Paraquat-dichloride

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1978

Report date:

1978

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

GLP compliance:

not specified

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Paraquat-dichloride

EC Number:

217-615-7

EC Name:

Paraquat-dichloride

Cas Number:

1910-42-5

Molecular formula:

C12H14N2.2Cl

IUPAC Name:

1,1’-dimethyl-4,4’-bipyridyldiylium dichloride

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

other: Wistar-derived

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 to 10 weeks.
- Weight at study initiation: 150 to 200 g.
- Assigned to test groups randomly: yes.
- Housing: Four per cage. The cages were constructed of 19-gauge galvanised wire mesh (1 cm2) on three sides and floor with a solid back, the overall measurements being 33 x 27.5 x 13.5 cm. They were suspended on racks over collecting trays lined with absorbent paper.
- Diet: Rat cubes, ad libitum.
- Water: Ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2
- Humidity (%): 45 to 45
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: aqueous solution of 0.5% ‘Tween’ 80.
- Amount of vehicle: 10 mL/kg bw/day.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in an aqueous solution of 0.5% ‘Tween’ 80 and stored throughout the treatment period in a screw-topped, opaque glass bottle at room temperature.

Duration of treatment / exposure:

The treatment period was 5 consecutive days.

Frequency of treatment:

Once per day.

Post exposure period:

The animals were killed 6 hours after receiving the last dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

6.5 mg/kg bw/day (actual dose received)

Remarks:

Group 2. Concentration for test substance cation.

Doses / concentrations 2

Dose / conc.:

12.5 mg/kg bw/day (actual dose received)

Remarks:

Group 3. Concentration for test substance cation.

Doses / concentrations 3

Dose / conc.:

19 mg/kg bw/day (actual dose received)

Remarks:

Group 4. Concentration for test substance cation.

No. of animals per sex per dose:

8 males per group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulpnonate (EMS) 98% pure.
- Route of administration: Gavage.
- Doses / concentrations: 200 mg/kg bw/day.
- A fresh aqueous solution was made each day.

Examinations

Tissues and cell types examined:

Bone marrow.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Doses for the treatment regime were determined by 5 day range-finding studies, the doses for which were selected on the basis of the LD50.

TREATMENT AND SAMPLING TIMES:
Three groups of rats were given the test substance, the positive control group was given EMS and the negative control group was given the vehicle alone. All groups were dosed by gavage for 5 consecutive days. A constant dose volume of 10 mL/kg bw was used. The animals were killed at 6 hours after receiving the last dose. Each rat received 3 mg/kg bw colchicine intraperitoneally two hours prior to sacrifice to arrest dividing cells in metaphase. The rats were killed by rapid asphyxiation with carbon dioxide; both femurs from each animal were removed and the bone marrow harvested by aspiration with Hanks’ basic salt solution.

DETAILS OF SLIDE PREPARATION:
The obtained bone marrow cells were treated with hypotonic solution (0.07 M potassium chloride at 37°C for 20 minutes), followed by fixation in glacial acetic acid and methanol in the ratio 1:3. Slides were prepared by air-drying and stained with Giemsa.

METHOD OF ANALYSIS:
The slides were coded, to avoid observer bias while being scored. 50 cells from each animal were examined. Any abnormality was assigned to the following category: chromatid or chromosome gaps, chromatid breaks, fragments, any other complex abnormality such as minutes or Robertsonian translocations.

Evaluation criteria:

Statistically significant proportion of cells with any abnormalities and statistically significant increase of proportion of cells with breaks.

Statistics:

