2 uvrA
- Other concentrations obtained by 1:2 dilutions to provide 6 concentrations for each strain

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Table 1 Dose finding experiment: Revertants per plate (mean of two plates)

Concentration (µg/plate)	TA 100		TA 1535		WP2 uvrA		TA 98		TA 1537
	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
0	134	128	23	14	19	27	18	39	16	23
1.2	137	131	21	13	20	30	19	35	16	26
4.9	127	142	18	16	24	26	16	33	17	22
20	114	127	28	16	20	34	18	32	19	17
78	132	130	18	13	29	30	18	31	14	22
313	46*	114	8*	8	21	18	11*	20	5*	12
1250	0*	125*	0*	4*	0*	17*	0*	10*	0*	4*
5000	0*	0*	0*	0*	0*	0*	0*	0*	0*	0*
Positive control	486	842	461	344	135	595	511	226	1767	82

* The growth inhibition of tested bacterium by the test substance was observed.

Table 2 Main experiment: Revertants per plate (mean of two plates)

Concentration (µg/plate)	TA 100		TA 1535		WP2 uvrA		TA 98		TA 1537
	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
0	117	137	17	13	27	26	17	43	8	22
10	137	NT	21	NT	NT	NT	15	NT	9	NT
20	124	NT	15	NT	NT	NT	21	NT	11	NT
39	124	125	9	11	22	27	22	37	9	23
78	125	136	15	10	27	31	11	37	12	15
156	116	133	16	17	20	26	17	39	6*	13
313	50*	115	6*	9	16	19	11*	32	6*	12
625	NT	106*	NT	4*	10*	21	NT	23*	NT	5*
1250	NT	108*	NT	2*	0*	13*	NT	3*	NT	5*
Positive control	496	810	451	321	160	563	588	200	1739	69

* The growth inhibition of tested bacterium by the test substance was observed.

Conclusions:

In a reliable study, conducted to Japanese guidelines on mutagenicity tests (notification nos. 77 and 653), no genotoxicity was seen in a bacterial mutagenicity assay at up to 1250 µg/plate. The study was performed in compliance with Japanese guideline notification no. 76.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

23-Aug-1983 to 16-Nov-1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

1.2 - 20 µl/ml in absence of S9 and 3 - 10 µl/ml in presence of S9.

Vehicle / solvent:

The solubility of the test article was determined in Acetone, DMSO, Ethanol and water, and water was selected at the solvent of choice.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9; 1.0 and 0.5 µl/ml

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with S9; 7.5 and 5 µg/ml

Details on test system and experimental conditions:

Agar plate method used to investigate effect of different expression periods and whether test compound allowed selective growth of pre-existing mutants. 
ADMINISTRATION Toxicity determined by relative suspension growth.  Cells cleansed of mutants before mutation study.  Cultures exposed to test substance for 4 hours in presence and absence of S9 mix. 
Expression periods of 0, 2 and 6 days used.  
Test performed on a single occasion, using triplicate plates for mutation and viability.  3 µg/ml trifluorothymidine used as selective agent.  Total of 3 x 10E+06 cells plated for mutation frequency and 600 cells for viability plates.  
Incubated for 10-12 days.

Statistics:

Dose-response assessed by ANOVA on transformed data.  Test groups compared to controls on transformed data using 1-tailed students t-test.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Results reported to be positive but the increase in mutant frequency was less than the control value plus the global evaluation frequency for the plate method, so reviewer considers results are negative by currently accepted criteria.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

50 µl/ml -S9; 10 µl/ml +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

The report authors concluded that the results were positive at the standard day 2 expression time in the presence of S9 but not at day 0 or day 6. The increases in mutant frequency were less than the control value plus the global evaluation frequency for the plate method, so would not be considered positive by currently accepted criteria. It is concluded by the reviewer that the results should be considered negative.

The top dose
plated of 10 µl/ml gave a relative total growth of 24% and a mutant frequency of 133 x 10e-6 compared to a control
frequency of 55 x 10e-6.  Negative response in the absence of S9 on day 0 and day 6. Day 2 response in absence of S9
was inconclusive since a statistically significant dose response was seen but the mutant frequency was not
significantly greater than the control value (63 x 10E-06 for the top dose at a relative total growth of 21% v 40 x 10E-06
in controls).  The positive control in the absence of S9, EMS, was positive at day 2 and day 6 expression time and
inconclusive at day 0.  The positive control in the presence of S9, DMBA, was only positive at the standard day 2
expression time.

Dequest 2060 at pH7.0. No details in report. Assumed dosing was in terms of aqueous solution and doses have not been converted to total solids in this entry.

