Methyl O-(4-amino-3,5-dichloro-6-fluoropyridin-2-yloxy)acetate

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restriction No guideline statement, but in general accordance with OECD guideline 475 (1997), EEC method B.11 and US-EPA OPPTS 870.5358 No justification is given for the 5-day dosing regime.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1983

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

yes

Remarks:

; No justification is given for the 5-day dosing regime.

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5385 (In Vivo Mammalian Cytogenetics Tests: Bone Marrow Chromosomal Analysis)

Deviations:

yes

Remarks:

; No justification is given for the 5-day dosing regime.

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

; No justification is given for the 5-day dosing regime.

GLP compliance:

no

Remarks:

GLP was not compulsory during conduct of study.

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

Methylheptyl, 0-(4-amino-3,5-dichloro-6-fluoro-2-pyridyloxy) acetate

IUPAC Name:

Methylheptyl, 0-(4-amino-3,5-dichloro-6-fluoro-2-pyridyloxy) acetate

Constituent 2

Reference substance name:

Acetic acid [(4-amino-3,5-dichloro-6-fluoro-2-pyridinyl) oxy], 1-methylheptyl

IUPAC Name:

Acetic acid [(4-amino-3,5-dichloro-6-fluoro-2-pyridinyl) oxy], 1-methylheptyl

Constituent 3

Reference substance name:

1-methylheptyl [(4-amino-3,5-dichloro-6-fluoropyridin-2-yl)oxy]acetate

EC Number:

279-752-9

EC Name:

1-methylheptyl [(4-amino-3,5-dichloro-6-fluoropyridin-2-yl)oxy]acetate

Cas Number:

81406-37-3

IUPAC Name:

1-methylheptyl [(4-amino-3,5-dichloro-6-fluoropyridin-2-yl)oxy]acetate

Details on test material:

- Name of test material (as cited in study report): Dowco 433
- Physical state: off white powder
- Purity: not reported
- Lot/batch No.: 433T-1282-7

Test animals

Species:

hamster, Chinese

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bantin and Kingman Limited, Hull, England
- Age at study initiation: Young adult
- Weight at study initiation: Males: 19-37 g, females: 17-30 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Individually in polypropylene and stainless steel cages with white wood shavings as bedding
- Diet (e.g. ad libitum): Rat and Mouse Maintenance Diet No. 1 expanded ground (Special Diet services Limited, Witham, essex, England), ad libitum.
- Water (e.g. ad libitum): Tap water, ad libitum
-Acclimation period: at least 11days quarantine.


ENVIRONMENTAL CONDITIONS
Temperature: about 20°C;
humidity: about 50%;
12 hour light-dark cycle


IN-LIFE DATES: from November 1982 to February 1983

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

see any other information on materials and methods incl. tables

Duration of treatment / exposure:

5 consecutive days

Frequency of treatment:

see any other information on materials and methods incl. tables

Post exposure period:

see any other information on materials and methods incl. tables

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100, 300, 900, 2700, or 5000 mg/kg bw/day
Basis:
actual ingested
Dose finding test

Doses / concentrations 2

Remarks:

Doses / Concentrations:
750, 1500, or 2500 mg/kg bw/day
Basis:
actual ingested
Main toxicity test

Doses / concentrations 3

Remarks:

Doses / Concentrations:
73.5, 235, or 735 mg/kg bw/day
Basis:
actual ingested
Cytogenetic test

No. of animals per sex per dose:

Dose finding test: 1
Main toxicity test: 5
Cytogenetic test: 5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide;
- Route of administration: oral, gavage
- Doses / concentrations:
concentration 3 mg/mL;
dosing volume was 10 mL/kg bw,
dose 30 mg/kg bw/day

Examinations

Tissues and cell types examined:

bone marrow from femora

Details of tissue and slide preparation:

