1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 January 2014 to 19 May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese guidelines in accordance with METI, MHLW and MAFF

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine

EC Number:

227-367-1

EC Name:

1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine

Cas Number:

5807-14-7

Molecular formula:

C7H13N3

IUPAC Name:

1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine

Test material form:

solid

Details on test material:

- Appearance: Off-white solid
- Storage Conditions: room temperature in the dark, in a closed container under nitrogen

Method

Species / strain

Species / strain / cell type:

other: Human lymphocytes

Details on mammalian cell type (if applicable):

CELLS
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in the testing laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

CELL CULTURE
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 °C with 5 % CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 43.5, 87, 174, 348, 696 and 1392 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test material was insoluble in culture media

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

EXPERIMENT 1
Two exposure groups were used for Experiment 1:
i) 4-hour exposure to the test material without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest.
ii) 4-hour exposure to the test material with S9-mix (2 %), followed by 20-hour culture in treatment-free media prior to cell harvest.

EXPERIMENT 2
Two exposure groups were used for Experiment 2:
i) 24-hour continuous exposure to the test material without S9-mix prior to cell harvest.
ii) 4-hour exposure to the test material with S9-mix (1 %) followed by 20-hour culture in treatment-free media prior to cell harvest.

CULTURE CONDITIONS
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture: 9.05 - 9.15 mL MEM, 10 % (FBS); 0.1 mL Li-heparin; 0.1 mL phytohaemagglutinin; and 0.65 - 0.75 mL heparinised whole blood.

WITH METABOLIC ACTIVATION (S9) TREATMENT
After approximately 48 hours incubation at approximately 37 °C, 5 % CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test material was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1 mL of 20 % S9-mix (i.e. 2 % final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.
In Experiment 2, 1 mL of 10 % S9-mix (i.e. 1 % final concentration of S9 in standard co-factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL.
After 4 hours at approximately 37 °C, 5 % CO2 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 °C in 5 % CO2 in humidified air.

WITHOUT METABOLIC ACTIVATION (S9) TREATMENT
In Experiment 1, after approximately 48 hours incubation at approximately 37 °C with 5 % CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then re-suspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL of the appropriate vehicle control, test material solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37 °C, 5 % CO2 in humidified air the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.
In Experiment 2 the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test material dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 °C, 5 % CO2 in humidified air for 24 hours.

The preliminary toxicity test was performed using both of the exposure conditions as described for Experiment 1 and for Experiment 2 in the absence of metabolic activation only.

CELL HARVEST
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075 M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 °C to ensure complete fixation prior to slide preparation.

PREPARATION OF METAPHASE SPREADS
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry.

STAINING
When the slides were dry they were stained in 5 % Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

QUALITATIVE SLIDE ASSESSMENT
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test material. These observations were used to select the dose levels for mitotic index evaluation.

MITOTIC INDEX
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

SCORING OF CHROMOSOME DAMAGE
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were at least 30 to 50 % of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44 - 48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985).
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. The incidence of cells with endoreduplicated chromosomes was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

Evaluation criteria:

The following were used to determine a valid assay:
- Negative Control
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures will normally be within the laboratory's historical control data range.
- Positive Control
All the positive control chemicals must induce a clear positive response (p<=0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.

A test material can be classified as non-genotoxic if:
- The number of induced chromosome aberrations in all evaluated dose groups is within the range of the laboratory's historical control data.
- No toxicologically or statistically significant increase of the number of structural chromosome aberrations is observed following statistical analysis.

A test material can be classified as genotoxic if:
- The number of induced structural chromosome aberrations is not in the range of the laboratory's historical control data;
And
- Either a concentration-related or a statistically significant increase of the number of structural chromosome aberrations is observed. Marked increases only observed in one dose level will be assessed on a case by case basis.

Biological relevance of the results will be considered first. Statistical methods will be used to analyse the increases in aberration data as recommended in the OECD guideline. However, statistical significance will not be the only determining factor for a positive response.

A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).

Results and discussion

Test results

Key result

Species / strain:

other: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
A pink colouration of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 348 µg/mL, in the 4(20)-hour exposure groups and at and above 174 µg/mL in the continuous exposure group.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1392 µg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was also 1392 µg/mL. The test material induced some evidence of toxicity in the absence of metabolic activation 24 hour exposure group only. Increases in mitotic index values over the vehicle control in the 4(20)-hour group in the absence of S9 may indicate cell cycle delay induced by test material toxicity.

MAIN TEST - EXPERIMENT 1
The control, 174, 348, 696 and 1392 µg/mL dose levels were selected for metaphase analysis (along with the positive controls) in the assays both with and without S9.
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 1392 µg/mL in both exposure groups.
Observations were made at the end of exposure and a pink colouration was noted at and above 348 µg/mL in both exposure groups.
The mitotic index data are given in Table 1. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 39 % mitotic inhibition was achieved at 1392 µg/mL in the absence of S9 and 17 % mitotic inhibition was achieved at 1392 µg/mL in the presence of S9.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

MAIN TEST - EXPERIMENT 2
The control, 87, 174 and 348 µg/mL dose levels in the absence of metabolic activation and control, 348, 696 and 1392 µg/mL dose levels in the presence of metabolic activation were selected for metaphase analysis (along with positive controls).
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test material dose level of 1392 µg/mL in the absence and presence of S9.
A pink colouration of the test material was observed at the end of exposure, at and above 348 µg/mL in the 4(20)-hour exposure group and at and above 696 µg/mL, in the 24-hour continuous exposure group.
The mitotic index data are given in Table 2. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 43, 72 and 79 % mitotic inhibition was achieved at 348, 696 and 1392 µg/mL, respectively, in the absence of S9. In the presence of S9 a dose-related inhibition of mitotic index was observed and 43 % mitotic inhibition was achieved at 1392 µg/mL.
The maximum dose level selected for metaphase analysis was the same as Experiment 1 in the presence of metabolic activation; however it was 348 µg/mL in the 24-hour continuous exposure group and was based on toxicity. Dose levels above 348 µg/mL in the 24-hour exposure group were considered to exhibit excessive toxicity.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.
The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Any other information on results incl. tables

