Mecoprop

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

Study period:

13 June 2000 to 10 August 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UKEMS Sub-Committee on Guidelines for Mutagenicity Testing, Report, Part 1 revised

Version / remarks:

Basic Mutagenicity Test: UKEMS recommended procedures, 1990

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: US, EPA, TSCA and FIFRA Guidelines

Version / remarks:

Not specified

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Mecoprop

EC Number:

230-386-8

EC Name:

Mecoprop

Cas Number:

7085-19-0

Molecular formula:

C10H11ClO3

IUPAC Name:

2-(4-chloro-2-methylphenoxy)propanoic acid

Test material form:

solid

Remarks:

Light brown

Details on test material:

Storage: Room temperature in the dark.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately eight to ten weeks old.
- Weight at study initiation: 225 to 270g
- Assigned to test groups randomly: Yes. The animals were selected at random and given a number unique within the study by ear punching and a number written on a colour coded cage card.
- Housing: The animals were housed in groups of up to seven by sex in solid-floor polypropylene cages with woodflakes bedding.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: Minimum acclimatisation period of seven days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): approximately fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was freshly prepared as required as a suspension at the appropriate concentration in arachis oil.
- Dose volume: 10 mL/kg
- Concentration:
125 mg/L group: 12.5 mg/mL
250 mg/L group: 25 mg/mL
500 mg/L group: 50 mg/mL

Duration of treatment / exposure:

Single oral administration

Frequency of treatment:

Once

Post exposure period:

The animals were sacrificed between 24 - 48 hours after test material administration.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Mid dose

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

Vehicle control: 14 males total (Kill Time 48 and 24 hours)
Positive control: 5 males
Test material low and mid dose: 7 males
Test material high dose: 14 males (Kill time 48 and 24 hours)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide:
- Doses / concentrations: 25 mg/kg. The positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water.

Examinations

Tissues and cell types examined:

100 metaphase cells of adequate quality were scored, if possible, from the slides prepared from each animal for both numerical and structural chromosome aberrations.

Details of tissue and slide preparation:

DOSE SELECTION RATIONALE:
A range-finding toxicity study was performed to determine a suitable dose level and route of administration for the chromosome aberration study. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2 000 mg/kg. The range-finding toxicity study was also used to determine if the main study was to be performed using both sexes or males only. Using toxicity data supplied by the sponsor it was considered unnecessary to investigate the intraperitoneal route. The supplied data also showed no marked differences between the sexes and therefore only male rats were used to determine the maximum tolerated dose level for use in the main study.
All animals were dosed once only at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of dosing.
Animals were observed one hour after dosing and subsequently once daily for two days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.
In the main study, groups, each of seven rats, were dosed once only via the oral route with the test material at 125, 250 or 500 mg/kg. One group of rats from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed at 500 mg/kg was killed after 48 hours. In addition, three further groups of rats were included in the study; two groups (seven rats) were dosed via the oral route with the vehicle alone (arachis oil) and a third group (five rats) was dosed orally with cyclophosphamide, a positive control material known to produce chromosome aberrations under the conditions of the test. The vehicle controls were killed 24 and 48 hours following treatment and positive control group animals were killed 24 hours following treatment.
All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.

TREATMENT AND SAMPLING TIMES:
Animals were injected i.p. with a solution of colchicine at 4 mg/kg approximately 2 to 3 hours prior to bone marrow harvest. At the scheduled time, animals were killed by cervical dislocation and one femur was extracted from each animal and cleaned of muscle and connective tissue. The bone marrow was aspirated into 5 mL of Hank's balanced salt solution (HBSS) and spun down in a centrifuge. The supernatant was removed and the cell pellet resuspended in 0.075 M KCl at room temperature for 15 minutes (including five minutes centrifugation). The cells were recentrifuged and all but 1 mL of the supernatant removed. After resuspension of the cell pellet, the cells were fixed by the addition of freshly prepared fixative (methanol:glacial acetic acid, 3:1). The fixative was changed several times and the cells stored at 4 °C for at least 4 hours.

