Reaction products of cyclohexylamine and 4-alkylaniline and 1,1'-methanediylbis(4-isocyanatobenzene)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

One Ames study in 1991 shows negative to S. typhimurium (TA1535, TA1537, TA98 and TA100) and one Ames study in 2009 shows negative to E. coli (WP2uvrA) strains.

One in vitro mammalian chromosome aberration test in 1996 resulted that the test substance did not show any evidence of clastogenic activity incultured human lymphocytes.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2008-12-22 to 2009-01-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

EC Commission Directive 2000/32/ECAnnex 4D

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Appearance: White powder
Storage conditions: Refrigerated in the dark
Batch number: 2008-9-3
Expiry date: 30 September 2009
Purity: >95.0%

Target gene:

Trypophan dependancy

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

Test 1: 5, 15, 50, 150, 500, 1500, 5000 µg per plate
Test 2: 50, 150, 500, 1500, 5000 µg per plate

Vehicle / solvent:

It has been shown in a previous study conducted in this laboratory that test item is insufficiently soluble in solvents compatible with the test system (Life Sciences Research report no. 91/NNG003/0816). Suspensions of test item in water (purified in house by reverse osmosis) containing 0.15% bacteriological agar were, therefore, used in this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

aqueous 0.15% agr solution

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

First test
Aliquots of 0.1 mL of the test substance suspensions (seven concentrations up to 5000 μg/plate), positive control or negative control were placed in glass vessels. The negative control was the chosen vehicle, aqueous 0.15% agar solution. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labeled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).


Second test
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5000 μg/plate, but only five concentrations were used.

Positive controls:
In the absence of S9 mix
Identity: 4-Nitroquinoline-1-oxide
CAS No.: 56-57-5
Solvent: DMSO (A.C.S. spectrophotometric grade)
Concentration: 2 μg/plate

In the presence of S9 mix
Identity: 2-Aminoanthracene
CAS No.: 613-13-8
Solvent: DMSO (A.C.S. spectrophotometric grade)
Concentration: 10 μg/plate

Evaluation criteria:

For a test to be considered valid, the mean of the vehicle control revertant colony numbers should lie within or close to the 99% confidence limits of the current historical control range of the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 1E+09/mL.

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results obtained with test item and positive control compounds are presented in Table 1 (first test) and in Table 2 (second test).
The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test substance formulation.
The total colony counts on nutrient agar plates (see Table 3) confirmed the viability and high cell density of the cultures of the individual organisms.
The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory (Appendix 1). Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

First test
No evidence of toxicity was obtained following exposure to test item. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test.
No substantial increases in revertant colony numbers over control counts were obtained following exposure to test item at any concentration up to 5000 μg/plate in either the presence or absence of S9 mix.

Second test
No evidence of toxicity was obtained following exposure to test item.
No substantial increases in revertant colony numbers over control counts were obtained following exposure to test item at any concentration up to 5000 μg/plate in either the presence or absence of S9 mix.

Conclusions:

The test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

In this in vitro assessment of the mutagenic potential of test item in complied with OECD 471, a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), was exposed to test item suspended in aqueous 0.15% agar solution, which was also used as a negative control. Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of test item up to 5000 μg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration. No signs of toxicity were observed towards the tester strain in either mutation test following exposure to test item. No evidence of mutagenic activity was seen at any concentration of test item in either mutation test. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory. It is concluded that test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 1991-07-16 to 1991-08-07

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study in accordance with recognised test guideline but: Ecoli not tested Test substance Lot/batch No.: not stated Test substance expiration date of the lot/batch: not stated NOTE: study deemed acceptable because spectral data for A-3622 are available, covering approximately before and after the test period - see section 1.4 Analytical Information.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

First issued 1985, amended 1987

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Physic state: white powder
Purity: approximately 100%
Storage conditions: at room temperature in the dark

Target gene:

Histdine dependency

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Cultures of the histidine-dependent strains of Salmonella typhimurium were derived from cultures provided by Prof. Bruce Ames, University of California. The characteristics of the individual strains are as follows:
TA 1535 - contains a histidine missense mutation but is also deficient in a DNA repair system (uvr B) and has a defective lipopolysaccharide coat on the cell wall. It is reverted by many agents causing base-pair substitutions, but is not sensitive to frameshift mutagens.

TA 100 - is the same as TA 1535 but contains a resistance transfer factor conferring ampicillin resistance and increasing sensitivity to some mutagens (plasmid pKM 101). In addition to base-pair substitutions, it is also able to detect certain frameshift mutagens.

