Nitrapyrin

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 November 1981 to 05 January 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.18 (DNA Damage and Repair - Unscheduled DNA Synthesis - Mammalian Cells In Vitro)

Deviations:

no

GLP compliance:

yes

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Test material

Test material information

Constituent 1

Reference substance name:

Nitrapyrin

EC Number:

217-682-2

EC Name:

Nitrapyrin

Cas Number:

1929-82-4

Molecular formula:

C6H3Cl4N

IUPAC Name:

2-chloro-6-(trichloromethyl)pyridine

Test material form:

not specified

Method

Species / strain

Species / strain / cell type:

hepatocytes: Primary cultures of rat hepatocytes

Details on mammalian cell type (if applicable):

PREPARATION OF PRIMARY RAT HEPATOCYTE CULTURES
Primary cultures of rat hepatocytes were prepared by slight modification of the method described by Williams et al. (1977).
The rat was anaesthetised with methoxyfluorane and the liver exposed by an abdominal incision. Sodium heparin (200 units) was injected into the inferior vena cava and the hepatic portal vein cannulated. The liver was then perfused for approximately 2-3 minutes with 0.5 mM ethylene glycol -bis(ß-amino ethyl ether)N,N’-tetraacetic acid (EGTA) in Ca++, Mg ++ free Hank's balanced salt solution (37 °C) at pH 7.4.
The liver was subsequently perfused with 100 units/mL of collagenase in Williams Medium E (WE; 37 °C) buffered with 0.05 M N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) to pH 7.4. An antibiotic-antimycotic solution was also added to all WE medium in the experiment so that the final concentration of penicillin, streptomycin and fungizone was 100 units, 100 µg and 0.25 µg per mL of medium, respectively.
After a 12-15 minute perfusion the liver was removed from the rat and the cells dispersed in WE with 10 % foetal calf serum (WE + FCS). The cells were sedimented by centrifugation a t 50 x g for approximately 30 seconds and re-suspended in WE + FCS. Cellular viability (exclusion of trypan blue) and the total number of cells were assessed by mixing equal volumes of cell suspension and 0.5 % trypan blue (prepared in 0.9 % saline) and counting with a haemocytometer. The viability of rat liver cells obtained by this experiment was approximately 65 %.
One ml of the cell suspension (8 x 10⁵ viable cells) was inoculated into a 35 x 17.8 mm tissue culture dish containing a plastic coverslip and 2 mL of WE + FCS. The cultures were placed in a humidified 37 °C incubator and maintained in an atmosphere of 95 % air: 5 % CO₂ for 1.5 h. During this interval viable cells attach to the coverslip. Nonviable cells were removed by washing the cultures with 3 mL of WE at the end of the attachment interval.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Metabolic activation system:

The rat hepatocyte UDS assay has the advantage that the primary hepatocyte cultures have sufficient metabolic capacity (activation/deactivation) to eliminate the need of an exogenous enzyme source.

Test concentrations with justification for top dose:

- The test material was prepared at concentrations of 1 x 10^-4, 3.16 x 10^-5, 1 x 10^-5, 3.16 x 10^-6, 1 x 10^-6, 3.16 x 10^-7 and 1 x 10^-7 M in WE + FCS containing 0.1 % (v/v) dimethylsulfoxide (DMSO).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO at 0.1 % in the media was used because the test material has low aqueous solubility.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Cultures exposed to media + 0.1 % DMSO served as the negative control.

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

The positive control employed was 2-AAF at concentrations of 10^-5, 10^-6 and 10^-7 M. Due to limited solubility, 2-AAF was prepared in WE + FCS containing 0.1 % of DMSO.