DETAILS OF SLIDE PREPARATION:
After storage the cell suspensions were recentrifuged and the fixative removed to leave a sufficient amount to give a milky suspension on resuspension of the cell pellet. A few drops of each cell suspension was dropped onto clean, wet slides and air-dried. When completely dry the slides were stained in 5 % Giemsa for 10 minutes and rinsed in tap water and distilled water. When dry the slides were coverslipped using a mounting medium.


Groups, each of seven rats, were dosed once only via the oral route with the test material at 125, 250 or 500 mg/kg. One group of rats from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed at 500 mg/kg was killed after 48 hours. In addition, three further groups of rats were included in the study; two groups (seven rats) were dosed via the oral route with the vehicle alone (arachis oil) and a third group (five rats) was dosed orally with cyclophosphamide, a positive control material known to produce chromosome aberrations under the conditions of the test. The vehicle controls were killed 24 and 48 hours following treatment and positive control group animals were killed 24 hours following treatment.

Evaluation criteria:

SLIDE EVALUATION
The stained slides were coded and examined 'blind' using light microscopy at x 100 and x 1 000 magnifications.
If the cell had 40 to 44 or more chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1990 UKEMS Guidelines for Mutagenicity Testing. Aberrations recorded by the slide scorers were checked by a Senior Cytogeneticist.

Statistics:

Comparisons were made between the negative control groups and each corresponding treatment dose group, with a chi-squared test, using observed numbers of cells with aberrations.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In animals dosed with test material via the oral route, clinical signs were observed at and above 500 mg/kg as follows: hunched posture, ataxia, lethargy, loss of righting reflex, decreased and laboured respiration, ptosis, pile-erection and splayed gait. When observations were made approximately 24 hours after dosing there was some concern with the condition of the animals dosed with 750 mg/kg. These animals were observed again at approximately 26 hours and, due to the severity of the toxicity of the test material, they were killed in extremis.
Adequate evidence of test material toxicity was demonstrated via the oral route of administration, therefore, this was selected for use in the main study. A maximum tolerated dose (MTD) of the test material, 500 mg/kg, was selected for use in the main study, with 125 and 250 mg/kg as the lower dose levels.

RESULTS OF DEFINITIVE STUDY
Evaluation of bone marrow slides:
- There were no clear statistically significant reductions in the mean mitotic index in any of the test material treatment groups when compared to their concurrent vehicle control groups. The mean mitotic index of the positive control group was sharply depressed compared to that of the 24-hour vehicle control group.
- All the negative control animals gave values of chromosome aberrations within the expected range. The mean frequency of cells with aberrations was consistent between the two vehicle control groups, the highest frequency (0.3 % cells with aberrations excluding gaps) being seen in the 24 hour group.
- The positive control group animals showed highly significant increases in the frequency of cells with aberrations indicating that the test method itself was operating as expected.
- The test material was seen to induce no significant, dose-related increases in the frequency of cells with aberrations in any of the treatment groups.
- The test material did not induce a significant increase in the numbers of polyploid cells in any of the treatment groups.

Historical Aberration Ranges for Vehicle and Untreated Control Cultures
Experiments with rat bone marrow cells have established a range of aberration frequencies acceptable for control animals, these are commonly in the range of 0 to 3 % cells with structural aberrations.
A positive response was recorded for a particular treatment if the % cells with aberrations markedly exceeded that seen in the concurrent control. For modest increases in aberration frequency, appropriate statistical tests may be applied in order to record a positive response.

Any other information on results incl. tables

Mortality and Clinical Results

There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at and above 125 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: lethargy, hunched posture, ataxia, decreased and laboured respiration, ptosis, loss of righting reflex, splayed gait, stained urine and diuresis. The severity of the clinical signs increased with the increase in dose of the test material.