Table 1 Mutant frequency (without metabolic activation

Concentration	Day 0		Day 2		Day 6
	MF*	%TG**	MF*	%TG**	MF*	%TG**
20	1.4	1	++		0.7	5
	0.4	7	++		0.6	4
	1.2	0	++		0.5	2
17.2	0.3	8	0.6	25	0.6	2
	0.2	3	0.6	30	0.5	27
	0.2	6	0.7	7	0.6	12
14.8	0.3	0	0.6	47	0.5	9
	0.4	3	0.6	19	0.5	78
	0.3	20	++		0.5	34
11.9	0.2	30	0.6	36	0.4	90
	0.3	27	0.6	61	0.5	77
	0.3	26	0.5	66	0.6	84
9.4	0	51	0.5	50	0.6	74
	0.3	31	0.4	54	0.5	73
	0.2	41	0.5	68	0.4	71
6.4	0.3	46	0.3	66	0.6	70
	0.2	60	0.5	52	0.6	69
	0.2	50	0.4	44	0.6	60
3.6	0.1	63	0.4	87	0.4	73
	0.2	52	0.2	72	0.7	64
	0.2	56	0.4	74	0.6	53
1.2	0.2	89	0.4	105	0.5	129
	0.2	90	0.4	138	0.7	108
	0.3	91	0.4	149	0.8	105
0	0.2	-	0.5	-	0.6	-
	0.2	-	0.4	-	0.9	-

MF* Mutant frequency per 10 E+04 cells

%TG** % total growth

++ Too toxic to clone

Table 2 Mutant frequency (with metabolic activation)

Concentration	Day 0		Day 2		Day 6
	MF*	%TG**	MF*	%TG**	MF*	%TG**
10	1.7	3	1.3	20	1.1	42
	0.4	14	1.5	24	1.1	63
	0.7	10	1.2	27	0.9	50
9	0.5	31	0.7	64	1.1	102
	0.3	47	0.7	83	0.5	134
	0.4	41	0.7	75	0.7	130
8	0.4	40	0.8	76	0.8	115
	0.6	28	0.7	77	0.7	113
	0.5	46	0.8	74	0.7	125
7	0.4	65	0.7	83	0.9	119
	0	58	0.7	83	0.7	156
	0.5	34	0.8	71	1.1	104
6	0.4	60	0.6	83	0.6	134
	0.4	60	0.6	91	0.7	143
	0.7	17	0.8	81	0.8	129
5	0.5	60	0.6	86	0.8	80
	0.3	74	0.6	70	1.1	80
	0.3	75	0.6	74	1.1	64
4	0.3	77	0.5	87	1.1	69
	0.4	78	0.6	81	1.1	58
	0.3	78	0.6	69	1.1	83
3	0.4	79	0.5	83	1.2	75
	0.4	89	0.4	84	1	91
	0.4	86	0.5	87	1	82
0	0.3	-	0.6	-	0.9	-
	0.4	-	0.5	-	1	-

MF* Mutant frequency per 10 E+04 cells

%TG** % total growth

Conclusions:

In a reliable study, conducted according to a protocol similar to OECD standard guideline 476 and in compliance with GLP, mutagenicity was reported in mouse lymphoma L5178Y cells when tested to the limit of toxicity in the presence of metabolic activation. However, the increases in mutant frequency were less than the control value plus the global evaluation frequency for the plate method, so would not be considered positive by currently accepted criteria.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

31-Aug-1983 to 08-Feb-1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

inadequacies with respect to concentrations of submission substance tested

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

(some omissions from study report with respect to test system itself)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: F12
- 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:

other: CHO-K1-BH4

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 preparation at 2%

Test concentrations with justification for top dose:

3000, 5000, 6000, 7000, 8000 µg Dequest 2060/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none (test substance diluted in F12 culture medium)

Untreated negative controls:

yes

Remarks:

F12 culture medium only

Negative solvent / vehicle controls:

no

Remarks:

test substance diluted in F12 culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

200 µg/ml without metabolic activation

Untreated negative controls:

yes

Remarks:

F12 culture medium only

Negative solvent / vehicle controls:

no

Remarks:

test substance diluted in F12 culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

100 µg/ml with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 19 hours
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

To be considered positive if:
a) The mean mutation frequencies of at least one of the 3 highest test article concentrations, with a mean survival rate of at least 10% are significantly
greater than that of the solvent control (p<=0.01 based on pooled inter-group variance); and,
b) The change in mean mutation frequency with increasing test article concentration exhibits a significant (p<=0.01) linear component of the dose-response relationship up to a maximum toxicity level of 90%."
To be considered negative if:
a) None of the mean mutation frequencies of any of the 3 highest test article concentrations with a mean survival rate of at least 10% are significantly
greater than that of the solvent control (p<=0.01) and,
b) The change in mean mutation frequency with increasing test article concentration does not exhibit a statistically significant (p<=0.01) linear
component of the dose-response relationship up to a maximum toxicity level of 90%.

Statistics:

Statistical analysis: Students t-test for group mean comparison and ANOVA on transformed data (performed on second test only).

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Relative survival at 8000 µg/ml: -S9 65%, +S9 44%

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: no data

RANGE-FINDING/SCREENING STUDIES: Preliminary test to establish optimum level of S9 preparation

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: Two preliminary cytotoxicity tests were performed
1. no cytotoxicity at up to 1000 µg/ml with up to 10% S9 preparation
2. 84% survival at 6000 µg/ml in the absence of S9; 58, 62, 44 and 52% survival at 6000 µg/ml in the presence of 1, 2, 5 and 10% S9 preparation

Top dose is higher than the value of 5000 µg/ml normally considered the maximum for this test.

