Diamminediisocyanatozinc

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro: Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538, ± S9, OECD TG 471 of 1981: negative ± S9, all strains

In vitro: Chromosome aberration test; Human lymphocytes, ± S9, equivalent to OECD 473: positive -S9, negative +S9

In vitro: Gene mutation (Gene mutation in mammalian cells, TK assay): mouse lymphoma L5178Y cells, ± S9, OECD 490, EU B.17: positive ±S9

Link to relevant study records

Reference

Reference 1

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.

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

Target gene:

his-

Species / strain / cell type:

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

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

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

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

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

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

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

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

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".

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

until 1986-04-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Remarks:

in vivo follow-up

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

n/a

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
Human blood was collected aseptically and diluted with RPMI 1640 tissue culture medium. Lymphocytes were separated by centrifugation on a Histopaque-1077 gradient (Sigma London. Chemical Company Limited) for 30 minutes at 400 x g. After 3 washes and sedimentation by centrifugation for 10 minutes at 200 x g the cells were suspended at a concentration of 1 x 10exp6 cells per ml in RPMI 1640 + 20% Foetal Calf Serum (Gibco) + 2% phytohaemagglutinin (Wellcome). 1 ml aliquots of the cell suspension were placed in the wells of a Falcon multiwell tissue culture plate and incubated at 37°C in a humid atmosphere containing 5% CO2 for 48 hours.
- Suitability of cells:
Lymphocytes obtained from human blood are easy to culture in vitro and are readily available. However they do not divide and grow unless stimulated to do so. This is usually achieved by adding phytohaemagglutinin (PHA) to the culture which results in a high mitotic yield.

For lymphocytes:
- Whether whole blood or separated lymphocytes were used: Lymphocytes were separated by centrifugation
- Mitogen used for lymphocytes: 2% phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: 37°C in a humid atmosphere containing 5% CO2.
RPMI 1640 tissue culture medium + 20% Foetal Calf Serum (Gibco)

Cytokinesis block (if used):

After 70 hours incubation mitotic activity was arrested by addition of colchicine to each culture at a final concentration of 0.25 pg/ml.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
Species: Rat.
Strain: CD rats of Sprague-Dawley origin.
Source: Charles River, UK Limited, Manston Road, Margate, Kent, England.
Age range: 6 - 8 weeks.
Weight range: 180-200 g.
Diet: Labsure Laboratory Animal Diet (LAD)
Number used: 10 males
- method of preparation of S9 mix
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 after treatment# following an overnight starvation, the rats were killed and their livers aseptically removed.
Preparation of liver homoqenate 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 3 volumes of 0.15 M KC1 and homogenised in an MSE homogeniser. This homogenate was centrifuged for 10 minutes at 9000 x g and the supernatant divided into 5 ml aliquots. These were stored at -80°C and tested before use, with the carcinogen, 7,12-dimethylbenz(a)anthracene. For this assay, previously prepared aliquots from a single batch of S-9, which had been stored deep frozen, were thawed and used to prepare the S-9.

Each ml S-9 mix contained:
S-9 fraction 0.1 ml
0.4 M MgCl2 0.02 ml
0.2 M Na2HPO4 (pH 7.4) 0.5 ml
1.0 M glucose-6-phosphate 0.005 ml
0,1 M NADP 0.04 ml
Distilled water 0.335 ml
All the above solutions were mixed and then filter-sterilised (apart from the S-9 fraction which was added after filter-sterilisation of the other components).
- concentration or volume of S9 mix and S9 in the final culture medium
250 ml of S-9 mix per ml
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
tested before use, with the carcinogen, 7,12-dimethylbenz(a)anthracene.