TA 1537 - bears a histidine frameshift mutation. Like TA 1535, it is defective in a DNA repair system and lipopolysaccharide coat. It is sensitive to agents causing frameshift mutations involving insertion or deletion of a single base-pair.

TA 98 - contains another histidine frameshift mutation. Again it has a defective DNA repair system and lipopolysaccharide coat but also contains the pKM 101 plasmid. It is reverted by agents causing deletion of two adjacent base-pairs (double frameshift mutations), but not by simple alkylating agents causing base-pair substitutions.

Cultures of all organisms were prepared by overnight incubation of nutrient broth (Oxoid No.2) freshly inoculated from a frozen culture stock.

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

NADPH-generating system and hepatic microsomes from rats that had been pre-treated with Aroclor 1254

Test concentrations with justification for top dose:

50, 158, 500, 1580, 5000 µg per plate
No visible thinning of the background lawn of non-revertant cells was obtained following exposure to test item in the Preliminary toxicity test. A top exposure level of 5 mg per plate was therefore selected for use in the main tests.

Vehicle / solvent:

The test item was found to be insufficiently soluble in solvents compatible with the test system (water, ethanol, dimethyl sulphoxide and acetone). Suspensions of test item in purified water (obtained by reverse osmosis) containing 0.15% agar, freshly prepared immediately before use with the aid of an Ultra-Turrax high-shear mixer, were therefore employed in this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Purified water containing 0.15% agar

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

PRELIMINARY TOXICITY TEST:
A suspension of test item was prepared at 25 mg/mL in purified water containing 0.15% agar, and an aliquot of this suspension (0.2 mL) was transferred to a sterile tube containing molten, histidine-deficient top-agar (2.0 mL) maintained at 45°C. An additional aliquot (0.1 mL) of the test material suspension was similarly transferred to another tube of molten top-agar (2.0 mL). Three serial ten-fold dilutions in molten top-agar were prepared from each of the above preparations, giving a series of eight different concentrations of test material from 2.5 µg to 5 mg per plate. All tubes were inoculated with an overnight culture of strain TA 98 (0.1 mL) and overlaid onto minimal medium plates. Control plates were prepared containing top-agar and culture alone, top-agar, 0.15% agar (0.2 mL) and culture, and top-agar and test material (0.1 mL) without bacterial culture.

The plates were incubated at 37°C for 2 days and were then examined for the presence of a background lawn of non-revertant colonies; toxicity of the test material is shown by absence or thinning of the background lawn. The level of test material chosen as the top level for pour-plate tests is normally the lowest level causing visible thinning of the lawn. (In the absence of such thinning, a top level of 5 mg/plate was selected).

The control plates were checked for the absence of growth on sterility checks or normal counts on negative controls in the presence and absence of the vehicle.

POUR-PLATE ASSAY FOR MUTAGENESIS:
A suspension of test item was prepared at 50 mg/mL in purified water containing 0.15% agar, and four half-log dilutions were prepared from this suspension. An aliquot (0.1 mL) of each concentration of test item was placed in a sterile tube. Molten, histidine-deficient top-agar (2 mL) and bacterial suspension (0.1 mL), maintained at 45°C, were then added. The tubes were mixed by inversion and 0.5 mL rat liver microsomal preparation (S-9 mix) was added where appropriate. The tubes were again inverted to mix thoroughly and the contents poured onto plates containing solidified minimal medium (20 mL).

Further plates were prepared without the inclusion of the test organisms to verify the sterility of the S-9 mix and the test material. A control series of plates was prepared to confirm the inability of purified water containing 0.15% agar (0.1 mL) to induce reversion in the bacterial strains, and to provide a measure of the spontaneous mutation rates.

Aliquots (0.1 mL) of a 1E-6 dilution of culture were spread on the surface of plates of complete medium to measure the viability and cell-density of each culture.

All plates were prepared in triplicate, allowed to solidify and incubated at 37°C for 2 days. After incubation, numbers of revertant colonies were counted, either manually or with a Biotran II automatic colony counter. Total colonies on nutrient plates were counted in the same way. Growth of the background lawn of non-revertant cells on minimal plates was verified.

Each test, in each strain, was conducted on two separate occasions.

All plates and tubes were identified by the use of numbers indelibly marked on the plates and test tube racks.

POSITIVE CONTROLS:
Appropriate positive control plates were included for each strain. All positive control compounds were prepared as solutions in DMSO, except sodium azide, which was dissolved in purified water.

Evaluation criteria:

Increase in revertant colony numbers over control counts

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Main test:
The results obtained with test item and positive control compounds are presented in Tables 1 to 4 in attached background information. Note: the mean values quoted have been corrected to the nearest whole number.