Details on test system and experimental conditions:

SUMMARY OF PROCEDURE
In the UDS assay, rat hepatocytes were isolated by perfusing the liver in situ with a solution of collagenase. After harvesting the cells, primary cultures were established and the cultures were treated with the test material and ³H-thymidine. After chemical treatment, the cells were processed for micro-autoradiography. The cells were coated with a thin layer of photographic emulsion. The emulsion is designed so that passage of ionising radiation (³H) through the emulsion results in exposure of individual silver grains which can be visualised microscopically after photographic development. The cells were then stained and the grains in the nucleus were counted. If there was repairable damage to the DNA, ³H-thymidine would be incorporated during the repair process. The amount of ³H-thymidine incorporated is detected and quantified by micro-autoradiography.

ANIMALS
Male CDF Fischer 344 rats were obtained from a pathogen free colony and acclimated to the laboratory for at least 10 days. The rats were housed in a room designed to maintain a temperature of approximately 72 °F, a relative humidity of approximately 50 % and a 12 hour lighting cycle. Food and water were provided ad libitum. One male rat weighing 247 g was used.

CHEMICAL TREATMENT
Two mL of medium supplemented with dexamethasone (1 µM in 0.1 % ethanol, final concentration) containing the test material and ³H-thymidine (10 µCi/mL) was applied to triplicate cultures at each dose level. The cultures were then incubated for 18 hours after which they were washed for three 30 minute intervals with 1 mM non-labelled thymidine in WE at 37 °C. The nuclei of the cells were then swelled for 10 minutes (room temperature) with 1.0 % (w/v) sodium citrate and subsequently fixed with a solution of ethanol:acetic acid (3:1) for 30 minutes, (room temperature). The coverslips were allowed to air dry and were then mounted on glass slides.

MICRO-AUTORADIOGRAPHY
The slides were dipped in NTB nuclear track photographic emulsion and stored refrigerated for 10 days. At the end of the exposure period the slides were developed with Kodak D-19 developer and stained with haematoxylin and eosin. UDS was quantitated by counting the number of grains in the nucleus and subtracting the mean number of grains in 3 nuclear sized areas adjacent to the nucleus in the cytoplasm. The grains were counted using an Artek Counter (Model 880, Artek Systems Co., Farmingdale, NY). Those cells undergoing semi-conservative DNA synthesis (DNA replication) were easily visualised because of their completely blackened nuclei and were not counted. Fifteen cells on each of two slides were evaluated per dose level.
Frequently the cytoplasmic background of radioactivity is slightly greater than the nuclear labelling in control or unaffected treated cultures. This results in a negative value for the net number of nuclear grains. Grain counts were reported as the mean ± the standard deviation.

Evaluation criteria:

Net nuclear labelling is often observed in control cells but rarely exceeds a mean of 5 grains per nucleus. Therefore, a mean of 6 or more net grains per nucleus (this may vary with experimental conditions) and statistical significance from control at p ≤0.05 is required for an unequivocal positive result (Williams, 1977). In addition to statistical significance from control, a dose response relationship should exist.

Statistics:

The net number of nuclear grains in treated cells is compared to the appropriate control using a computer program that performs Bartlett's test for homogeneity of variance (Winer, 1971). If variances are homogeneous, a one-way analysis of variance (ANOVA) is performed, followed by Dunnett's test if the ANOVA is significant. If variances are not homogeneous, treatment groups are compared using a nonparametric Kruskal-Wallis ANOVA followed by multiple comparisons as described by Hollander and Wolfe (1973).

Results and discussion

Test results

Species / strain:

hepatocytes: Primary cultures of rat hepatocytes

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 1 x 10^-4 and 3.16 x 10^-5 M

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test material was observed to be toxic to the hepatocyte cultures at concentrations of 1 x 10^-4 and 3.16 x 10^-5 M, as indicated by detachment of the cells from the coverslip and/or an un-flattened, granular appearance.
Cultures exposed to 3.16 x 10^-6 to 1 x 10^-7 M test material matched the appearance of the negative control cultures. Thus, a wide spectrum of concentrations was evaluated ranging from toxic to nontoxic.
The test material failed to elicit UDS at any concentration tested compared to the negative control (media + 0.1 % DMSO). In contrast, 2-acetylaminofluorene (2-AAF) elicited a significant, dose-related increase in UDS at all concentrations tested when compared to the media + 0.1 % DMSO control. The positive response of 2-AAF, a known genotoxic chemical, demonstrates the responsiveness of the assay in the present study.