 

Group Mean Results of Chromosome Aberration Assay

Treatment Group	Animals	Numbers of Cells Scored	Polyploid Cells	Total Gaps	Chromatid		Chromosome		Others	Total Aberrations (+ Gaps)	Total Aberrations (- Gaps)	Cells with  Aberrations (+ Gaps)	Cells with  Aberrations (- Gaps)
					Breaks	Exchanges	Breaks	Exchanges
Vehicle Control 48 hours	Males	700	1 (0.1)	3 (0.4)	1 (0.1)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	4 (0.6)	1 (0.1)	3 (0.4)	1 (0.1)
Vehicle Control 24 hours	Males	700	1 (0.1)	1 (0.1)	1 (0.1)	0 (0.0)	1 (0.1)	0 (0.0)	0 (0.0)	3 (0.4)	2 (0.3)	3 (0.4)	2 (0.3)
Positive Control	Males	500	0 (0.0)	61 (12.2)	71 (14.2)	58 (11.6)	20 (4.0)	2 (0.4)	16 (3.2)	212 (42.4)	151 (30.2)	113 *** (22.6)	94 *** (18.8)
Test Material 500 mg/kg 48 Hours	Males	700	0 (0.0)	2 (0.3)	3 (0.4)	0 (0.0)	1 (0.1)	0 (0.0)	0 (0.0)	6 (0.9)	4 (0.6)	5 (0.7)	3 (0.4)
Test Material 500 mg/kg 24 Hours	Males	700	0 (0.0)	5 (0.7)	3 (0.4)	0 (0.0)	5 (0.7)	0 (0.0)	0 (0.0)	13 (1.9)	8 (1.1)	9 (1.3)	5 (0.7)
Test Material 250 mg/kg 24 Hours	Males	700	1 (0.1)	1 (0.1)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.1)	0 (0.0)	1 (0.1)	0 (0.0)
Test Material 125 mg/kg 24 Hours	Males	700	1 (0.1)	5 (0.7)	2 (0.3)	0 (0.0)	1 (0.1)	0 (0.0)	0 (0.0)	8 (1.1)	3 (0.4)	8 (1.1)	3 (0.4)

X = > 10 aberrations per cell (not included in total aberrations)

Figures in brackets = aberrations per 100 cells

*** = p < 0.001

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study the test material did not induce any significant increases in the frequency of chromosome aberrations in rat bone marrow under the conditions of the test. The test material was considered to be non-clastogenic to rat bone marrow cells in vivo.

Executive summary:

The potential of the test material to produce damage to chromosomes or the mitotic apparatus when administered to rats was assessed according to OECD Test Guideline 475 and EU Method B.11, in compliance with GLP. 

A range-finding study was performed to find suitable dose levels of the test material. Adequate oral toxicity data in rats was supplied by the sponsor which showed that there was no marked differences in test material toxicity between the sexes. Therefore, the main study was performed using only male rats. The chromosome aberration study was conducted using the oral route in groups of seven rats (males) at the maximum tolerated dose (MTD) 500 mg/kg with 250 and 150 mg/kg as the lower dose levels. Further groups of rats were given a single oral dose of arachis oil (7 rats) or dosed orally with cyclophosphamide (5 rats), to serve as vehicle and positive controls respectively. Animals were killed 24 or 48 hours later, the bone marrow extracted and slide preparations made and stained. Bone marrow cells were scored for the presence of chromosome aberrations.

No statistically significant decreases in the mitotic index mean values were observed in the 24 or 48 hour test material groups when compared to their concurrent vehicle control group. However, the observation of clinical signs indicated that systemic absorption, and consequently, exposure to the target tissue, had occurred.

There was no evidence of a statistically significant increase in the incidence of chromosome aberrations in animals dosed with the test material when compared to the concurrent vehicle control groups. The positive control material produced a marked increase in the frequency of chromosome aberrations.

Under the conditions of this study the test material did not induce any significant increases in the frequency of chromosome aberrations in rat bone marrow under the conditions of the test. The test material was considered to be non-clastogenic to rat bone marrow cells in vivo.