Note however this may represent the test solution as supplied, rather than the active acid.

Table 1 Preliminary mutagenicity assay - Dequest 2060 1% activation

Concentration µg/ml	+/-S9	Post treatment survival	Number of replicates	Mutation frequency (Mutants/10x6)
Solvent Control	-	97	2	1.7
	-	102	1
	+	98	1	3.6
	+	97	6
2500	-	101	2	1.2
	-	99	0
6000	-	76	2	2.4
	-	72	2
7500	-	26	15	15.1
	-	24	14
2500	+	88	0	1.2
	+	85	2
6000	+	71	2	2.6
	+	49	3
7500	+	40	5	3.9
	+	39	2
Positive control	-	60	193	325.2
	-	57	183

 

Table 2 Preliminary mutagenicity assay - Dequest 2060 2%, 5%, 10% activation

Concentration µg/ml	+/-S9	Post treatment survival	Number of replicates	Mutation frequency (Mutants/10x6)
Solvent Control	-	97	2	1.7
	-	102	1
	+	100	5	6.1
	+	95	6
2500	2% +	85	0	1.9
	2% +	92	3
6000	2% +	74	4	4.9
	2% +	76	5
7500	2% +	36	12	10.2
	2% +	38	7
Positive control	2% +	19	126	298.4
	2% +	22	108
Solvent Control	+	107	7	4.7
	+	87	1
2500	5%+	77	2	2.6
	5%+	81	3
6000	5%+	72	4	6
	5%+	76	7
7500	5%+	33	6	9
	5%+	35	9
Solvent Control	+	102	1	2.6
	+	91	4
2500	10%+	100	0	0
	10%+	94	0
6000	10%+	72	7	9.5
	10%+	63	8
7500	10%+	42	6	6.4
	10%+	42	5

 

Table 3 Mammalian cell forward gene mutation assay

Concentration µg/ml	+/-S9	Post treatment survival	Number of replicates	Mutation frequency (Mutants/10x6)
Solvent Control	-	100.6.	1	0.4
	-	99.4	0
	-	97.2	0
3000	-	95	0	0
	-	107.2	0
	-	99.4	0
5000	-	98.8	0	0
	-	103.5	0
	-	87.2	0
6000	-	97.8	3	1.8
	-	99.3	0
	-	103.2	1
7000	-	91.9	0	2.5
	-	71.2	6
	-	82.8	1
8000	-	59.6	0	0.5
	-	70.3	1
	-	65.9	0
Positive control	-	69	129	310.3
	-	58.4	222
	-	57.3	216
Control	+	103.2	0	2.6
	+	102.1	4
	+	93.2	2
3000	+	79.4	1	0.6
	+	65.1	0
	+	97.5	0
5000	+	86.4	0	6.4
	+	84.5	12
	+	62.8	2
6000	+	80.5	12	6.5
	+	66.8	3
	+	71.1	0
7000	+	48.9	0	6.5
	+	54.3	14
	+	52.6	1
8000	+	53.2	2	2.9
	+	38.4	0
	+	43.7	5
Positive control	+	17.1	136	345
	+	21	109
	+	25.4	121

 

Conclusions:

In a reliable study, conducted using a protocol similar to the OECD guideline 476, no evidence of mutagenic potential was detected in Chinese hamster ovary cells in vitro in the absence or presence of an exogenous metabolic activation system in an assay evaluating mutation at the HGPRT locus. The study was performed in compliance with GLP.

Reference 6

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:

comparable to guideline study with acceptable restrictions

Remarks:

not tested without metabolic activation

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

(not tested without metabolic activation)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fischer's medium for leukemic cells of mice
- 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

Metabolic activation:

with

Metabolic activation system:

Aroclor 1254-induced male rat liver S9

Test concentrations with justification for top dose:

Final concentrations tested were 500, 1000, 1250 and 1500 µl/ml of the 50% solution, equivalent to 350, 700, 875 and 1050 µg/ml of 158-276-08

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

+S9, 5 µg/ml

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

reviewer considers result to be negative when assessed by currently accepted criteria

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

> 1500 µl/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Mutations were induced by the test substance in a dose-related manner (up to 4-fold control levels at the top
dose at a relative total growth of 27%).  These were all demonstrated to be stable by their growth on TFT plates
after suspension growth in the absence of TFT and no thymidine kinase activity was detectable.

Concentration μg/ml	Relative total growth (%)	Mutation Frequency (x 10e-6)
Solvent control	100	34
Positive control	48	249
0.35	88	49
0.7	70	77
0.875	68	111
1.05	27	157

Conclusions:

A study was conducted according to a protocol similar to OECD guideline 476 and in compliance with GLP in order to evaluate the possiblity that the mutagenicity reported in an earlier study (Mitchell, 1983) was not an artefact of pH. The neutralised test substance was tested in mouse lymphoma L5178Y cells tested to the limit of toxicity in the presence of metabolic activation. The study authors reported that the result was positive in the presence of metabolic activation. However, the increases in mutant frequency were less than the control value plus the global evaluation frequency for the plate method, so would not be considered positive by currently accepted criteria.