Each hamster was injected i.p. with 2 mg/kg bw colchicine dissolved in HBSS 2 h before each scheduled kill by ether asphyxiation and neck dislocation.
Both femora from each animal were dissected out, cleaned of adherent tissue and the marrow aspirated into a plastic heparinised tube containing HBSS at room temperature.
After centrifuging the cells at 1500 r.p.m. for 5 min and decanting the supernatant fluid, the cells were treated with 0.075 M KCl at 37.C for 20 min. After further centrifugation freshly prepared fixative (methanol:glacial acetic acid 3:1) was added. The fixative was changed and the suspension stored for 2 days at 4°C. The fixative was changed again prior to making the slides. Five slides were prepared from each animal. The slides were air dried, stained with 5% Giemsa (Gurr R66), dehydrated in alcohol and cleared in xylene. Coverslips were sealed with D.P.X. mountant.

Slide Reading
Slides were assigned numbers corresponding to the coded tube numbers, unrelated to the animal or group number, and were read "blind- in a random order with Leitz Dialux 20 binocular microscopes.
From each animal, 50 metaphases with a minimum of 20 well spread chromosomes were examined and scored. The locations of all spreads examined were recorded using the microscope stage Vernier scale.
The number of abnormalities (gaps, breaks, acentric fragments, exchanges, dicentrics, rings. translocations (within the limitations of the staining methods), pulverisations and multiple aberrations) was recorded.

Evaluation criteria:

All chromosome aberrations, as well as being recorded separately, were converted into lesions (or breaks) in the following manner: chromatid and chromosome (isochromatid) gaps, breaks and fragments were counted as one lesion each, while Markers, i.e. exchanges, rings and dicentrics were counted as 2 lesions each (Bauchinger et al, 1976). Pulverised chromosomes were given the artificial value of five lesions, whereas cells with chromosomes showing multiple and extensive damage were given the artificial maximum value of 10 lesions per metaphase.

The clastogenic potential of a compound was evaluated by calculating the lesion/cell ratio and the percentages of aberrant cells including and excluding gaps. For comparison, the incidence of metaphases showing heteroploidy, i.e. hypodiploidy (< 22 chromosomes), hyperdiploidy (>22 chromosomes) or polyploidy (ca.33, ca. 44 etc. chromosomes) also was noted.

A positive response was recorded if a doubling over the corresponding vehicle control value was observed in any of the aberration frequencies and if the result was in good dose agreement.

Statistics:

Chi-square and Fischer exact tests with significance levels adjusted for multiple comparisons were carried out using Bonferroni's inequality (Cox. 1970 ). Parameters examined were:

1) Percent metaphases including cells with gaps alone
2) Percent metaphases excluding cells with gaps alone
3) Percent metaphases with numerical chromosome alterations

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

mortality was observed from doses of 1500 mg/kg bw on

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

see remarks on results including tables and figures

Any other information on results incl. tables

DOSE FINDING TEST

Ten Chinese hamsters (5 males + 5 females) were dosed for five consecutive days with test compound concentrations ranging between 100 and 5000 mg/kg bw/day. Animal deaths were recorded at dose levels of 2700 and 5000 mg Dowco 433/kg bw/day. Clinical signs observed in the dosed animals were piloerection, sedation, prostration, coma, hunched, hypothermia and ataxia. Based on the results from the dose finding test the dose levels for the main toxicity test were set as followed: 750, 1500 and 2500 mg/kg bw/day.

MAIN TOXICITY TEST

Three groups of Chinese hamsters (5 males + 5 females per dose group) were treated for five consecutive days with Dowco 433 at concentrations of 750, 1500 and 2500 mg/kg bw/day. Animal deaths following treatment at the three dose levels used were 0/10, 1/10 and 10/10, respectively. Clinical signs observed this time, were hypokinesia, piloerection, soiled coat, hunched, ataxia, sedation, prostration, coma and hypothermia.

On the basis of these results, the 5-day oral LD₅₀ value for the test material was calculated. The method of analysis used was the probit transformation of mortality (Finney, 1971). Due to the large difference in mortality between the 1500 mg and 2500 mg/kg bw/day dose levels, the standard errors for the calculation of the regression line were high.

