Benzoyl chloride, 3-[[[2,3-bis(acetyloxy)propyl]amino]carbonyl]-2,4,6-triiodo-5-[(2-methoxyacetyl)amino]-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

study conducted according to OECD test guideline 471 (1983); result: negative

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-07-05 to 1995-11-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Principles of method if other than guideline:

plate incorporation method

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

His locus

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9-liver mix
- source of S9: CCR
- method of preparation of S9 mix: The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male Wistar rats, strain HanIbm (BRL, CH-4414 Füllinsdorf, weight approx. 220 - 320 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-76275 Ettlingen, F.R.G.) in olive oil 5 days previously. After cervical dislocation the livers of the animals were removed, washed in 150 mM KCI and homogenised. The homogenate, was diluted 1+3 in KCI and centrifuged at 9,000 g for
10 minutes at 4° C. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 mL and stored at -80° C. Small numbers of the ampoules are kept at -20° C for up to several weeks before use.
- concentration or volume of S9 mix and S9 in the final culture medium: The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-80939 Mtinchen: Bio-Rad protein assay, Catalogue 500 000 6. The protein concentration in the S9 preparation was 30.6 mg/mL (lot 250795). The amount of S9 supernatant was 15% v/v.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The metabolic activity of the S9 preparation was checked with benzo (a) pyrene.

Test concentrations with justification for top dose:

10.0; 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate, tested up to limit concentration

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqueous solvents (water or saline or culture medium) and DMSO

- Justification for choice of solvent/vehicle: As requested by the sponsor

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Rationale for test conditions:

According to OECD test guideline

Evaluation criteria:

A test article is considered positive if either a dose related increase in the number of revertants or a biological relevant increase for at least one test concentration is induced.
A test article producing neither a dose related increase in the number of revertants nor a biological relevant positive response at any one of the test points is considered non-mutagenic in this system.

A significant response is described as follows:
A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 100 and TA 102 or thrice on TA 1535, TA 1537, and TA 98. Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