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17-Jan-1997 to 25-Feb-1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The test substance was not tested up to the maximum concentration required by guideline.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(not tested to maximum concentration required by guideline)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI-1640
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital- and beta-naphthoflavone-induced male rat liver S9

Test concentrations with justification for top dose:

Phase 1: 266-2128 µg active acid/ml
Phase 2: 265-2121 µg active acid/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: test substance supplied in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9; 750 µg active acid/ml

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with S9; 3 µg active acid/ml

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): no data

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: duplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: other: cell growth

Evaluation criteria:

- Criteria for a positive response: a statistically significant, dose related increase in mutant frequency is required, but not only at concentrations eliciting excessive toxicity. An associated absolute increase in mutant number above the solvent control values is a further requirement. Such a response must be reproducible in an independent experiment for the test substance to be described as positive in this assay.
- CrIteria for a negative response: A negative response is obtained when there is no reproducible statistically significant dose related increase in mutant frequency. When reproducible significant increases in mutant frequency are seen only at levels of excessive toxicity, or when such increases are not accompanied by an increase in absolute numbers of mutants over solvent control values, then the effect is considered not to be indicative of a positive response in this assay.

Statistics:

- If considered necessary, the data are considered by logit regression, using a complimentary log-log link function. The dependent variable is the number of empty wells. This procedure provided maximum likelihood estimates of log mutant frequencies. Variances are inflated by the between duplicate heterogeneity factor.
- Tests for trend in log mutant frequency with actual concentration are obtained separately for each experiment, in the presence and absence of S9-mix. In addition, an overall test for trend combining data across experiments is performed.
- Intergroup comparisons of log mutant frequency comparing each treated group with the solvent control are performed within each experiment. In addition, tests for trend in log mutant frequency with actual concentration are obtained both within each experiment and combined across all experiments.
- All tests are one-sided. Similar analyses are carried out separately for the positive controls.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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 solutions neutralised to ~pH7
- Effects of osmolality: "higher concentrations [than 2200 µg mixed sodium salts/ml] produced excessive increases (up to 70 mM/kg) in the osmolality of the treatment medium. Such increases in osmolality have been associated with artefactual responses in genotoxicity assays ... and these concentrations were therefore considered not to be appropriate for testing."
- Evaporation from medium: no data
- Water solubility: soluble in water
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: no data


ADDITIONAL INFORMATION ON CYTOTOXICITY:

No increases in mutant frequency were seen in any of the tests and it was not considered necessary to conduct statistical analysis.  All the positive control cultures had elevated mutant frequencies as expected.

The maximum concentration tested was 2200 µg/ml.  Higher concentrations were claimed to give excessively high osmolarity, although the values given for 4256 and 4242µg/ml in subsequent tests only resulted in increases to 354 and 334 mOsm/kg respectively. Since no increases in mutant frequency were seen in the first test at a dose producing 330 mOsm/kg it could be argued that the dose of 4242 could have been tested.  All concentrations below 5000 µg/ml are <10 mM, the upper limit defined by OECD for this assay.  A toxicity limit was not reached in these tests - top levels had >75% survival.  Therefore the upper limit defined for this assay was not reached.

Conclusions:

In a reliable study, conducted according to OECD guideline 476, no genotoxicity was seen in an in vitro mouse lymphoma L5178Y cell mutagenicity assay in the presence or absence of S9. However, the highest concentration selected for testing was below the maximum required by the guideline. The study was performed in compliance with GLP.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

No in vivo genetic toxicity data are available for DTPMP-xNH4, however, information is available for DTPMP-H. The use of read-across data between members of the category is in accordance with the rationale outlined in the read-across justification included in Section 13.

Bone marrow chromosome study in rats (oral gavage administration): DTPMP-H was negative (similar to OECD TG 475)

According to ECHA decision number CCH-D-2ii4495836-29-01/F, an in vivo mammalian alkaline comet assay with the registered substance DTPMP 5-7 Na-salt is currently being conducted according to OECD 489 and in compliance with GLP. When the study is completed, the substance will be updated accordingly.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15-Aug-1983 to 04-Nov-1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

There is some inaccuracy with respect to test substance identity.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

(insufficient cells scored for aberrations and for mitotic index)

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, New York, USA
- Age at study initiation: 64 days
- Weight at study initiation: 275-286 g males, 198-204 g females
- Assigned to test groups randomly: yes, via computer-generated random numbers
- Fasting period before study: no data
- Housing: individually in wire mesh cages
- Diet (e.g. ad libitum): Purina Lab Meal #5001, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 74-83
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 15-Aug-1983 To: 17-Aug-1983

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data, but well-known vehicle
- Concentration of test material in vehicle: 20, 66 and 197 mg active acid/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
- Purity: distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Freshly prepared on the day of administration

Duration of treatment / exposure:

Single oral gavage dose; post-treatment sampling times were 6, 12, 24 and 48 hours

Frequency of treatment:

Once

Post exposure period:

6, 12, 24 and 48 hours

Dose / conc.:

200 other: mg active acid/kg bw

Remarks:

Calculated from nominal concentration of test substance

Dose / conc.:

660 other: mg active acid/kg bw

Remarks:

Calculated from nominal concentration of test substance

Dose / conc.:

1 970 other: mg active acid/kg bw

Remarks:

Calculated from nominal concentration of test substance

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): no data, but well-known clastogen
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Selected on the basis of a range-finding test in which no effect on mortality or mitotic index and minimal clinical signs were observed at the top dose of 1970 mg active acid/kg bw only; all 3 dose levels analysed.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Colchicine administered 4, 10, 22 and 46 hours after treatment; animals sacrificed 2 hours later.