These circumstances led to the calculated line of best fit rotating upwards to accommodate the uneven spread in the mortality data. Hence, the calculated LD₅₀value was unexpectedly low at 1469 mg/kg bw/day, with 95% confidence limits of +99 mg and -93 mg/kg bw/day.

CYTOGENETIC TEST

Dosing observations

No abnormal findings were recorded in the low or high dose treatment groups. In the middle dose group, one female showed a fit of clonic convulsion on days 3 and 4 of dosing, but otherwise no abnormalities were detected. In the vehicle control group of hamsters, one animal showed a red-stained ear on the last day of dosing.

No appreciable fluctuations in bodyweights were in evidence in any of the treatment groups during the 5-day dosing regime.

 

Cytogenicity test

There was no evidence to suggest increased chromosomal aberration frequencies when bone marrow from the male and female hamsters dosed with Dowco 433 was analysed for clastogenic effects. The two chromatid breaks (one in the low and one in the middle dose group) and one chromosome break (in the low dose group) recorded, although each from a separate animal, did not in any way constitute a significant response and were, therefore, judged random events of no biological importance (see tables).

There was no evidence to suggest an increased incidence of aneuploidy in the test compound treated animals when the male and female hamsters were analysed for such occurrences. There was, however, some indication of polyploidy induction in the low dose Dowco 433 treated male hamsters. No significant increases in the numbers of polyploid cells were observed in the middle or high dose groups of animals (see Table 3).

Male and female hamsters dosed with the positive control substance, cyclo¬phosphamide, on the other hand, showed clear and significant (P<0.001) increases (females only, or males and females together) in the yield of chromosomal aberrations, both when cells including and excluding gaps were analysed for damage. There was also some suggestion of polyploidy induction in the positive control male animals.

 

Table 1: Structural aberrations in male and female Chinese Hamsters after 5-day treatment with Dowco 433

Treatment	Sex	No. of analysed cells	Observed structural aberrations
			Chromatid			Chromosome			Marker			Other	Multiple
			G	B	F	G	B	F	E	D	R
Vehicle control 10 mL 0.5% CMC/kg /day	♂	206	0	0	0	0	0	0	0	0	0	0	0
	♀	250	1	0	0	0	0	0	0	0	0	0	0
	♂+♀	456	1	0	0	0	0	0	0	0	0	0	0
Positive control 30 mg cyclophosphamid/kg/day	♂	250	8	5	1	1	1	0	2	0	0	0	0
	♀	223	6	11	6	1	1	3	1	0	0	0	1
	♂+♀	473	14	16	7	2	2	3	3	0	0	0	1
Low Dose 73.5 mg Dowco/kg/day	♂	250	0	1	0	0	1	0	0	0	0	0	0
	♀	250	0	0	0	0	0	0	0	0	0	0	0
	♂+♀	500	0	1	0	0	1	0	0	0	0	0	0
Middle Dose 235 mg Dowco/kg/day	♂	250	1	0	0	0	0	0	0	0	0	0	0
	♀	250	0	1	0	0	0	0	0	0	0	0	0
	♂+♀	500	1	1	0	0	0	0	0	0	0	0	0
High Dose 735 mg Dowco/kg/day	♂	250	0	0	0	0	0	0	0	0	0	0	0
	♀	250	0	0	0	0	0	0	0	0	0	0	0
	♂+♀	500	0	0	0	0	0	0	0	0	0	0	0

G = Gap: achromatic lesion, B = Break: with and without associated fragment, F = Fragment: unassociated acentric fragment; E = Exchange: triradials, quadriradials, multiradials, D = Dicentric: chromosome with 2 centromers, R = Ring: centric or acentric circular chromosome

 

Table 2: Structural aberration frequencies in Chinese Hamster bone marrow cells after 5-day treatment with Dowco 433