Test concentrations with justification for top dose:

Final concentrations of the test compound in both series of cultures were 0.2, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 µg/ml, based on the results of a preliminary toxicity test, dissolved in DMSO immediately before use

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO, 12.5 µl/ml

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Two wells were used for each concentration with four wells for the solvent control.
- Number of independent experiments :
1 (cytotoxicity test)
2 (main study)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 x 10exp6 cells / ml
- Test substance added to medium. 250 µl of S-9 mix was added to one set of cultures followed by 12.5 µll of various dilutions of the test compound. 10 µl of the dilutions of test compound were added to the remaining cultures

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2h with S9, than further 22h after removal; 24h treatment without S9 can be assumed
- Harvest time after the end of treatment (sampling/recovery times): 70h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.: 0.25 µg/ml colchicine for 2h
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
After 2 hours incubation with colchicine each cell suspension was transferred to a conical centrifuge tube where the solutions were made hypotonic. After an 11-minute period of hypotonic incubation the suspensions were centrifuged at 110 x 'g' for 10 minutes and the cell pellets fixed by addition of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid). The pellets were allowed to fix slowly. Prior to slide preparation the pellets were aspirated through a hypodermic needle, then centrifuged at 200 x 'g1 for 10 minutes and finally resuspended in a small volume of fixative. Two or three drops of the cell suspensions were dropped onto pre-cleaned slides which were then allowed to air-dry. The slides were stained using Giemsa solution and, when dry, mounted in DPX.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 100 per experiment
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
To clarify the results obtained, part of the study was repeated using final concentrations of the test item of 50 and 75 µg/ml in the absence of S-9 mix and 100 µg/ml in its presence. A statistically significant increase in the number of cells containing chromosomal aberration's would be indicative of a positive response.
A gap is defined as an achromatic region occurring in one or both chromatids, the gap size being no greater than the width, of a single chromatid. The separated regions are not displaced.
A break is defined as an achromatic region occurring on one or both chromatids that is greater than the width of a single chromatid. The
accompanying fragment is usually, but not always, displaced from the main chromosome.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI); r

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Lymphocyte cultures were prepared as described above and placed in 5 ml aliquots (10exp6 cells/ml) in 10 ml culture plates. After 48 hours incubation 50 µl aliquots of technical FIC 30/5 were added to one set of duplicate cultures to give final concentrations of 5, 25, 50 and 75 µg/ml. Domethylsulphoxide in 50 µl aliquots was added to four cultures and ethylmethylsulphonate, the positive control, at a final concentration of 500 µg/ml was added to two cultures. For the test in the presence of metabolic activation 1.25 ml S-9 mix was added to each culture followed by 62.5 µl. aliquots of various dilutions of the giving final concentrations of 1, 10, 25, 50 and 100 µg/ml. Dimethylsulphoxide and sterile distilled water (62.5 µl) were added to four and two cultures respectively as the solvent controls and cyclophosphamide at a final concentration of 20 µg/ml added to another two cultures as the positive control. Two hours after dosing the cultures containing S-9 mix were centrifuged and the cell pellets resuspended in fresh medium. The cells were incubated for a fur*ther 20 hours, arrested in metaphase by addition of colchicine for a further 2 hours, then made hypotonic and fixed as described above. Five slides were prepared from each culture, stained with. Giemsa and mounted in DPX. The slides were then coded and wherever possible, a total of 100 metaphase spreads were examined from each culture at a magnification of x1000 using an oil immersion objective. In the presence of S-9 mix only slides from cultures exposed to 10, 50 and 100 µg/ml were examined.

Evaluation criteria:

A statistically significant increase in the number of cells containing chromosomal aberrations would be indicative of a positive response.

Species / strain:

lymphocytes: human

Remarks:

1st experiment

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

at 50µg/ml

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

50% mitotic index at 50µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Remarks:

1st experiment

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

at 50µg/ml

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

more than 50% mitotic index at 50µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Remarks:

repetition

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

at 50µg/ml

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

50% mitotic index at 50µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human

Remarks:

repetition

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

at 100µg/ml

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

more than 50% mitotic index at 50µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
none stated

RANGE-FINDING/SCREENING STUDIES (if applicable):
See "Overall remarks"

STUDY RESULTS
- Concurrent vehicle negative and positive control data
appropriate results

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: mitotic index (MI) : 50% at 50 µg/ml
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps
A gap is defined as an achromatic region occurring in one or both chromatids, the gap size being no greater than the width, of a single chromatid. The separated regions are not displaced.
A break is defined as an achromatic region occurring on one or both or chromatids that is greater than the width of a single chromatid. The
accompanying fragment is usually, but not always, displaced from the main chromosome.
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps : 100 cells per repetition

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
no data

Conclusions:

The study was conducted related to OECD guideline 473 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of the test item to induce chromosome mutations in human lymphocytes.
When tested in dimethylsulphoxide at dose levels extending into the toxic range, the test item showed evidence of clastogenic activity in the absence of metabolic activation but not in its presence.
Consequently, an in vivo test was conducted to show he relevance of these results, and it turned out that they do not have any relevance in vivo, so no classification based on chromosome mutations is triggered.