DETAILS OF SLIDE PREPARATION: Bone marrow cells collected from femurs by aspiration into Hank's Balanced Salt Solution; after centrifugation, supernatant decanted and cells suspended in warm 0.075 M KCl for 25 minutes; cells fixed using 3:1 methanol:acetic acid fixative; chilled then dispersed on glass microscope slides (2/animal) and air dried; stained with Giemsa and mounted with glass coverslips.

METHOD OF ANALYSIS: Slides coded; attempted to examine >=60 metaphases from 5/6 rats per sex per group (if not possible, 6th animal also analysed); 48-hour timepoint not analysed; for each animal, data recorded included numbers and types of chromosome aberrations, mitotic index (500 cells/animal), modal number for each metaphase; chromosome aberrations were classified as chromatid breaks, chromosome breaks, chromatid and chromsome gaps, exchanges, cells with >10 aberrations, pulverized cells.

Evaluation criteria:

No data

Statistics:

Mean mitotic index, mean modal number, percent aberrant cells and mean number of aberrations per cell analysed by Kruskall-Wallis non-parametric non-parametric analysis of variance and non-parametric pairwise group comparisons. Body weight analysed by analysis of covariance. All tests one-tailed.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

body weight loss, clinical observations

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 200, 660, 1970 mg active acid/kg bw
- Solubility: miscible with water
- Clinical signs of toxicity in test animals: no mortality; "a few abnormal clinical observations were observed at the highest dose"
- Evidence of cytotoxicity in tissue analyzed: no effect on mitotic index
- Rationale for exposure: slight toxicity at the highest dose
- Harvest times: observed 1 day after dosing
- High dose with and without activation: not applicable
- Other:

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): none
- Appropriateness of dose levels and route: no data
- Statistical evaluation:
- no statistically significant increase in chromosome damage at any dose at any timepoint in either sex; positive control (24 hours) statistically significantly incresased
- no statistically significant change in mitotic index
- no statistically significant change in mean modal number

3/12 animals died when treated with 1970mg/kg.  Mild clinical signs seen at this dose.  Loss of body weight in top dose animals (both sexes) over 48 hours observed. No evidence of mitotic delay so 48 hour animals not analyzed.

Table 1 Results of Chromosome aberration study

Treatment (mg/kg bw)	Treatment time (hrs)	Number of animals analyzed per group	Number of cells analyzed	% aberrant cells per group
Control	6	12	600	0.5
200	6	11	600	0
660	6	11	600	0.3
1970	6	11	600	0.5
Control	12	10	600	0
200	12	11	600	0
660	12	11	561	0.2
1970	12	10**	600	0.2
Control	24	11	540	0
Positive control	24	12	280	7.9
200	24	11	540	0.2
660	24	11	540	0
1970	24	10**	540	0.6

** Animals found dead prior to sacrifice

Conclusions:

In a reliable study, conducted using a protocol similar to OECD guideline 475, no evidence of clastogenicity was seen in rat bone marrow following a single oral gavage administration at doses up to the maximum tolerated dose of 1970 mg active acid/kg bw. The study was performed in compliance with GLP.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

No genetic toxicity data are available for DTPMP-xNH4, however, information is available for DTPMP-H and DTPMP-7Na. The use of read-across data between members of the category is in accordance with the rationale outlined in the read-across justification included in Section 13.

Bacterial mutagenicity

The results of all the bacterial mutagenicity studies were negative.

In a recent well-conducted assay an aqueous solution containing DTPMP-7Na (pH 6-8) was tested for the induction of gene mutations in Salmonella typhimurium strains TA98, 100, 1537 and 1535 and in Escherichia coli strain WP2 uvr- (Japan Oilstuff Inspectors Corporation, 2001a). The study was conducted using the preincubation modification in the presence and absence of S9 mix on two occasions. The test substance did not induce mutations in either strain at concentrations up to 5000 µg/ plate, the accepted upper limit for this assay.

In another study, conducted to Japanese guidelines on mutagenicity tests (notification nos. 77 and 653), no genotoxicity was seen in a bacterial mutagenicity assay for DTPMP-H at concentrations up to 1250 µg/plate when tested with or without metabolic activation in Salmonella typhimurium TA98, TA100, TA 1535, TA 1538 and E. coli WP2 uvrA (BMI Inc, 2003).

The mutagenic potential of a 50% aqueous solution of DTPMP-H was investigated in a well conducted standard guideline test using the plate incorporation method with strains TA98, TA100, TA1535 and TA 1537 (Monsanto, 1981). The top doses used were 10 µl/plate with S9 mix and 0.3 µl/plate without S9 mix and toxicity was induced at the highest doses, consistent with the high toxicity of phosphonic acids in this test system. No statistically significant increase in mutant numbers was seen at any concentration. This negative result is confirmed with the results of a briefly reported study with strains TA98, 100, 1535, 1537 and 1538 with the same test substance (Monsanto, 1977).