Sterility checks, spontaneous reversion rate and viability checks:
The absence of colonies on test item and S-9 mix sterility check plates indicates that these preparations were free of microbial contamination. The total colony counts on plates number 18 confirmed the viability and high cell density of the cultures of the individual organisms. The counts recorded on appropriate negative control plates confirmed the characteristically low spontaneous reversion rates of the tester strains and the absence of effects of inclusion of purified water containing 0.15% agar on these rates.

Mutagenic activity of positive control chemicals:
Appropriate positive control chemicals (with S-9 mix where required) induced marked increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S-9 mix.
Action of test item
No increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test item at levels from 50 to 5000 µg per plate.

Preliminary toxicity test results:

Test material (µg per plate)	TA 98
	Background lawn			Revertant colonies
	Plate A	Plate B	Plate C	Plate A	Plate B	Plate C
2500 (sterility check)	A	A	A	0	0	0
5000 500 50 5	P P P P	P P P P	P P P P	10 16 14 20	16 22 19 28	24 15 14 23
2500 250 25 2.5	P P P P	P P P P	P P P P	16 19 24 25	16 17 19 13	14 23 26 17
0  0 (0.2ml vehicle)	P P	P P	P P	31 19	24 21	22 23

 

A  Absent

P   Present and normal

Conclusions:

The test material was devoid of mutagenic activity under the conditions of the test.

Executive summary:

The test item was examined for mutagenic activity in four histidine-dependent auxotrophs of Salmonella typhimurium, strains TA 98, TA 100, TA 1535 and TA 1537, using pour-plate assays, according to OECD 471. Each test, in each strain, was conducted on two separate occasions. The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a range of levels of test item (tested as a suspension) from 50 to 5000 µg per plate, selected following a preliminary toxicity test in strain TA 98. All tests included vehicle (0.15% agar in aqueous solution) controls with and without S-9 mix. No increases in reversion to prototrophy were obtained with any of the four bacterial strains at the test item levels tested, either in the presence or absence of S-9 mix. Marked increases in the number of revertant colonies were induced by the known mutagens when examined under similar conditions. It was concluded that test item was devoid of mutagenic activity under the conditions of the test.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 1996-01-08 to 1996-02-20

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study in accordance with recognised test guideline but: Lot/batch No.: not stated Expiration date of the lot/batch: not stated NOTE: study deemed acceptable because spectral data for test item are available, covering before and after the test period - see section 1.4 Analytical Information.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test) (migrated information)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Substance type: Organic
- Physical state: fine cream cohesive powder
- Storage condition of test material: ambient temperature in the dark

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: cultured human lymphocytes

Details on mammalian cell type (if applicable):

Human peripheral blood was obtained by venepuncture from a healthy, non-smoking,
male, human volunteer not currently taking any medication, and collected in heparinised
vessels.

The doubling time for lymphocytes from the donor was assessed in June 1995 and found
to be 13 hours.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Refer to attached background information Appendix 1 Preparation of culture medium, post-mitochondrial fraction and S-9 liver enzyme mix

Test concentrations with justification for top dose:

Preliminary toxicity test: 8, 40, 200, 1000, 5000 µg/mL
First and second cytogenetic test: 250, 500, 1000, 2000 µg/mL

Vehicle / solvent:

The test material was found to be relatively insoluble in purified water, dimethyl sulphoxide and acetone, but to form a fine and apparently homogeneous suspension in culture medium at concentrations up to 6.25 mg/mL. Accordingly, suspensions of the test material for use in testing were prepared in culture medium immediately before addition to test cultures. Suspensions prepared to give final treatment concentrations of 200 µg/mL and above were prepared individually, suspensions prepared to give final concentrations of 8 and 40 µg/mL were prepared by serial dilution in culture medium. All concentrations cited in this report are stated in terms of the material as supplied; no correction has been made for purity or activity below 100%.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Chlorambucil

Details on test system and experimental conditions:

Culture establishment and treatment:
Small inocula of whole blood (0.5 mL) were added to tubes containing culture medium (9.0 mL) and phytohaemagglutinin solution (0.5 mL) to stimulate lymphocytes to divide. The tubes were sealed and incubated at 37°C with occasional shaking to prevent clumping.
After approximately 48 hours of incubation, the cultures were centrifuged, the supernatant was removed and the cell pellet was resuspended in culture medium. Freshly prepared S-9 mix (1.0 mL) was then added to the appropriate cultures and an aliquot of test material suspension or vehicle (8.0 mL) or positive control solution (50 µg) was added to the relevant cultures. The final volume in each culture was 10 mL. Two cultures were treated at each test point in the preliminary toxicity and main cytogenetic tests.
All cultures were incubated at 37°C, in a shaking water bath, for three hours. After this initial exposure period, the cultures treated in the absence of S-9 mix were transferred to a 37°C incubator for the remainder of the scheduled exposure period. Cultures treated in the presence of S-9 mix were centrifuged and the cells washed twice with Hanks' Balanced Salt Solution (5 mL) to remove the test material and S-9 mix. The cells were then resuspended in culture medium (9.5 mL), and the cultures incubated at 37°C, under static conditions, until scheduled harvesting.
All culture vessels were indelibly marked with unique identification using colour coding and numbers.