Executive summary:

The test item was tested for its ability to induce chromosomal aberrations in human lymphocytes cultured in vitro.

Cultured human lymphocytes, stimulated to divide by addition of phytohaemagglutinin were exposed to the test compound in both, the presence and absence of a metabolic activation system (S-9 mix) derived, from rat livers- Cell division was then arrested using colchicine and after hypotonic treatment, fixation and staining, metaphase spreads were examined for chromosomal damage.

Based on the results of a preliminary toxicity test the dose levels used for metaphase analysis were 5, 25 and 50 µg/ml in the absence of metabolic activation and 10, 50 and 100 µg/ml in its presence. In a second test, dose levels of 50 and 75 µg/ml were tested without S-9 mix and 100 µg/ml was tested with S-9 mix.

In the absence of S-9 mix cells treated with the test item showed a statistically significant increase in the incidence of chromosome damage at a dose level of 50 µg/ml in both tests. This concentration caused a decrease of approximately 50% in the mitotic index in both cases. No increases in the levels of chromosomal damage were observed at any other dose level.

In the presence of S-9 mix, there was a slight but statistically significant increase in the proportion of cells with aberrant chromosomes at a concentration of 100 µg/ml in the first test. This response was not reproduced in the second test and no other dose levels showed any such increase,

When tested in dimethylsulphoxide at dose levels extending into the toxic range, the test item showed evidence of clastogenic activity in the absence of metabolic activation but not in its presence.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-10-10 - 2020-03-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

OECD Guideline for the Testing of Chemicals, Part 490, adopted 29. Jul. 2016 ”In vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene“

Deviations:

yes

Remarks:

not affecting the validity and reliability o the study

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

EU-Method B.17 of the Commission Regulation (EC) No. 440/2008, adopted 30 May 2008: “Mutagenicity – In vitro Mammalian Cell Gene Mutation Test”

Deviations:

yes

Remarks:

not affecting the validity and reliability o the study

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

tk+/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: The cells were purchased by ATCC (Wesel, Germany) and were sold under the name L5178Y TK+/- clone (3.7.2C) [TK+/- (clone 3.7.2C)] (ATCC® CRL-9518™).
- Suitability of cells: Reasons for the Choice of the Cell Line L5178Y
The L5178Y is a murine T-cell lymphoma cell line, which grows as single or aggregated round cells in suspension. This cell line is characterized by a high sensitivity to chemical mutagens, by a high proliferation rate (doubling time 10-12 h in stock cultures), a high cloning efficiency (CE) and a stable spontaneous mutant frequency. The L5178Y consists of a stable karyotype and shows a diploid chromosome number (40 ± 2).
- Normal cell cycle time (negative control): 10-12 h

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: n/a
- Methods for maintenance in cell culture: Cleansed and for mycoplasma contamination screened stocks of cells were stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
Cells were thawed 6 d prior treatment and cultivated in RPMI 1640 complete culture medi-um with 5 % HS (pre-test) or 10 % HS (experiment I and I_2) in cell culture flasks at 37.0 ± 1.0 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
- Cell cycle length, doubling time or proliferation index : doubling time 10-12 h in stock cultures
- Modal number of chromosomes: 40 ± 2
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Culture base Medium RPMI 1640, Supplier Biochrom AG, 12247 Berlin, Germany, and Supplier PAN-Biotech, 94501 Aidenbach, Germany serving as base for:
Complete Culture Medium RPMI 1640 with 10 % HS
Heat inactivated horse serum 10%
Penicillin/Streptomycin (per mL: 10000 Units Pen/ 10 mg Strep) in H2O 1 %
Sodiumpyruvate (100 mM) 2 %
Viability Medium RPMI 1640 with 15 % HS
Heat inactivated horse serum 15 %
Penicillin/Streptomycin (per mL: 10000 Units Pen/ 10 mg Strep) in H2O 1 %
Sodium pyruvate (100 mM) 2 %
Selection Medium RPMI 1640 with TFT
Heat inactivated horse serum 15 %
Penicillin/Streptomycin (per mL: 10000 Units Pen/ 10 mg Strep) in H2O 1 %
Sodium pyruvate (100 mM) 2 %
Trifluorothymidine 5 µg/mL