In vitro cytogenicity

In the key cytogenicity assay, which was conducted to the appropriate Japanese guideline an aqueous solution containing DTPMP-7Na was tested for the induction of chromosomal aberrations in CHL IU cells. (Japan Oilstuff Inspectors Corporation, 2001b). A delay in the cell cycle was observed in the continuous test with 24-hours treatment, so a 48 hour treatment (3 cell cycles) was carried out. The 4-hour treatment was conducted in the presence and absence of phenobarbitone and 5,6-benzoflavone induced S9 mix. A dose-related increase in the number of structural aberrations was observed in the 48-hour treatment group. It was concluded that the test substance has a clastogenic potential in the CHL/ IU cell line. The study is not fully compliant with the current test guideline for in vitro cytogenicity: the number of cells per treatment was lower than the updated guideline; cytotoxicity was not evaluated by the criteria now required; the level of cytotoxicity observed at the top two concentrations in the 48 hour treatment was greater than that required by the guideline.

Mammalian mutagenicity

Results are available from four mammalian mutagenicity studies on DTPMP-H. In some of the studies the test substance was neutralised.

In the oldest study, DTPMP-H was tested in a mammalian mutagenicity study conducted according to a protocol similar to the OECD Test Guideline 476 and in compliance with GLP (SRI International, 1982). The authors of the report concluded that mutagenicity was seen in mouse lymphoma L5178Y cells tested up to the limit of toxicity in both the presence and absence of S9. However, in the absence of metabolic activation the toxicity in one of the dose levels reported to be positive was too great to be acceptable under the current guideline, and the increase in mutant frequency at the dose with acceptable toxicity was less than the control value plus the global evaluation frequency for the plate method, so would not be considered positive by currently accepted criteria. In the presence of metabolic activation, the positive result in the first two experiments was probably caused by reduction in pH. The study report referred to a follow up study using neutralised test solution and it was concluded by the study authors that the results were not due to pH but there is insufficient detail provided to assess whether pH was adequately controlled. Since the pH was likely to have been low at high concentrations the result is considered to be unreliable and the study is disregarded.

A second study is available which was conducted according to a protocol similar to OECD Test Guideline 476 and in compliance with GLP in order to evaluate the possibility that the mutagenicity reported in an earlier study was not an artefact of pH (SRI International, 1983). The neutralised test substance was tested in mouse lymphoma L5178Y cells tested to the limit of toxicity in the presence of metabolic activation. The study authors reported that the result was positive in the presence of metabolic activation. However, the increases in mutant frequency were less than the control value plus the global evaluation frequency for the plate method, so would not be considered positive by currently accepted criteria.

The third study in mouse lymphoma L5178Y cells was conducted according to a protocol similar to OECD Test Guideline 476 (1997) and in compliance with GLP, mutagenicity was reported when tested to the limit of toxicity in the presence metabolic activation (Microbiological Associates, 1983). The study was conducted in compliance with GLP. However, the increases in mutant frequency were less than the control value plus the global evaluation frequency for the plate method, so would not be considered positive by currently accepted criteria.

The potential for DTPMP-H to induce gene mutations in mammalian cells has also been investigated in CHO cells using forward mutation at the hprt locus (Pharmakon Research International, 1984). This wellconducted study exceeded OECD Test Guidelines. The top dose (8000 µg/ml) exceeded that normally considered the maximum for this assay and resulted in moderate cytotoxicity (relative survival 65% and 44% in presence and absence of S9, respectively). However, it was not clear whether the test solution, or active acid was tested. No evidence of mutagenic potential was seen at the hprt locus in CHO cells in the absence or presence of S9 at four different levels.

An aqueous solution of DTPMP-xNa (CAS 22042-96-2), neutralized with NaOH, was tested in the mouse lymphoma L5178Y thymidine kinase assay using the microwell variant of the assay (Central Toxicology Laboratory, 1997). The maximum concentration tested was 2200 µg/ml. Higher concentrations were not used as the study authors considered that they gave excessively high osmolarity. A toxicity limit was not reached in these tests as the relative toxicity was >75%. No increases in mutation frequency were seen in any of these tests. Nevertheless a higher dose level could have been employed in order to satisfy the criteria for a convincing assay.

Osmolarity was not assessed in the in vitro studies on the acid; it is an unlikely cause of the positive response because positive responses are only seen consistently in the presence of metabolic activation. These substances are strong chelators and there is some unpublished evidence that other chelators may produce false positive responses in this assay, presumably due to the removal of essential metal ions such as Zn, Mg etc. Genetic toxicity studies conducted with the structurally analogous phosphonate ATMP are available. It has been observed that DTPMP acid produces a positive response in this assay yet ATMP does not (SIAR, 2005), which is consistent with their relative chelation potentials. Thus there is considerable evidence that the positive response with the acid is an isolated and artefactual response resulting from pH. The elevated mutant frequency with neutralised acid is below the frequency at which the study would be considered to be positive according to currently accepted criteria.

