Diamminediisocyanatozinc

Genetic toxicity: in vivo

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1982

Deviations:

no

Test guideline 2

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

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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

86 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

172 mg/kg bw (total dose)

Doses / concentrations 3

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

Examinations

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)

Results and discussion

Test results

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:

Applicant's summary and conclusion

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.