TA1535 / without S9
Concentration mg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	11	16	5	11	5.5
Solvent control	8	15	14	12	3.8	1.0
Positive control#	712	725	735	724	11.5	58.7
10.0	14	18	11	14	3.5	1.2
33.3	8	11	14	11	3.0	0.9
100.0	15	11	13	13	2.0	1.1
333.3	17	10	15	14	3.6	1.1
1000.0	10	15	10	12	2.9	0.9
2500.0	14	13	14	14	0.6	1.1
5000.0	11	7	14	11	3.5	0.9
TA1535 / with S9
Concentration mg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	15	14	16	15	1.0
Solvent control	17	14	11	14	3.0	1.0
Positive control##	244	274	245	254	17.0	18.2
10.0	18	14	17	16	2.1	1.2
33.3	22	14	10	15	6.1	1.1
100.0	16	13	22	17	4.6	1.2
333.3	17	14	12	14	2.5	1.0
1000.0	16	18	19	18	1.5	1.3
2500.0	16	17	19	17	1.5	1.2
5000.0	14	16	7	12	4.7	0.9
#= sodium azide 10 µg/plate; ##= 2-aminoanthracene 2.5 µg/plate
TA 1537 without S9
Concentration mg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	7	6	5	6	1.0
Solvent control	12	13	9	11	2.1	1.0
Positive control#	39	48	46	44	4.7	3.9
10.0	11	12	13	12	1.0	1.1
33.3	12	16	6	11	5.0	1.0
100.0	13	6	12	10	3.8	0.9
333.3	14	16	13	14	1.5	1.3
1000.0	10	10	12	11	1.2	0.9
2500.0	13	10	12	12	1.5	1.0
5000.0	10	17	10	12	4.0	1.1
TA 1537 with S9
Concentration mg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	15	8	15	13	4.0
Solvent control	16	16	14	15	1.2	1.0
Positive control##	93	79	100	91	10.7	5.9
10.0	19	13	21	18	4.2	1.2
33.3	13	13	19	15	3.5	1.0
100.0	13	17	18	16	2.6	1.0
333.3	15	20	5	13	7.6	0.9
1000.0	18	19	16	18	1.5	1.2
2500.0	15	18	13	15	2.5	1.0
5000.0	19	18	22	20	2.1	1.3
#= 4-nitro-o-phenylene-diamine 10 µg/plate; ##= 2-aminoanthracene 2.5 µg/plate
TA 98 without S9
Concentration mg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	32	40	31	34	4.9
Solvent control	42	44	40	42	2.0	1.0
Positive control#	135	137	143	138	4.2	3.3
10.0	28	37	50	38	11.1	0.9
33.3	42	43	46	44	2.1	1.0
100.0	42	37	54	44	8.7	1.1
333.3	42	36	30	36	6.0	0.9
1000.0	50	34	34	39	9.2	0.9
2500.0	48	35	54	46	9.7	1.1
5000.0	24	44	37	35	10.1	0.8
TA 98 with S9
Concentration mg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	51	47	29	42	11.7
Solvent control	49	34	47	43	8.1	1.0
Positive control##	214	197	184	198	15.0	4.6
10.0	32	41	49	41	8.5	0.9
33.3	45	36	41	41	4.5	0.9
100.0	42	43	37	41	3.2	0.9
333.3	45	34	40	40	5.5	0.9
1000.0	41	34	48	41	7.0	0.9
2500.0	38	39	34	37	2.6	0.9
5000.0	40	46	49	45	4.6	1.0
#= 4-nitro-o-phenylene-diamine 10 µg/plate; ##= 2-aminoanthracene 2.5 µg/plate
TA 100 without S9
Concentration mg/plate	Plate	Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	130	119	131	127	6.7
Solvent control	115	144	130	130	14.5	1.0
Positive control#	765	695	688	716	42.6	5.5
10.0	112	107	129	116	11.5	0.9
33.3	146	137	139	141	4.7	1.1
100.0	142	120	121	128	12.4	1.0
333.3	130	122	121	124	4.9	1.0
1000.0	116	130	141	129	12.5	1.0
2500.0	138	135	126	133	6.2	1.0
5000.0	119	118	122	120	2.1	0.9
TA 100 with S9
Concentration mg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	139	128	166	144	19.6
Solvent control	141	144	137	141	3.5	1.0
Positive control##	1462	1468	981	1304	279.5	9.3
10.0	145	151	146	147	3.2	1.0
33.3	152	161	133	149	14.3	1.1
100.0	150	157	139	149	9.1	1.1
333.3	167	165	135	156	17.9	1.1
1000.0	130	141	150	140	10.0	1.0
2500.0	129	127	137	131	5.3	0.9
5000.0	142	125	125	131	9.8	0.9
#= sodium azide 10 µg/plate; ##= 2-aminoanthracene 2.5 µg/plate
TA 102 without S9
Concentration mg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	200	211	192	201	9.5
Solvent control	217	217	210	215	4.0	1.0
Positive control#	1765	1755	1647	1722	65.4	8.0
10.0	177	192	207	192	15.0	0.9
33.3	234	259	257	250	13.9	1.2
100.0	164	181	186	177	11.5	0.8
333.3	165	186	223	191	29.4	0.9
1000.0	209	224	230	221	10.8	1.0
2500.0	215	254	241	237	19.9	1.1
5000.0	242	254	254	250	6.9	1.2
TA 102 with S9
Concentration mg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	238	240	254	244	8.7
Solvent control	254	278	246	259	16.7	1.0
Positive control##	1168	1171	1276	1205	61.5	4.6
10.0	319	325	310	318	7.5	1.2
33.3	326	367	327	340	23.4	1.3
100.0	252	246	250	249	3.1	1.0
333.3	291	309	289	296	11.0	1.1
1000.0	297	284	294	292	6.8	1.1
2500.0	324	322	333	326	5.9	1.3
5000.0	336	319	323	326	8.9	1.3
#= methyl methane sulfonate 5 µL/plate; ##= 2-aminoanthracene 2.5 µg/plate
* enhancement factor = Sum revertants/concentration test article/ sum revertants/ solvent control

Conclusions:

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

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD test guideline 471 (1983), strains (TA 98, TA 100, TA 102, TA 1535, and TA 1537) of S. typhimurium were exposed to TIP-Diaminchlorid (100 % a.i.), in water at concentrations of 10.0, 33.3, 100.0, 333.3, 1000.0, 2500.0, 3333.3, 5000.0 µg/plate in the presence and absence of mammalian metabolic activation with the plate incorporation method.

TIP-Diaminchloird was tested up to limit concentration (5000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

TIP-Diamidchlorid did not show a mutagenic potential in a bacterial reverse mutation assay (Ames test in 5 strains of S. typhimurium (TA98, TA100, TA102, TA1535 and TA1537) when tested up to the highest recommended dose level of 5.0 mg/plate in the absense or presense of extrinsic metabolic activation (liver S9 mix from Aroclor 1254 -treated rats).

 

Justification for classification or non-classification

Based on the available information TIP-Diamidchlorid does not need to be classified according to Regulation (EC) No. 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).