In vivo chromosome aberration

While there is evidence for potential to induce chromosome aberrations in vitro, there is no evidence of clastogenicity in vivo. Evidence for a lack of genotoxic potential of DTPMP-H, and its salts, in vivo is provided by a well-conducted chromosome aberration study in rat bone marrow following gavage with doses up to 1970 mg DTPMP-H/kg bw (Hazleton Laboratories, 1983). The number of cells scored for aberrations and mitotic index was lower than that required in the current guideline. The top dose resulted in 25% mortality and loss of body weight. Metaphases were analysed from 4-6 animals per group. No evidence is presented in the study for the test substance reaching bone marrow. The mitotic index was unaffected by the administration of the test substance. However, there is sufficient evidence from other sources (Table 1) to provide information on exposure and toxicity to bone marrow, so in this respect the study does not stand alone. In the interests of animal welfare, use should be made of historical data and it is considered that in view of the clear negative result from the in vivo chromosome aberration assay, and a negative carcinogenicity study with the sodium salt (CAS 22042-96-2), it can be concluded that DTPMP-H is not clastogenic.

 

The measurement of mitotic index might not be considered conclusive because an insufficient number of cells were scored. The mitotic index is a measure of toxicity, therefore the insufficient number of cells scored affects the measure of toxicity to bone marrow. There is sufficient evidence from other sources (Table 1) to provide information on exposure and toxicity to bone marrow, so in this respect the study does not stand alone. In the interests of animal welfare, use should be made of historical data and it is the lead registrant’s opinion that in view of the clear negative result from the in vivo chromosome aberration assay, this study should be considered sufficient to follow up the positive in vitro chromosome aberration result. The sample size for in vivo chromosome aberration tests was increased in order to increase the statistical power of the assay (OECD, 2015). The sample size in the study was 100 cells per animal with 6 animals per group, and therefore exceeded the size required when the guideline was updated in 1997 (100 cells per animal with 5 animals per dose), though it falls short of that required in the 2014 and 2016 OECD 475 guidelines. The positive in vitro result was observed in a study which was statistically less robust than a new study would be, because the in vitro chromosome aberration study does not comply with the updated guideline (OECD 473, 2016). In the interests of animal welfare and in view of:

- the lack of positive results in mammalian mutagenicity studies

- the deficiencies in the in vitro cytogenicity study,

- clear negative result from the in vivo chromosome aberration assay, the existing in vivo study is considered adequate to follow up the in vitro results. However, ECHA required an additional in vivo mammalian alkaline comet assay (OECD 489) which is contracted and ongoing.

 

 Evidence for systemic exposure

 Table 1: Toxicokinetic data for DTPMP

Endpoint	Reference
Absorption and distribution of DTPMP following oral and dermal administration	Procter and Gamble, 1978
Distribution of  DTPMP           following           intravenous administration	Subramanian et al, 1975
Distribution of DTPMP following intravenous administration	Goeckeler, 1987

 

Although there is no evidence from the in vivo chromosome aberration study that the substance reached the bone marrow, evidence of systemic exposure was reported in the in vivo chromosome aberration study in which test substance was administered at 200, 600 and 1970 mg/kg bw (Hazleton Laboratories, 1983). Clinical signs and three mortalities were recorded in the high dose group.

Though absorption of DTPMP has been shown to be low (approximately 2%) after oral dosing (Procter and Gamble, 1978), the data indicate that a small proportion of an oral dose is systemically available and this would primarily result in distribution to bone, with the remainder distributed to other tissues including the bone marrow and germ cells. Systemic availability following oral administration and affinity for bone was demonstrated following oral gavage with DTPMP, radiolabelled in the carbon atoms of the phosphonomethyl groups (Procter and Gamble, 1978). The proportion of radiolabel in the tissues was low as a result of the low absorption; the concentration in bone, (2.9 ± 0.79 μg/g) was nine times greater than any other tissue. Radioactivity was not detected in bone marrow. Very low levels were observed in blood (0.05 ± 0.05 μg/ g), plasma (0.03 ± 0.02 μg/g) and testes (0.05 ± 0.007 μg/g). The limit of detection of the method was not stated, however it is likely that the differences between bone marrow, blood and testes are not significant. Both bone marrow and testes have very good blood supply, so would be exposed to substances present in the blood.

Further information comes from evidence from published literature (Subramanian et al, 1975; Goeckeler, 1987), which demonstrates through the use of medical imaging in rats, that DTPMP (in the form of radiochemical complexes) is preferentially distributed to the outer bone rather than the bone marrow (following intravenous administration).

Intravenous administration of radiolabelled DTPMP salts to rabbits was followed by imagining and distribution analysis. DTPMP was excreted in the urine (70% at 4 hours), and DTPMP localised in the skeleton (Subramanian et al, 1975). At 4 hours after dosing with indium-113-labelled DTPMP, the ratio of % dose/1% body weight was as shown below. Whilst the radioassay analysis detects the complexed radionuclide, the observation of different distribution patterns and clearance rates when different phosphonate complexes of the same nuclide were compared in this study supports the authors’ conclusion that the properties of the phosphonate are important.