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : obtained from a specialized company (Trinova Biochem GmbH, Gießen) and stored at – 80°C ± 5 °C, Batch nos: 3974, 4046
- method of preparation of S9 mix : produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.
- concentration or volume of S9 mix and S9 in the final culture medium :
Phosphate buffer for S9-Mix 10 mL
NADP-solution 1.25 mL
Glucose-6-phosphate solution 0.175 mL
Salt solution for S9-Mix 0.6 mL
S9 1 mL
The S9 mix preparation was performed according to Ames et al. 1975.

Test concentrations with justification for top dose:

Dilutions experiment I:
Test Item 1:3200
Test Item 1:6400
Test Item 1:12800
Test Item 1:25600
Test Item 1:51200
Test Item 1:102400
Test Item 1:204800

Dilutions experiment I_2:
Test Item 1:6400
Test Item 1:7360
Test Item 1:8464
Test Item 1:9734
Test Item 1:11194
Test Item 1:12873
Test Item 1:14804
Dilutions based on cytotoxicity.
According to the OECD guideline 490 the highest concentration should be 0.01 M or 2 mg/mL or 2 µL/mL (whichever is lowest), unless limited by the solubility or toxicity of the test item. RCE values below 20 % are considered toxic. In case of toxic effects, the highest test item concentration of the main experiment should reduce the RSG value to 10 - 20 %.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: actetone

- Justification for choice of solvent/vehicle:
Since the test item Diammono-diisocyanato-Zink is reactive in water what is the main component of cell culture medium, RPMI 1640 was not the adequate solvent. Therefore, the solubility of the test item in DMSO and acetone was determined.
Two non-GLP preliminary tests were performed.
Acetone: 367 mg/mL nominal load Diammono-diisocyanato-Zink solution in acetone were used. The solution was shaken for 3 days, membrane filtrated via 0.2 µm PP filter. The filtrate was diluted 1:200 in culture medium. No turbidity or precipitation was visible.
DMSO: 1000 mg/mL nominal load Diammono-diisocyanato-Zink solution in DMSO were used. The solution was shaken for 3 days, membrane filtrated via 0.2 µm PTFE filter. Turbidity and precipitation were visible in the filtrated solution of the test item in DMSO in culture medium (1:200)
Based on these results, acetone was used as solvent, as it turned out to be most suitable.

- Justification for percentage of solvent in the final culture medium:
The solvent control acetone was used in a final concentration of 1 % during treatment.
Acetone is used as solvent control for the test item in a final concentration of 1 % during treatment. Since no historical control data are available for acetone as solvent, an un-treated control was carried along to demonstrate, that no deleterious or mutagenic effects are induced by the chosen solvent acetone. No effects were noted.

Untreated negative controls:

yes

Remarks:

negative control for the solvent

Negative solvent / vehicle controls:

yes

Remarks:

Acetone is used as solvent control for the test item in a final concentration of 1 % during treatment. 0.9 % NaCl was used as solvent control for the positive control CPA in a final concentra-tion of 0.5 % during treatment.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
Cytotoxicity pre-test: duplicate
Main experiment: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10exp7 cells suspended in 10 mL RPMI 1640 medium
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: n/a
- Exposure duration/duration of treatment: 4h
- Harvest time after the end of treatment (sampling/recovery times): see below