Table 1: Experiment 1 - Mitotic Index

Dose Level (µg/mL)	4(20)-Hour Without S9				4(20)-Hour With S9
	A	B	Mean	% of Control	A	B	Mean	% of Control
0	8.30	4.50	6.40	100	7.15	6.90	7.03	100
43.5	-	-	-	-	-	-	-	-
87	-	-	-	-	-	-	-	-
174	3.95	4.15	4.05	63	3.50	5.35	4.43	63
348	9.30 P	6.85 P	8.08	126	10.10 P	5.45 P	7.78	111
696	4.10 P	5.35 P	4.73	74	8.70 P	11.95 P	10.33	147
1392	5.85 P	1.95 P	3.90	61	6.30 P	5.40 P	5.85	83
MMC 0.4	2.90	1.55	2.23	35	NA	NA	NA	NA
CP 5	NA	NA	NA	NA	1.60	1.60	1.60	23

MMC = Mitomycin C

CP = Cyclophosphamide

P = Pink colouration observed at end of exposure period

NA = Not applicable

- = Not assessed for mitotic index

 

Table 2: Experiment 2 - Mitotic Index

Dose Level (µg/mL)	24-Hour Without S9				4(20)-Hour With S9
	A	B	Mean	% of Control	A	B	Mean	% of Control
0	4.85	5.85	5.35	100	3.25	3.45	3.35	100
43.5	-	-	-	-	-	-	-	-
87	2.10	4.45	3.28	61	-	-	-	-
174	4.00	2.05	3.03	57	-	-	-	-
348	2.90	3.25	3.08	57	3.90 P	2.55 P	3.23	96
696	1.70 P	1.25 P	1.48	28	3.15 P	2.25 P	2.70	81
1392	0.85 P	1.40 P	1.13	21	1.85 P	2.00 P	1.93	57
MMC 0.4	2.40	1.05	1.75	32	NA	NA	NA	NA
CP 5	NA	NA	NA	NA	0.90	0.85	0.88	26

MMC = Mitomycin C

CP = Cyclophosphamide

P = Pink colouration observed at end of exposure period

NA = Not applicable

- = Not assessed for mitotic index

Table 3: Summary of Chromosome Aberration Data for Experiment 1

Treatment	Dose Level (µg/mL)	Cells Scored	No. of Cells with Aberrations Including Gaps)	No. of Cells with Aberrations Excluding Gaps	Frequency of Aberrant Cells Including Gaps (%)	Frequency of Aberrant Cells Excluding Gaps (%)
Without S9	Vehicle	200	1	1	0.5	0.5
	174	200	1	0	0.5	0.0
	348	200	0	0	0.0	0.0
	696	200	5	3	2.5	1.5
	1392	200	2	1	1.0	0.5
	MMC 0.4	100¹	59	55	39.0	36.0***
With S9 (2 %)	Vehicle	200	2	2	1.0	1.0
	174	200	0	0	0.0	0.0
	348	200	2	2	1.0	1.0
	696	200	2	1	1.0	0.5
	1392	200	5	4	2.0	2.0
	CP 5	100¹	67	57	39.0	36.0***

Results are the mean values from replicates A and B

MMC = Mitomycin C; CP = Cyclophosphamide

¹Slide evaluation was terminated at 50 cells as at least 30 % cells with aberrations had been observed

***Significantly different from control group P<0.001

 

Table 4: Summary of Chromosome Aberration Data for Experiment 2

Treatment	Dose Level (µg/mL)	Cells Scored	No. of Cells with Aberrations Including Gaps)	No. of Cells with Aberrations Excluding Gaps	Frequency of Aberrant Cells Including Gaps (%)	Frequency of Aberrant Cells Excluding Gaps (%)
Without S9	Vehicle	200	4	3	2.0	1.5
	87	200	1	0	0.5	0.0
	174	200	0	0	0.0	0.0
	348	200	3	0	1.5	0.0
	MMC 0.2	100¹	54	41	27.3	22.7***
With S9 (1 %)	Vehicle	200	0	0	0.0	0.0
	348	200	1	1	0.5	0.5
	696	200	2	1	1.0	0.5
	1392	200	0	0	0.0	0.0
	CP 5	100¹	43	36	34.0	30.0***

Results are the mean values from replicates A and B

MMC = Mitomycin C; CP = Cyclophosphamide

¹Slide evaluation was terminated at 50 cells as at least 30 % cells with aberrations had been observed

***Significantly different from control group P<0.001

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Under the conditions of this study, the test material is considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

An in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells was conducted in accordance with the standardised guidelines OECD 473, EU Method B.10, EPA OPPTS 870.5375, 40 CFR 799.9537 TSCA and the Japanese guidelines published by METI, MHLW and MAFF under GLP conditions.

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; in Experiment 1, 4 hours in the presence of metabolic activation (S9) at a 2 % final concentration with cell harvest after a 20-hour expression period, and a 4 hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hour exposure with addition of S9 was repeated (using a 1 % final S9 concentration) whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows: 0, 43.5, 87, 174, 348, 696 and 1392 µg/mL. The vehicle used was DMSO.

All vehicle controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequencies of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50 % mitotic inhibition, in either the absence or presence of a liver enzyme metabolising system.

Under the conditions of this study, the test material is considered to be non-clastogenic to human lymphocytes in vitro.