Culture harvesting:
Three hours before harvesting, cell division was arrested by the addition of the spindle poison, Colcemid, to each culture (to a final concentration of 0.4 µg/mL). The cultures were incubated for a further three hours. The cells were then harvested by low speed centrifugation (1400 r.p.m. for 5 minutes) and the pellets of cells were resuspended in hypotonic potassium chloride solution (0.56%) for ten minutes, centrifuged again and then fixed in freshly prepared methanol: glacial acetic acid fixative (3:1 v/v).

Slide preparation:
After two further changes of fixative, the tubes were centrifuged, the supernatant removed and the cell pellet resuspended in a few drops of fresh fixative. Single drops of the cell suspension were transferred to clean, moist, grease-free glass slides, and the slides were left to air-dry. Two or four slides (for the preliminary toxicity test or main cytogenetic tests respectively) were made from each culture, stained for ten minutes in Giemsa stain (1 in 10 in Sorensen's buffer, pH 6.8), washed in buffer and left to air-dry. The slides were cleared in xylene and coverslips were applied using DPX mountant.

Evaluation criteria:

The test material is considered to be clastogenic in this test if the following conditions are met:
- statistically significant increases in the frequency of metaphases with aberrant chromosomes (excluding gap-type aberrations) are observed at one or more test concentrations
- the increases exceed the historical negative control range at this laboratory
- the increases are reproducible between replicate cultures and between tests
- the increases are not associated with large changes in pH or osmolarity of the treatment medium or extreme toxicity (which can cause non-specific effects)
- evidence of a dose-response relationship, or increases at both sampling times will be considered to support the conclusion.

The biological significance of gap-type aberrations is questionable and increases observed only when gaps are included in the analysis will not be considered to be conclusive evidence of clastogenic activity.

Statistics:

The Fisher Exact Probability test is a useful technique for analysing data when comparing two independent samples. It is used when the observed events all fall into one or other of two mutually exclusive classes. The test determines whether the two groups differ in the proportions with which they fall into the two classifications.
The frequency of aberrant metaphases for each treatment group was compared with the corresponding vehicle control group value using a one-tailed test.

Species / strain:

lymphocytes: cultured human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Preliminary toxicity test:
The concentrations of test item tested were 8, 40, 200, 1000 and 5000 µg/mL. Slides were prepared and stained from all cultures. A precipitate was observed on the slides from cultures treated at the higher concentrations of the test material; on slides from cultures treated at 5000 µg/mL the precipitate obscured the cells and metaphases and the mitotic index could not be determined.

- First cytogenetic test:
On the basis of the results of the preliminary toxicity test, concentrations of test item in the range 250 - 2000 µg/mL were selected for use in the first cytogenetic test. A precipitate was observed on all slides from cultures treated with test item. The highest concentration tested (2000 µg/mL) was the highest practicable concentration; although some metaphases were obscured by the precipitate it was possible to find 100 scoreable metaphases.

Mitotic index - initially, mitotic indices were scored from all cultures.
On the basis of these results, slides from cultures treated at the following concentrations of test item were selected for the scoring of chromosomal aberrations (in the presence and absence of S-9 mix):
- 19 hour sampling time: 500, 1000 and 2000 µg/mL
- 43 hour sampling time: 2000 µg/mL.
The highest concentration of test item scored for chromosomal aberrations (2000 µg/mL) was the highest practicable concentration.

Incidence of chromosomal aberrations: comparison of treated and control cultures - one hundred metaphases were scored from each culture.
Treatment with test item did not produce biologically or statistically significant increases in the frequency of metaphases with aberrant chromosomes at any concentration tested, compared to vehicle control values (p>0.05 both including and excluding gap-type aberrations), at either sampling time, either in the presence or absence of S-9 mix. The numbers of cells with polyploidy or endoreduplication were within the normal range.