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): After 4 h, the test item was removed by centrifugation (600 * g, 5 min) and the cells were washed twice with the buffer solution saline G. Subsequently, the whole cell pellet was cultured in 30 mL complete culture medium containing 10 % horse serum and incubated at 37.0° +/- 1.5 °C in 5.0 ± 0.5 % CO2 for an expression and growth period of in total 48 h. During this expression and growth period, the cell density was determined after 22 h and after 48 h. After 22 h the cell density was additionally adjusted to 3*105 cells/mL to ensure stable culture conditions. After the expression period, the cultures were selected.
- Selection time (if incubation with a selective agent): Cells from each experimental group were seeded into two microtiter plates so that each well contained 4*10exp3 ± 100 cells in selective medium containing TFT. The viability was determined by seeding about 2 cells per well into 2 microtiter plates (using complete culture medium with 15 % horse serum). The plates were incubated at 37.0 +/- 1.5 °C in 5.0 ± 0.5 % CO2 for 11 d before they were evaluated.
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: For experiment I the cytotoxicity is given as the relative total growth (RTG). A reduction of the RTG below 20 % is defined as a cytotoxic effect.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Experiment I
The exposure date of experiment I was 29. Oct. 2019. 107 cells suspended in 10 mL RPMI 1640 medium with 10 % HS were exposed to the chosen concentrations of the test item in the presence and in the absence of metabolic activation each. Positive and solvent con-trols were performed in parallel. After 4 h, the test item was removed by centrifugation (600 * g, 5 min) and the cells were washed twice with the buffer solution saline G. Subse-quently, the whole cell pellet was cultured in 30 mL complete culture medium containing 10 % horse serum and incubated at 37.0°+/- 1.5 °C in 5.0 ± 0.5 % CO2 for an expression and growth period of in total 48 h. During this expression and growth period, the cell density was determined after 22 h and after 48 h. After 22 h the cell density was additionally adjusted to 3*105 cells/mL to ensure stable culture conditions. After the expression period, the cultures were selected.
Cells from each experimental group were seeded into two microtiter plates so that each well contained 4*103 ± 100 cells in selective medium containing TFT. The viability was determined by seeding about 2 cells per well into 2 microtiter plates (using complete culture medium with 15 % horse serum). The plates were incubated at 37.0 +/- 1.5 °C in 5.0 ± 0.5 % CO2 for 11 d before they were evaluated. For experiment I the cytotoxicity is given as the relative total growth (RTG). A reduction of the RTG below 20 % is defined as a cytotoxic effect.

Experiment I_2
None of the tested test item concentrations showed an RTG value between 10 % - 20 % in experiment I. Therefore, a further experiment I_2 was performed in order to find out the highest possible concentration by adjusting the test item concentrations. The exposure date of experiment I_2 was 19. Nov. 2019. It was performed in the same manner as experiment I (only one difference: cell density was adjusted to 3*10exp5 cells/mL to ensure stable culture conditions after 23 h instead of 22 h). Evaluation was performed after 11 d incubation at 37.0°+/- 1.5 °C in 5.0 ± 0.5 % CO2.

Rationale for test conditions:

As stipulated by the guideline

Evaluation criteria:

Evaluation

Cell Numbers for Determination of RSG
Cell numbers in suspension were determined with a cell counter. Afterwards, the total suspension growth (TSG) and the relative suspension growth (RSG) were calculated.

Viability
Number of empty wells per microwell plate was counted manually. All generated data of each experiment (test item, solvent controls and positive control) were recorded in the raw data. Afterwards the relative cloning efficiency (RCE) and the relative total growth (RTG) were calculated.

Mutant Colonies
Colonies were counted manually under a binocular magnifying glass. In accordance with their size, the colonies were classified into two groups:
Less than 25 % of the well’s diameter = small colony
More than 25 % of the well’s diameter = large colony
All generated data of each experiment (test item, solvent controls and positive control) were recorded in the raw data. Afterwards the mutant frequency (MF) was calculated.

