N,N,N',N'-tetrakis(2-hydroxypropyl)adipamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

It is concluded that test material can not cause any genetic toxicity under the experimetal conditions.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from March 27, 1996 to July 3, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

salmonella typhimurim histidine(his) reversion system

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

TA1537: his C 3076; rfa-; uvrB': frame shift mutations
TA 98. his D 3052; rfa-; uvrB; R-factor; frame shift mutations
TA1535: his G 46, rfa-; uvrB': base-pair substitutions
TA 100: his G 46; rfa-; uvrB';R-factor: base-pair substitutions

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

microsoma liver activation(s9)

Test concentrations with justification for top dose:

33.3; 100.0; 333.3; 1000.0, 2500.0 and 5000.0 µg/plate

Vehicle / solvent:

aqua bidest

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

aqua bidest

True negative controls:

not specified

Positive controls:

yes

Remarks:

10 µg/plate

Positive control substance:

sodium azide

Remarks:

without S9 for TA 1535, TA 100

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

aqua bidest

True negative controls:

not specified

Positive controls:

yes

Remarks:

2.5 µg/plate

Positive control substance:

other: 2-aninoanthracene, 2-AA

Remarks:

with s9 for all strains

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

aqua bidest

True negative controls:

not specified

Positive controls:

yes

Remarks:

10 µg/plate

Positive control substance:

other: 4-nitro-0-phenylene-diamine

Remarks:

without S9 for TA 1537, TA 98

Details on test system and experimental conditions:

The bacterial strains TA 1535, TA 98, and TA 100 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen). The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5% DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.
Mammalian Microsomal Fraction S9 Mix: The bacteria used in these assays do not possess the enzyme systems which, in mammals, are known to convert pro-mutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.
S9 (Preparation by C C R): The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male Wistar rats, strain Hanlbm (BRL, CH-4414 Fullinsdorf, weight approx. 220 - 320 g) which received a triple i.p. injection of 80 mg/kg bw phenobarbital (Desitin, D-22335 Hamburg) and p-naphtoflavone (Aldrich, D-895 55 Steinheim) orally, in corn oil 1 - 3 days previously.
After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCl 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. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-80939 München: Bio-Rad protein assay, Catalogue 500 000 6.The protein concentration in the S9 preparation was 25.2 mg/ml (150296).
S9 Mix: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v. The composition of the co-factor solution was chosen to yield the following concentrations in the S9 mix: 8 mM MgCl, 33 mM KCl, 5 mM Glucose-6-phosphate and 5 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.
Experimental Performance: For each strain and dose level, including the controls three plates were used as a minimum.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µl Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µl Overlay agar
In the pre-incubation assay 100 µl test solution, 500 µl S9 mix / S9 mix substitution buffer and 100 µl bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

Evaluation criteria:

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

Statistics:

The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GmbH, D-61184 Karben; F.R.G.). The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates.

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

Positive controls validity:

valid

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

Positive controls validity:

valid

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

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

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not specified

Additional information on results:

No toxic effects, evidenced by a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.
No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with test article at any concentration level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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.
Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay (negative with and without metabolic activation).

Executive summary:

This study was performed to investigate the potential of test substance to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA 1537, TA98, and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 µg/plate.

No toxic effects, evidenced by a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with test article at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Under the experimental condition, the test article does not need to be classified in accordance with CLP (Regulation EC No. 1272/2008) for mutagenicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity/mutagenicity

No human data is available.

In vitro data: There are five study reports covering three study endpoint including in vitro gene mutation in bacteria, in vitro gene mutation and cytogenicity in mammalian cells. The Ames test (Dr. Hans-Eric Wollny, 1996) following OECD guideline 471 and GLP principles presented no evidence of gene mutation toxicity in the Salmonella typhimurium reverse mutation assay. In another two key studies (Huntingdon Research Centre Ltd., 1983; Paul C. Brooker, et al.1990), it is demonstrated that test article caused no mutagenic potential in the vitro test system, one of which was used as read-across since test article is structurally similar to target substance. Other read-across studies (J. L. Sames and J. P. Frank, 1989; Kashima Laboratory of Mitsubishi Chemical Safety Institute Ltd., 1998) also supported the negative results.

An in vivo study does not need to be conducted in absence of any positive in vitro findings for mutagenicity.

In conclusions, the test article does not cause any genetic toxicity as assessed under the present experimental conditions.

Justification for classification or non-classification

Based on the existing mutagenicity data, the test article does not require classification for mutagenicity according to CLP (Regulation EC No. 1272/2008).