Diamminediisocyanatozinc

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985-09-13 to 1985-09-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well-documented study according to OECD 471 with minor deviations: only four strains of S. typhimurium (TA1535, TA1537, TA98, TA100) as indicated in the latest guideline were used, data on E.coli WP2 strains or S. typhimurium TA102 are lacking. However, since these strains were mainly included in the recent version of OECD 471 because the four formerly only recommended S. typhimurium strains TA1535, TA1537, TA98 and TA100 may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of the test item, this restriction is considered to be negligible.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guideline for testing of chemicals no. 471:- Genetic Toxicology : Salmonella tvphimurium. Reverse Mutation Assay. Presumed of 1981.

Deviations:

no

GLP compliance:

yes

Remarks:

self-declaration

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diamminediisocyanatozinc

EC Number:

401-610-3

EC Name:

Diamminediisocyanatozinc

Cas Number:

122012-52-6

Molecular formula:

C2H6N4O2Zn

IUPAC Name:

copper(2+) bis(carbonylazanide) diamine

Test material form:

solid: particulate/powder

Method

Target gene:

his-

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-Mix, induced by Aroclor 1254

Method of preparation of S9 mix
Species: Rat
Strain: CD (Sprague-Dawley-derived).
Source: Charles River UK Limited, Manston Road, Margate, Kent, England.
Age range: 7-8 weeks on arrival.
Weight range: 180-200 g on arrival.
Diet: Spratt's Laboratory Diet No. 1.
Number of rats used: 8
Stimulation of rat liver enzymes: Mixed-function oxidase systems in the rat liver were stimulated following a single i/p injection of Aroclor 1254 (diluted in Arachis oil to 200 mg/ml) at a dosage of 500 mg/kg. On the fifth day of induction, following an overnight starvation, the rats were killed and their livers aseptically removed.
Preparation of liver homogenate S-9: All steps were at 0-4°C using sterile solutions and glassware. The livers were placed in beakers containing 0.15 M KCl. After weighing, livers were transferred to a beaker containing 0.15 M KCl (the volume of KCl in ml was equivalent to 3 times the weight of liver in gram), minced with a sterile scalpel and homogenized in an MSE top-drive homogenizer. This homogenate was centrifuged for 10 minutes at 9000 x 'g' and the supernatant divided into 15 ml aliquots. These were frozen on dry ice and stored at -80°C, and tested with the carcinogen 7,12-dimethylbenzanthracene before use. For this assay, previously prepared aliquots of S-9 which have been deep frozen were thawed and used to prepared the S-9 mix as follows:
S-9 mix contains: S-9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors are filter-sterilized before use.

Test concentrations with justification for top dose:

Dose range finding test: 5000, 500, 50, 5 µg/plate
Mutation tests: 5000, 1500, 500, 150, 50 µg/plate

Vehicle / solvent:

Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Remarks:

buffer or S-9 mix but without solvent or test compound

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene (TA 1535. TA 1537), 2-Aminoanthracene (TA 1538, TA 98, TA 100)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- 5 strains per concentration
- 3 individual revertant colony counts per dose level and strain

METHOD OF TREATMENT/ EXPOSURE:
- 0.1 ml overnight bacterial culture containing approximatey 2x10^9 cells/ml
- 0.5 ml S-9-Mix or 0.5 ml 0.1 M sodium phosphate buffer (pH 7.4)
- 0.1 ml test solution
- 2 ml his- agar
- shaken and overlaid onto prepared plates containing 15 ml minimal agar.

TREATMENT AND HARVEST SCHEDULE:
- incubation at 37 °C for 72 h
- After this period plates were examined for the appearance of a complete bacterial lawn. Revertant colonies were counted using a Biotran Automatic Colony Counter. Any toxic effects of the test substance were detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn.

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for negative and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group.
A compound is deemed to provide evidence of mutagenic potential if
(1) a statistically significant dose-related increase in the number of revertant colonies is obtained in two separate experiments, and
(2) the increase in the number of revertant colonies is at least twice the concurrent solvent control value

Results and discussion

Test results

Test results 1

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

Remarks:

no vast cytotoxicity noted

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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

Remarks:

no vast cytotoxicity noted

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

no vast cytotoxicity noted

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

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

Remarks:

no vast cytotoxicity noted

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

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

Remarks:

no vast cytotoxicity noted

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The study is regarded as a valid guideline study with self-reported GLP compliance. The test item did not show mutagenic effects in both experiments. The revertants were not increased in comparison with the spontaneous revertants (solvent only). Therefore it can be stated, that under the test conditions, the test item is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and TA 1538.

Executive summary:

This study was performed in order to evaluate the mutagenic potential of Diaminodiisocyanatozinc.

The study was performed under GLP and conducted in accordance with the following guideline: OECD Guidelines for the Testing of Chemicals „Bacterial Reverse Mutation Test" Part 471, presumed 1981.

In this in vitro assessment of the mutagenic potential of the test item, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) were exposed to the test material, diluted in dimethylsulphoxide, which was also used as a negative control.

Two independent mutation tests were performed using agar plates, in the presence and absence of liver preparations from Aroclor 1254 -induced rats.

In the preliminary dose range finding study no toxicity was observed at concentrations of up to 5000 µg/plate. Therefore 5000 µg/plate was chosen as the highest dose level for the subsequent mutation study. Other dose levels used in the mutation assays were: 1500, 500, 150, 50 µg/plate.

The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the metabolic activation system.

No evidence of mutagenic activity was seen at any dose level of the test item in either mutation test.

It is concluded that, when tested at dose levels up to 5000 µg/plate in dimethylsulphoxide, the test item was not mutagenic in either the presence or absence of metabolic activation. 

THE TEST ITEM DIAMINODIISOCYANATOZINC CAN BE STATED AS "NOT MUTAGENIC UNDER THE CONDITIONS OF THE TEST".