Any other information on results incl. tables

Table 1: The Effects of the test material in the UDS Assay

Treatment	Concentration (moles/litre)	UDS* (net nuclear grains)
Media Control	-	-2.0 ± 3.3
Media + 0.1 % DMSO Control	-	-2.7 ± 4.9
Test Material	1 x 10^-4	-1.1 ± 2.9 ∆
	3.16 x 10^-5	-2.2 ± 5.0 ∆
	1 x 10^-5	-4.0 ± 4.1
	3.16 x 10^-6	-2.4 ± 3.4
	1 x 10^-6	-1.1 ± 3.1
	3.16 x 10^-7	-2.8 ± 3.0
	1 x 10^-7	-1.0 ± 2.7
2-AAF (positive control)	1 x 10^-5	54 ± 32 ∆†
	1 x 10^-6	37 ± 27†
	1 x 10^-7	22 ± 21†

*Net nuclear grains = total nuclear grains - background cytoplasmic grains

∆ = Test chemical toxicity observed

† = Positive response; 6 or more net nuclear grains and statistical significance (p≤0.05)

Applicant's summary and conclusion

Conclusions:

Interpretation of results: Negative

Under the conditions of the study, the test material has been determined to give a negative response when evaluated using the UDS assay.

Executive summary:

The genotoxic potential of the test material was evaluated in the rat hepatocyte unscheduled DNA synthesis (UDS) assay under GLP conditions. The methodology employed was equivalent to that outlined in the standardised guidelines OECD 482 and EU Method B.18.

Primary cultures of rat hepatocytes were prepared by slight modification of the method described by Williams et al. (1977). The test material was prepared at concentrations of 1 x 10^-4, 3.16 x 10^-5, 1 x 10^-5, 3.16 x 10^-6, 1 x 10^-6, 3.16 x 10^-7 and 1 x 10^-7 M in Williams Medium E with 10 % foetal calf serum containing 0.1 % (v/v) dimethylsulfoxide (DMSO). Concurrent positive (2-acetylaminofluorene, 2-AAF), solvent and media controls were run.

Two mL of medium supplemented with dexamethasone (1 µM in 0.1 % ethanol, final concentration) containing the test material and ³H-thymidine (10 µCi/mL) was applied to triplicate cultures at each dose level. The cultures were then incubated for 18 hours after which they were washed for three 30 minute intervals with 1 mM non-labelled thymidine in WE at 37 °C. The nuclei of the cells were then swelled for 10 minutes (room temperature) with 1.0 % (w/v) sodium citrate and subsequently fixed with a solution of ethanol:acetic acid (3:1) for 30 minutes, (room temperature). The coverslips were allowed to air dry and were then mounted on glass slides. The slides were then examined by micro-autoradiography, with 15 cells on each of two slides being evaluated per dose level.

The test material was observed to be toxic to the hepatocyte cultures at concentrations of 1 x 10^-4 and 3.16 x 10^-5 M. Cultures exposed to 3.16 x 10^-6 to 1 x 10^-7 M test material matched the appearance of the negative control cultures. Thus, a wide spectrum of concentrations was evaluated ranging from toxic to nontoxic.

The test material failed to elicit UDS at any concentration tested compared to the negative control (media + 0.1 % DMSO). In contrast, 2-AAF elicited a significant, dose-related increase in UDS at all concentrations tested when compared to the media + 0.1 % DMSO control.

Under the conditions of the study, the test material has been determined to give a negative response when evaluated using the UDS assay.