Benzoic acid, 3-amino-5-[[[2,3-bis(acetyloxy)propyl]amino]carbonyl]-2,4,6-triiodo-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse muation assay/AMES test, GLP): negative with and without metabolic activation
[Schering AG, Report No. AL91; 1996-06-06]

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:

August 1995

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:

Histidine gene locus

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

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:

33.3, 100.0; 333.3; 1000.0; 2500.0; and 5000.0 ~g/plate

Vehicle / solvent:

DMSo, aqua dest.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9-mix: NaN3; 4-NOPD, MMS; with S9-mix: 2-AA

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:

other: TA 98, TA 100, TA 102, TA 1535, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

TA1535 / without S9
Concentration mg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	16	12	13	14	2.1
Solvent control	11	14	15	13	2.1	1.0
Positive control#	866	653	891	803	130.8	60.3
33.3	16	15	14	15	1.0	1.1
100.0	9	11	14	11	2.5	0.9
333.3	13	13	12	13	0.6	1.0
1000.0	10	7	15	11	4.0	0.8
2500.0	7	12	17	12	5.0	0.9
5000.0	11	8	16	12	4.0	0.9
TA1535 / with S9
Concentration mg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	18	16	16	17	1.2
Solvent control	19	19	20	19	0.6	1.0
Positive control##	302	232	236	257	39.3	13.3
33.3	12	19	10	14	4.7	0.7
100.0	12	13	10	12	1.5	0.6
333.3	19	19	8	15	6.4	0.8
1000.0	18	17	14	16	2.1	0.8
2500.0	18	20	15	18	2.5	0.9
5000.0	10	18	19	16	4.9	0.8
#= 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	13	18	19	17	3.2
Solvent control	12	10	19	14	4.7	1.0
Positive control#	55	62	58	58	3.5	4.3
33.3	13	13	10	12	1.7	0.9
100.0	15	14	19	16	2.6	1.2
333.3	9	13	12	11	2.1	0.8
1000.0	7	14	17	13	5.1	0.9
2500.0	14	15	10	13	2.6	1.0
5000.0	16	18	14	16	2.0	1.2
TA 1537 with S9
Concentration mg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	10	17	16	14	3.8
Solvent control	18	17	17	17	0.6	1.0
Positive control##	100	100	96	99	2.3	5.7
33.3	21	15	19	18	3.1	1.1
100.0	16	18	13	16	2.5	0.9
333.3	14	17	18	16	2.1	0.9
1000.0	16	13	16	15	1.7	0.9
2500.0	15	14	10	13	2.6	0.8
5000.0	18	20	24	21	3.1	1.2
#= 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	38	39	44	40	3.2
Solvent control	40	43	51	45	5.7	1.0
Positive control#	171	191	166	176	13.2	3.9
33.3	51	74	53	59	12.7	1.3
100.0	51	58	73	61	11.2	1.4
333.3	59	43	52	51	8.0	1.1
1000.0	53	55	53	54	1.2	1.2
2500.0	40	51	-	46	7.8	1.0
5000.0	55	48	-	52	4.9	1.2
TA 98 with S9
Concentration mg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	46	53	63	54*	8.5
Solvent control	62	66	75	68*	6.7	1.0
Positive control##	537	656	609	601	59.9	8.9
33.3	47	43	63	51	10.6	0.8
100.0	48	49	41	46	4.4	0.7
333.3	42	48	53	48	5.5	0.7
1000.0	63	56	53	57	5.1	0.8
2500.0	60	64	59	61	2.6	0.9
5000.0	60	73	71	68	7.0	1.0
#= 4-nitro-o-phenylene-diamine 10 µg/plate; ##= 2-aminoanthracene 2.5 µg/plate; * These numbers of revertants are above the eorresponding values of the historieal eontrol data without S9. Since the range of spontaneous revertant colonies is frequently wider in the presenee of metabolie aetivation this does not interfere with the validity of the experiment.
TA 100 without S9
Concentration mg/plate	Plate	Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	168	155	172	165	8.9
Solvent control	168	178	173	173	5.0	1.0
Positive control#	966	1030	1012	1003	33.0	5.8
33.3	145	143	189	159	26.0	0.9
100.0	147	134	163	148	14.5	0.9
333.3	133	119	165	139	23.6	0.8
1000.0	134	131	124	130	5.1	0.7
2500.0	123	130	131	138	4.4	0.7
5000.0	138	146	140	141	4.2	0.8
TA 100 with S9
Concentration mg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	181	184	199	188	9.6
Solvent control	168	201	178	182	16.9	1.0
Positive control##	1138	1326	1417	1294	142.3	7.1
33.3	177	200	166	181	17.3	1.0
100.0	159	187	177	174	14.2	1.0
333.3	135	153	156	148	11.4	0.8
1000.0	164	159	165	163	3.2	0.9
2500.0	155	158	153	155	2.5	0.9
5000.0	167	172	155	165	8.7	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	216	251	257	241	22.1
Solvent control	272	277	302	284	16.1	1.0
Positive control#	899	745	1201	948	232.0	3.3
33.3	287	296	307	297	10.0	1.0
100.0	320	335	326	327	7.5	1.2
333.3	305	339	310	318	18.4	1.1
1000.0	272	297	312	294	20.2	1.0
2500.0	153	193	187	178	21.6	0.6
5000.0	191	192	195	193	2.1	0.7
TA 102 with S9
Concentration mg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative control	327	351	343	340*	12.2
Solvent control	341	368	345	351*	14.6	1.0
Positive control##	1469	1524	1468	1487	32.0	4.2
33.3	351	355	381	362	16.3	1.0
100.0	373	381	372	375	4.9	1.1
333.3	375	364	378	372	7.4	1.1
1000.0	389	388	418	398	17.0	1.1
2500.0	210	233	209	217	13.6	0.6
5000.0	222	221	212	218	5.5	0.6
#= methyl methane sulfonate 5 µL/plate; ##= 2-aminoanthracene 2.5 µg/plate
* enhancement factor = Sum revertants/concentration test article/ sum revertants/ solvent control

Conclusions:

Tamip diacetate is not genotoxic under the conditions of this test system and therefore classification is not required.

Executive summary:

Tamip diacetate (ZK 91248) did not show any mutagenic potential in a bacterial reverse mutation assay with S. typhymurium (TA 1535, TA 100, TA 1537, TA 102, TA 98) when tested up to 5.0 mg/plate in the absense and presense of intrinsic metabolic activation (liver mix from Aroclor 1254 -treated rats).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

 

Tamip diacetate (ZK 91248) did not show any mutagenic potential in a bacterial reverse mutation assay with S. typhymurium (TA 1535, TA 100, TA 1537, TA 102, TA 98) when tested up to 5.0 mg/plate in the absense and presense of intrinsic metabolic activation (liver mix from Aroclor 1254 -treated rats) (Schering AG, Report No. AL91; 1996-06-06)

 

Justification for classification or non-classification

 

Tamip diacetate (ZK 91248) was tested in a standard genotoxicity test and did not show any genotoxic potential. Thus, there is no sufficient evidence available to classify Tamip diacetate as genotoxic.

Classification according to Directive 67/548/EEC and Regulation (EC) 1272/2008 (CLP) is not required.