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

Experiment I_2

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

but above 20%, 24% resp. 32%

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH / Data on osmolality: None of the tested positive controls or test item concentrations provoked a critical change of the osmolality and the pH value compared to the solvent controls. Therefore, a negative influence of these parameters on the assay can be excluded.
- Possibility of evaporation from medium: none noted
- Water solubility: not exceeded
- Precipitation and time of the determination: none noted
- Other confounding effects: none stated

RANGE-FINDING/SCREENING STUDIES (if applicable):
Relevant cytotoxic effects were determined at all dilutions, only the highest dilution 1:6400 showed a mean RCE value of 27 % following 4 h treatment without metabolic activation and of 20 % following 4 h treatment with metabolic activation.
No precipitation was visible at the tested dilutions.
The mean RCE values of the positive controls were reduced to 59 % (CPA) and 60 % (MMS).
The pre-test was valid and could be used for the determination of the concentrations of the main experiment.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
valid, see details below

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : given

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency
see tables below

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures
o Number of cells plated in selective and non-selective medium
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency
o When using the thymidine kinase gene on L5178Y cells: colony sizing for the negative and positive controls and if the test chemical is positive, and related mutant frequency. For the MLA, the GEF evaluation.
see tables below / text above

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:
see tables below

Conclusions:

The study was conducted under GLP according to OECD guideline 490 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive (besides a minor deviation in the CPA positive control of experiment I, +S9) and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of the test item to induce gene mutations in mouse lymphoma cells.
A substantial dose dependent increase in mutant colony numbers was observed in experiment I_2 in the two highest dilutions in +S9 and -S9 (RTG > 20%). The mutation frequency exceeded the threshold of 126 above the corresponding solvent control.
In conclusion, it can be stated that under the experimental conditions reported the test item did induce gene mutations at the thymidine kinase locus (Tk1) in heterozygous mouse lymphoma L5178Y Tk+/- cells. Therefore, the test item Diammono-diisocyanato-Zink is considered to be “mutagenic under the conditions of the mouse lymphoma assay”.

Executive summary:

In a mammalian cell gene mutation assay (TK+/-) (OECD 490) under GLP, L5178Y Tk+/- cells cultured in vitro were exposed to Diammono-diisocyanato-Zink over 4h at dilutions ranging from 1:3200 to 1:204800 in a first experiment and 1:6400 to 1:14008 in the repetition in the presence and absence of mammalian metabolic activation (Aroclor 1254 induced rat liver S9). Top dose and dilutions were based on cytotoxicity.

The assay was performed in a pre-test and two independent experiments (experiment I and experiment I_2). The pre-test was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the two experiments were determined.

Both experiments were performed with (liver S9 mix from male rats, treated with Aroclor 1254) and without metabolic activation and a treatment period of 4 h. The highest nominal concentration applied was chosen with regard to the solubility of the test item in organic solvents and aqueous media and the cytotoxicity. Precipitation or turbidity of the test item was not visible in all experimental parts at the maximum concentration of the test item.

Methyl methanesulfonate (MMS) and Cyclophosphamide (CPA) as appropriate reference mutagens were used as positive controls. Both induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system.

A substantial dose dependent increase in mutant colony numbers was observed in the main experiment I_2. The mutation frequency exceeded the threshold of 126 above the corresponding solvent control.

Conclusion:In conclusion, it can be stated that under the experimental conditions reported the test item induced gene mutations at the thymidine kinase locus (Tk1) in heterozygous mouse lymphoma L5178YTk+/-cells. Therefore, the test item Diammono-diisocyanato-Zink is considered to be “mutagenic under the conditions of the mouse lymphoma assay”.

Genetic toxicity in vivo

Description of key information

In vivo: mammalian somatic cell study: erythrocyte micronucleus test: CD-1 mice, oral:gavage, 344 mg/kg bw, sampling times: 24, 48 and 72 hours, OECD 474, EU B.12: negative for micronuclei induction, 4 mice died and clinical signs of toxicity were observed in all animals

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987-11-17 to 1988-02-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1982

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Official Journal of the European Community No. L251 (19/9/1984).
B: Methods for determination of toxicity. B12: Other effects, Mutagenicity, Micronucleus test, pp.137 - 139.