A similar study has been carried out using samarium-153 labelled DTPMP acid (Goeckeler, 1987). After intravenous administration to rats, the majority of the substance was eliminated in the urine, and much of the remainder had distributed to the bone (Table 2). Although the analysis detected the radiolabel and not directly detect the complexing agent, DTPMP-H, the stability of the chelation of the radionuclide by DTPMP means that the result adds to the evidence for distribution of DTPMP to the bone. The differences between the results of the two studies reflect the different time after intravenous administration that samples were taken. Taking into account the differences resulting from route of administration and sampling time, the studies show similarities between the distribution of radionuclide complexed DTPMP and that of carbon-labelled DTPMP. The results of the two radionuclide studies support the assumption of comparable exposure of bone marrow and germ cells.

 

 Table 2: Distribution data for DTPMP

Tissue	Distribution following oral administration (μg/kg) Procter and Gamble (1978)    (species: rat)		Distribution following 2h intravenous administration (% dose/g) (2h) Goeckeler (1987) (species: rat)	Distribution following 4h intravenous administration (% dose/1% body weight) Subramanian (1975) (species: rabbit)	Distribution following 4h intravenous administration (% dose/1% body weight) Subramanian (1975) (species: rabbit)
Radiolabel	carbon		153-Sm	85-Sr	113m-In
Blood	0.05 ± 0.05		74	0.7	0.4
Plasma	0.03 ± 0.02		Not determined	Not determined	Not determined
Average bone	2.9 ± 0.79		30	8.1	4.8
Marrow	Below detection	limit	Not determined	0.3	0.2
Muscle	Below detection	limit	0.9	0.2	0.04
Kidney	0.17 ± 0.03		0.4	0.9	2.3
Liver	0.11 ± 0.02		0.3	0.3	0.2
Testes	0.05 ± 0.007		Not determined	Not determined	Not determined

The evaluation of genetic toxicity for REACH Registration is undertaken in order to make a decision on whether the registered substance should be classified for germ cell mutagenicity.

Taken together, the experimental data on absorption and distribution of DTPMP indicate that germ cells and bone marrow are exposed to similar very low levels of DTPMP. The exposure of workers and the general population would be expected to result in low levels of adsorption. The majority of the absorbed dose would be excreted via the kidneys, and the rest would distribute primarily to bone (Proctor and Gamble, 1978).

Although toxicity to bone marrow was not observed in the in vivo chromosome aberration study, systemic toxicity was seen indicating that DTPMP was available in the circulatory system. As the bone marrow has a very good blood supply bone marrow cells would have been exposed to levels that are relevant to germ cell mutagenicity. Therefore, it is considered that the lack of chromosome aberrations in the in vivo study are relevant to potential mutagenicity to germ cells.

 

 Discussion of trends in the DTPMP category

 

 Table 3: Summary of mutagenicity studies

Parameter	Results	Results	Reference and reliability
	15827-60-8	salts
Bacterial mutagenicity	Negative	Negative	Acid: Monsanto 1981a (Reliability 1) Salt:Japan       Oilstuff Inspectors corporation, 2001a (Reliability 1)
Mammalian cytogenicity	No data	Positive	Salt:Japan       Oilstuff Inspectors corporation, 2001b (Reliability 2)
Mammalian          gene mutation (L5178Y)	Negative	Negative	Acid: SRI International, 1983;   Microbiological Associates,          1983 (Reliabilities 2) Salt: Central Toxicology Laboratory           1997 (Reliability 2)
Mammalian          gene mutation (CHO/hprt)	Negative		Pharmakon   Research 1984 (Reliability 2)
Chromosome aberrations in vivo	Negative		Monsanto,            1983 (Reliability 2)

 

Conclusions:

Neither the acid nor the sodium salt induce mutations in well-conducted studies in bacterial or mammalian cells.

The conclusion that DTPMP-xNa is not mutagenic in L5178Y cells is substantiated by consideration of:

(1) Lack of structural alerts for mutagenicity

(2) Lack of evidence for gene mutation potential in sub-mammalian systems and

(3) Lack of potential to induce gene mutations in a well-conducted assay investigating mutations at thehprt locus in CHO cells.

While there is some evidence for the induction of chromosome aberrations in vitro, there is no evidence of clastogenicity in vivo. Considering the clear negative result from the in vivo chromosome aberration assay, and a negative carcinogenicity study with the sodium salt (CAS 22042-96-2), it is concluded that DTPMP acid is not clastogenic. Consequently, DTPMP and its salts are not considered to pose a genotoxic hazard.

Although no studies have been conducted on other salts, their genotoxic properties are expected to be related to the phosphonic ion and therefore the results on the acid and sodium salts can be used to predict a lack of genotoxicity of the potassium and ammonium salts. Potassium ions are not expected to contribute to the genetic toxicity of the potassium salt: potassium is an abundant mineral and plays an important role in metabolism. Ammonium ions are not expected to contribute to genetic toxicity of the ammonia salt of DTPMP as there is no indication for genetic toxicity in the data available for the ammonia category (OECD, 2007a).

Justification for classification or non-classification

Based on the reliable in vitro and in vivo data, DTPMP-xNH4 does not require classification according to Regulation (EC) No 1272/2008.