For each animal, the proportion of cells with any abnormalities has been transformed using a double arcsine function. The transformed values of the paraquat treated groups and the negative control group were analysed by one-way analysis of variance. The group means were then compared with the control group mean using a one-sided Student’s ‘t’ test based on the residual variance from the analysis of variance table. A comparison of the pooled treated group mean with the control mean was similarly conducted.
The positive control group (EMS) was compared directly with the negative control group by a one-sided Student's ‘t’ test. The above analysis was repeated considering only those cells with breaks. Breaks were also considered by comparing the proportion of animals in each group with any breaks. Fisher's exact test was used to compare the treated groups with the negative control group.
The value obtained from transforming the data depends on the number of cells examined. The variation in the number of cells examined for different animals can thus lead to difficulty in interpreting the results of this analysis. Also the probability of any breaks being seen for a given animal will also depend on the number of cells examined. All analyses were carried out twice:
1) using all the data and 2) using only those animals from which at least 20 cells had been examined.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels: There was no statistically significant increase in the percentage of cells with any abnormality in the treated groups (Table 1 in ‘Any other information on results incl. tables’). However, when animals from which more than 20 cells were analysed were examined, (Table 2), the difference in means between the negative control group and the group treated with 6.5 mg/kg bw was statistically significant at the 5% level but there was no evidence of a dose-related increase.
None of the treatment groups had statistically significant increase in the mean percentage of cells with breaks when compared with the negative control group, not even when the data from the treatment groups were pooled. There were no other statistically significant differences between the treated groups and the controls.
- Appropriateness of dose levels and route: The morphological changes present in the treatment groups indicated that the test substance had reached the bone marrow cells and, when in contact with mitotic cells, was affecting the morphology and staining characteristics of the metaphase chromosomes. It is not known whether this effect takes place in vivo or in vitro. A similar effect on staining characteristics to that observed in treated animals was found when untreated rat bone marrow cells were exposed to a 1 µM solution of the test substance either before fixation or after fixation. The presence of the test substance even in fixed cells leads to these effects which suggests, therefore, that the test substance interferes directly with staining of chromosomes. Therefore, this would seem to indicate that residual test substance in the processing system may be producing the morphological changes observed in the in vivo experiment.

Any other information on results incl. tables

Verification of concentration of the test substance in the dosing solutions

Samples of the dosing solutions used in this study were analysed. The actual test substance concentration were up to 14% below the intended concentrations. The actual concentrations were calculated from analysis of the dosing solutions to be 6.5, 12.5 and 19.0 mg/kg bw, respectively.

Table 1. Mean percentage of cells with abnormalities based on all (1) observations

Group dosed by gavage on 5 consecutive days	Mean (2) percentage of cells with any abnormalities	Mean (2) percentage of cells with any breaks	Number of animals
Treatment
6.5 mg/kg bw	8.8	2.2	6
12.5 mg/kg bw	4.7	1.0	6
19.0 mg/kg bw	5.1	2.4	7
Controls
0.5% 'Tween' 80	5.8	1.0	8
EMS 200 mg/kg bw	18.5***	5.0*	8

(1) - excluding animal dying during the study, preparations with no analysable metaphases and the animal in Group 3 from which only one cell was analysed. Animal 21 (6.5 mg/kg) died on day 4 of dosing due to misdosing.

(2) - calculated as a mean of the percentages for each animal.

* - statistically significantly different from negative control group, 5% level.

*** - statistically significantly different from negative control group, 0.1% level.

All statistics were based on analysis of transformed data.

 

 

Table 2. Mean percentage of cells with abnormalities based on animals for which more than 20 cells were examined.

Group dosed by gavage on 5 consecutive days	Mean (1) percentage of cells with any abnormalities	Mean (1) percentage of cells with any breaks	Number of animals
Treatment
6.5 mg/kg bw	10.6*	2.6	5
12.5 mg/kg bw	6.0	2.0	3
19.0 mg/kg bw	4.1	1.8	6
Controls
0.5% 'Tween' 80	6.6	1.1	7
EMS 200 mg/kg bw	18.5***	5.0*	8

(1) - calculated as a mean of the percentagesforeach animal.

* - statistically significantly different from negative control group, 5% level.

*** - statistically significantly different from negative control group, 0.1% level.

All statistics were based on analysis of transformed data.

Applicant's summary and conclusion

Conclusions:

In this non GLP compliant in vivo clastogenicity study, performed similar to OECD 475, it was concluded that the test substance did not induce biologically significant clastogenic effects in the bone marrow cells of rats.

Executive summary:

In this in vivo chromosome aberration study in mammalian cells, not under GLP and performed similar to OECD 475, the test substance was administered daily by gavage at doses equivalent to 6.5, 12.5 or 19 mg/kg bw to groups of 8 male Wistar-derived rats for 5 consecutive days. As negative control, the vehicle was used (0.5% 'Tween' 80 in water) and ethyl methanesulphonate (EMS) at 200 mg/kg bw was used as positive control group.

The test substance produced morphological changes in the chromosomes of the bone marrow cells, but preliminary studies suggested that these occurred due to altered staining affinity. There was also an increase in gaps and breaks which was not dose-related and was considered to be the result of an artefact due to the irregular staining of the chromosomes. EMS (the positive control) produced a clastogenic (chromosome-breaking) response.

It was concluded that the test substance did not induce biologically significant clastogenic effects in the bone marrow cells of rats.