Deviations:

no

GLP compliance:

yes

Remarks:

self-declaration

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River U.K. Limited, Margate, Kent, England on 13 November 1987 (preliminary toxicity test phase I), 20 November 1987 (preliminary toxicity test phase II), 27 November 1987 (preliminary toxicity test phase III), and from Charles River Inc., Portage, Michigan, USA on 4 December 1987 (main test)
- Age at study initiation: approx. 35 days old
- Weight at study initiation: 22-24 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes, overnight depravation of food prior dosing, food was withheld for two hours after dosing.
- Housing: each group of 2 or 5 mice was kept in a plastic disposable cage
- Diet (e.g. ad libitum): pelleted Labsure LAD 1 rodent breeding diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: approx. 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C
- Air changes (per hr): 30
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil (obtained from Brightstone Ltd.)
- Concentration of test material in vehicle:
- Amount of vehicle (intragastric gavage): The test compound and negative control were dosed at a dose volume of 10 ml/kg bodyweight. Mitomycin C, the positive control compound, was dosed at 20 ml/kg bodyweight.:

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

Duration of treatment / exposure:

animals were sacrificed 24, 48 and 72 h after dosing

Frequency of treatment:

single dosing

Post exposure period:

24 h, 48 h respective 72 h

Dose / conc.:

86 mg/kg bw (total dose)

Dose / conc.:

172 mg/kg bw (total dose)

Dose / conc.:

344 mg/kg bw (total dose)

No. of animals per sex per dose:

15 per sex per vehicle control
15+2* per sex per 86 mg/kg bw dose
15+3* per sex per 172 mg/kg bw dose
15+5* per sex per 344 mg/kg bw dose
5 per sex per 12 mg/kg positive control
* spare animals, dosed concurrently, to replace any that might die

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C
- Route of administration: gavage, solution in sterile 0.9 % w/v saline
- Doses / concentrations: 0.6 mg/ml, dosed at 20 ml/kg bw

Tissues and cell types examined:

femurs, epiphysis removed from each bone.

Details of tissue and slide preparation:

Five males and five females from the negative control and test compound groups were killed 24, 48 and 72 hours after dosing. The positive control group was sacrificed 24 hours after dosing. The animals were killed by cervical dislocation and both femurs dissected out from each animal. The femurs were cleared of tissue and one epiphysis removed from each bone. A direct bone marrow smear was made onto a slide containing a drop of calf serum. One smear was made from each femur. The prepared smears were air-dried and fixed in methanol (>10 minutes). The smears were then air-dried and stained for 10 minutes in 10% v/v Giemsa (prepared by a 1:9 dilution of standard Gurr's R66 Giemsa (BDH) with distilled water). Following rinsing in distilled water and differentiation in buffered distilled water (pH 6.8) for 10 minutes, the slides were air-dried and mounted with coverslips using DPX(5).
Only the smears prepared from the highest dosage group were analysed for the incidence of micronucleated cells. Smears from the lower two dose groups were prepared as a contingency, to be analysed if a positive response was found at the highest dose.
The stained smears were examined (under code) by light microscopy to determine:
(i) the incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal
(ii) the ratio of polychromatic to normochromatic erythrocytes for each animal by examination of at least 1000 erythrocytes
(iii) the number of micronucleated normochromatic erythrocytes.

Evaluation criteria:

A positive response would be indicated by a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes compared with the vehicle control. Similarly a statistically significant decrease in the ratio of polychromatoc to norchromatic erythrocytes would be indicative of bone marrow cell toxicity.

Statistics:

Wilcoxon's sum of ranks test (one-sided)

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Phase I: 250-8000 mg/kg, Phase II: 500-2048 mg/kg, Phase III: 100-800 mg/kg
- Solubility: 200 mg/ml in corn oil
- Clinical signs of toxicity in test animals: mortality, pilo-erection, hunched posture, wadling, lethargie, decreased respiration, ptosis, pallor of extremities, increased lacrimation, ataxia, paralysis of hind limbs, body tremor, coma, total paralysis
- Rationale for exposure: This part of the study was carried out in three phases. The dosages used in Phase I were used to give an approximate indication of the LD10. The dosages in Phase II were based on the outcome of Phase I and were used to confirm the results obtained. However the results for Phase II did not confirm the results for Phase I. Therefore a Phase III was carried out to clarify the estimation of the LD10.