Treatment	Sex	No. of cells analysed	Structural aberrations
			No. of lesions	lesion / cell	% aberrant cells incl. Gaps	% aberrant cells excl. Gaps
Vehicle control 10 mL 0.5% CMC/kg /day	♂	206	0	0.000	0.0	0.0
	♀	250	1	0.004	0.4	0.0
	♂+♀	456	1	0.002	0.2	0.0
Positive control 30 mg cyclophosphamid/kg/day	♂	250	20	0.080	3.2	2.4
	♀	223	40	0.179	7.6***	5.8***
	♂+♀	473	60	0.127	5.3***	4.0***
Low Dose 73.5 mg Dowco/kg/day	♂	250	2	0.008	0.8	0.8
	♀	250	0	0.000	0.0	0.0
	♂+♀	500	2	0.004	0.4	0.4
Middle Dose 235 mg Dowco/kg/day	♂	250	1	0.004	0.4	0.0
	♀	250	1	0.004	0.4	0.4
	♂+♀	500	2	0.004	0.4	0.2
High Dose 735 mg Dowco/kg/day	♂	250	0	0.000	0.0	0.0
	♀	250	0	0.000	0.0	0.0
	♂+♀	500	0	0.000	0.0	0.0

Significance levels (Fischer test): *, **, *** (p < 0.05, p < 0.01, p < 0.001)

 

Table 3: Numerical aberration frequencies in Chinese Hamster bone marrow cells after 5-day treatment with Dowco 433

Treatment	Sex	No. of cells analysed	Numerical aberrations
			No. of Heteroploid cells	No. of Hypodiploid cells	No. of Hyperdiploid cells	No. of Polyploid cells
Vehicle control 10 mL 0.5% CMC/kg /day	♂	206	29	14.1	0.0	0.0
	♀	250	19	7.2	0.0	0.4
	♂+♀	456	48	10.3	0.0	0.2
Positive control 30 mg cyclophosphamid/kg/day	♂	250	22	7.2	0.0	1.6**
	♀	223	20	8.5	0.4	0.0
	♂+♀	473	42	7.8	0.2	0.8
Low Dose 73.5 mg Dowco/kg/day	♂	250	37	13.6	0.0	1.2**
	♀	250	17	6.4	0.4	0.0
	♂+♀	500	54	10.0	0.2	0.6
Middle Dose 235 mg Dowco/kg/day	♂	250	16	6.4	0.0	0.0
	♀	250	24	8.8	0.0	0.8
	♂+♀	500	40	7.6	0.0	0.4
High Dose 735 mg Dowco/kg/day	♂	250	25	9.6	0.0	0.4
	♀	250	25	10.0	0.0	0.0
	♂+♀	500	50	9.8	0.0	0.2

Significance levels (Fischer test): *, **, *** (p < 0.05, p < 0.01, p < 0.001)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Based on the study results there is no evidence that Dowco 433 has either a clastogenic potential or is capable of inducing structural chromosomal damage in hamster bone marrow cells in vivo up to doses of 735 mg/kg bw/day.

Executive summary:

Dowco 433 was tested for its potential to induce structural or numerical chromosomal aberrations in bone marrow cells of male and female Chinese hamsters in vivo.

In a preliminary toxicity test an oral 5-day LD₅₀ value of 1469 mg Dowco 433/kg bw/day was determined.

The cytogenicity test was conducted with five consecutive oral daily doses of 73.5, 235, and 735 mg Dowco 433/kg bw/day. Negative control animals received vehicle (0.5% CMC), whereas positive control animals were treated with 30 mg Cyclophosphamid/kg bw/day. There were no biologically significant differences in the chromosomal aberration frequencies between the control and test substance groups. In the positive control group, a marked clastogenic response with increased yields of chromosomal damage was observed. Thus, Dowco 433 does not produce chromosomal aberrations in Chinese hamsters in vivo at doses up to 735 mg/kg bw/day.