[[(phosphonomethyl)imino]bis[hexamethylenenitrilobis(methylene)]]tetrakisphosphonic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are no in vitro genetic toxicity data for BHMT-H. Therefore, data are read-across from DTPMP-H and DTPMP-7Na.

Gene mutation (Bacterial reverse mutation assay / Ames test): read-across from DTPMP-H, negative with and without activation in Salmonella typhimurium TA98, TA100, TA 1535, TA 1538 and E. coli WP2 uvrA (OECD Test Guideline 471) (Italmatch, 2003)

Cytogenicity in mammalian cells: read-across from DTPMP-7Na (CAS number 22042-96-2), positive in Chinese hamster lung IU cells (OECD Test Guideline 473) (Japan Oilstuff Inspectors Corporation, 2001)

Mutagenicity in mammalian cells: read-across from DTPMP-H, negative in Chinese hamster ovary cells (similar to OECD Test Guideline 476) (Pharmakon Research International, 1984)

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

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 hours) and continuous treatment (24 hours 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 study, conducted according to OECD guideline 473, a dose related increase in the number of cells with aberrations was observed after 48 hours treatment in an in vitro chromosome aberration assay . The study was performed in compliance with GLP.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14-Jan-2003 to 17-Jan-2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Species / strain:

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

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 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Bone marrow chromosome study in rats (oral gavage administration): Read-across from DTPMP-H: Negative (similar to OECD Test Guideline 475) (Hazleton Laboratories, 1983).

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.

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

There are no in vitro genetic toxicity data for BHMT-H. Therefore, data are read-across from DTPMP-H and DTPMP-7Na.

DTPMP-H has been tested in a valid bacterial reverse mutation assay, conducted according to a guideline similar to OECD Test Guideline 471, In compliance with Japanese guideline notification no. 76, but not with GLP, using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or E. coli WP2 uvr A (Italmatch, 2003).

No increase in the number of revertant colonies was observed in any test strain, with or without metabolic activation, when tested up to cytotoxic concentration. Appropriate positive 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.

DTPMP-7Na has been tested for ability to cause chromosome aberrations in Chinese hamster lung cells, CHL/IU, conducted according to a guideline similar to OECD Test Guideline 473 and in compliance with GLP (Japan Oilstuff Inspectors Corporation, 2001). A delay in the cell cycle was observed in the continuous 24-hour treatment test, therefore a 48-hour treatment (3 cell cycles) was carried out. A dose-related increase in the number of cells with aberrations was observed following continuous 48-hour treatment without metabolic activation. No increase in the number of cells with aberrations was observed following pulsatile 6-hour treatment or continuous 24-hour treatment with and without metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is positive for the induction of chromosome aberrations under the conditions of the study. The study was 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.

DTPMP-7Na has been tested for mutagenicity in Chinese hamster ovary cells (CHO), conducted according to a protocol similar to OECD Test Guideline 476 and in compliance with GLP (Pharmakon Research International, 1984). No test-substance induced increase in the number of mutations was observed when tested with or without metabolic activation up to a cytotoxic concentration. 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 metabolic activation, respectively). However, it was not clear whether the test solution, or active acid was tested. Appropriate negative and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the study.

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. In respect of the clear negative result from the in vivo chromosome aberration assay DTPMP-H was concluded to be is not clastogenic.

In line with ECHA Final decision No CCH-D-2114495769-22-01/F, an in vivo mammalian alkaline comet assay is going to be conducted with DTPMP(5-7Na). The study will be conducted according to OECD Test Guideline 489 and in compliance with GLP. The conclusion on genetic toxicity of BHMT-H will be revised in line with the conclusions from the study.

Justification for classification or non-classification

Based on the available in vitro and in vivo genotoxicity data for the closely related substances DTPMP-H and DTPMP-xNa, BHMT-H is not classified according to Regulation (EC) No 1272/2008.