RESULTS OF DEFINITIVE STUDY:
- Induction of micronuclei (for Micronucleus assay):
Micronucleated polychromatic erythrocyte counts (PCE)
A dose of 344 mg/kg bw the test item did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes at any of the three kill times -(P>0.05) using Wilcoxon's sum of ranks test. Smears from the animals treated with the lower 2 doses were not analysed for micronucleated polychromatic erythrocytes. Mitomycin C caused large, highly significant increases (P<0.001) in the frequency of micronucleated polychromatic erythrocytes.
Micronucleated normochromatic erythrocytes (NCE)
The test item (344 mg/kg bwt) did not cause any meaningful increases in the incidence of micronucleated normochromatic erythrocytes at any of the three kill times.
- Ratio of PCE/NCE (for Micronucleus assay):
The test item at a dose level of 344 mg/kg bw did not cause any significant decreases in the ratio of polychromatic to normochromatic erythrocytes (P>0.05) using Wilcoxon's sum of ranks test. Therefore there was no evidence of toxicity to the bone marrow cells. Mitomycin C, the positive control, caused statistically significant decreases in the ratio (P<0.001).
- Appropriateness of dose levels and route:

Conclusions:

It is concluded that the test item, in corn oil, showed no evidence of mutagenic potential, or of bone marrow cell toxicity, when administered orally in this in vivo mouse micronucleus test.

Executive summary:

The purpose of this study was to assess the potential of the test item to induce micronuclei in the bone marrow cells of mice following acute oral administration. The study was performed under self-reported GLP and based on OECD guideline No. 474 (1982) and the EEC Annex V Committee Guideline B12 from 1984.

Method:

Based on the results of range finding tests, 344 mg/kg bw test item in corn oil was administered orally to 20 male and 20 female mice, by intragastric gavage. This dose level was expected to kill approximately 10% of the animals within 72 hours of dosing. Negative and positive control groups were also dosed by intragastric gavage with corn oil and mitomycin C (12 mg/kg), respectively.

Bone marrow smears were obtained from groups of 5 males and 5 females from the negative control and test compound groups at 3 sampling times: 24, 48 and 72 hours after dosing. Smears were taken from a similar sized group of positive control animals 24 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 1000 PCE. The ratio of PCE to NCE was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated NCEs was also kept.

Results

Four mice died and clinical signs of toxicity were observed in all animals after treatment with the test item.

None of the mice treated with the test item showed a significant increase in the frequency of micronucleated PCEs at any of the sampling times.

There was no significant decrease in the ratio of PCE/NCE after treatment with the test item i.e. no evidence of toxicity to the bone marrow cells.

The positive control compound, mitomycin C, produced large, highly significant increases in the frequency of micronucleated PCE together with decreases in the ratio of PCE/NCE.

Conclusion

It is concluded that the test item, in corn oil showed no evidence of mutagenic potential or bone marrow cell toxicity, when administered orally to mice at 344 mg/kg in this in vivo test procedure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the currently available data, no clear conclusion can be drawn for the necessity for classification. Given data indicates rather not the necessity for classification:

With regard to chromosome mutations, there are both an in vitro chromosome aberration test and an in vivo micronucleus test available. in vitro, positive results were only seen without metabolic activation, which are not relevant in in vivo situations. This conclusion can be proved by the negative in vivo micronucleus test, which did not reveal any positive results, even when tested up to the MTD and beyond. Hence, chromosome mutations can be neglected as a possible event indicating the necessity for classification as (germ cell) mutagen.

With regard to gene mutations, there are both a negative (±S9) Ames test with S. typhimurium TA 1535, TA 1537, TA 98, TA 100, and TA 1538 and an OECD 490 study in mouse lymphoma L5178Y cells available, the latter being positive ±S9, however in a cytotoxic range but above 20%, 24% resp. 32%. REACH foresees in this case to conduct further studies if deemed necessary by the registrant. So, a testing proposal was submitted in this dossier for an in vivo Comet Assay. Although not detecting gene mutations directly, it is an indicator for DNA damage and repair, and so detects previous events possibly leading to gene mutations. It is scientifically well accepted as a surrogate.

Data above from chromosome mutation tests indicate a minor relevance of genotoxic effects in vivo compared to in vitro data. This may be due to the fact that the substance hydrolyses rather fast in insoluble products and soluble products (see chapter “hydrolysis”), which are urea and most probably ammonia and carbon dioxide, all not known as genotoxins.

In consequence, it is aimed to prove via a Comet Assay, that the positive result in the OECD 490 study has no relevance in vivo, and so, no classification is needed so far.