- Second cytogenetic test:
On the basis of the results of the first cytogenetic test, concentrations of test item in the range 250 - 2000 µg/mL were selected for use in the second cytogenetic test, for the 19 hour sampling time only.
Mitotic index - initially, mitotic indices were scored from all cultures.
On the basis of these results, slides from cultures treated at the following concentrations were selected for the scoring of chromosomal aberrations (in the presence and absence of S-9 mix):
- 19 hour sampling time: 500, 1000 and 2000 µg/mL.
The highest concentration of test item scored for chromosomal aberrations (2000 µg/mL) was the highest practicable concentration.
Incidence of chromosomal aberrations: comparison of treated and control cultures - one hundred metaphases were scored from each culture.
Treatment with test item did not produce biologically or statistically significant increases in the frequency of metaphases with aberrant chromosomes at any concentration tested, compared to vehicle control values (p>0.05 both including and excluding gap-type aberrations), either in the presence or absence of S-9 mix. The numbers of cells with polyploidy or endoreduplication were within the normal range.

- Positive controls:
Treatment with the known clastogens, chlorambucil and cyclophosphamide, produced marked increases in the frequency of aberrant metaphases which were biologically and statistically significant at both sampling times in both main tests (p<0.001). These results demonstrate the efficacy of the S-9 mix employed, and the sensitivity of the test procedure.

Refer to attached background information for results tables as follows:

Table 1 Preliminary toxicity test Mitotic indices

Table 2 First main test Mitotic indices

Table 3 First main test Number of chromosomal aberrations and statistical analysis

Table 4 First main test Numbers of specific types of aberration

Table 5 Second main test Mitiotic indices

Table 6 Second main test Number of chromosomal aberrations and statistical analysis

Table 7 Second main test Numbers of specific types of aberration

Table 8 Summary of main cytogenetics test

Appendix 1 Preparation of culture medium, post-mitochondrial fraction and S-9 liver enzyme mix

Appendix 2 Historical negative control data (compiled July 1995)

Refer to attached full study report for:

Positive control data (not in test report)

Conclusions:

It is concluded that the test item, under the conditions of test, did not show any evidence of clastogenic activity.

Executive summary:

The effects on chromosomal structure of exposure to test item were investigated in cultured human lymphocytes according to OECD 473. Tests were conducted with and without the inclusion of a rat liver-derived metabolic activation system (S-9 mix): without S-9 mix cells were exposed continuously for 19 or 43 hours, with S-9 mix exposure was limited to three hours and cells were harvested 16 or 40 hours later. Treatments were established by the addition of suspensions of the test material (in culture medium) to 48-hour cultures established from whole, human blood. Cell division was arrested by the addition of the spindle poison, Colcemid, three hours before the cells were harvested; slides were then prepared for microscopic analysis. Mitotic indices were calculated for each culture: these were based on the number of metaphases observed per 1000 cells scored. Chromosome aberrations were scored by examination of 100 metaphases per culture, and the frequencies of cells with one or more aberrations were calculated both including and excluding gap-type aberrations. A preliminary test was performed to investigate the toxicity of test item to dividing lymphocytes. Subsequently, the cytogenetic tests were performed using concentrations of test item in the range 250 - 2000 µg/mL. The main tests also incorporated vehicle (culture medium) and positive (chlorambucil and cyclophosphamide) control cultures. Chlorambucil is a direct-acting clastogen; cyclophosphamide requires metabolic activation to achieve optimum activity. Two cultures were prepared at each test point. Slides from cultures treated at the following concentrations were scored for chromosomal aberrations (in the presence and absence of S-9 mix): - 19 hour sampling time, both main tests: 500, 1000 and 2000 µg/mL - 43 hour sampling time, first main test only: 2000 µg/mL. test item did not produce a marked reduction in mean mitotic index at any concentration tested. A precipitate was observed on slides from all cultures treated with the test material. The highest concentration of test item scored for chromosomal aberrations (2000 µg/mL) was the highest practicable concentration; although some metaphases were obscured by the precipitate it was possible to find 100 scoreable metaphases. One hundred metaphases were analysed from all selected cultures. No biologically or statistically significant increases in the frequency of metaphases with aberrant chromosomes, compared to vehicle control values, were seen in cultures treated with test item, including or excluding gap-type aberrations, in either cytogenetic test (p>0.05). The known clastogens, chlorambucil and cyclophosphamide, induced significant increases in the frequency of metaphases with aberrant chromosomes, compared to the solvent control values, at both sampling times in both cytogenetic tests (p<0.001 in all cases), thus demonstrating the sensitivity of the test procedure, and the metabolic activity of the S-9 mix employed. It is concluded that test item, under the conditions of test, did not show any evidence of clastogenic activity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Negative results in two Ames study and one in vitro mammalian chromosome aberration test.

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, the substance should not be classified for this endpoint.