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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Neurobehaviour examinations were not performed, but were not mandatory at the time the study was conducted.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

407-550-4

EC Name:

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

Cas Number:

69184-17-4

Molecular formula:

Hill formula: C8 H7 Cl2 F N2 O3 CAS formula: C8 H7 Cl2 F N2 O3

IUPAC Name:

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

Details on test material:

- Name of test material (as cited in study report): Fluroxypyr methyl ester
- Physical state: dark grey powder
- Analytical purity: 90.8%
- Purity test date: 28.08.1990
- Lot/batch No.: C-3B TOR 0020
- Expiration date of the lot/batch: August 1992
- Storage condition of test material: at 4°C in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Kent, England
- Age at arrival: 27-29 days
- Weight at arrival: 68-80 g
- Housing: in groups of up to five rats of the same sex
- Diet (ad libitum): Biosure LAD 2
- Water (ad libitum): tap water
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21.5
- Humidity (%): 43-56
- Air changes (per hr): approx. 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: plain diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Two pre-mixes were prepared each week by grinding the test substance directly into the diet and mixing in a Turbula mixer. The required concentrations were then prepared by direct dilution of the relevant pre-mix with further quantities of untreated diet; homogeneity was achieved by mixing in a double cone blender for a minimum period of 7 minutes.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with: untreated diet

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A representative sub-sample (10 g) of test diet, obtained by a standard riffling technique, was extracted (mechanical shaking, 30 minutes) with methanol (160 mL). The extract was filtered (Whatman CF/A) and appropriately diluted using methanol (Dilution A). A suitable volume (Volume A) of the diluted extract was evaporated to dryness (RFE, 40°C) and the residue dissolved in mobile phase (Volume B) to provide a solution, containing Fluroxypyr methyl ester at an expected concentration between 2 and 4 µg/mL.
The final solution was filtered (Acrodisc LC 13 PVDF 0.45 µm) and the concentration of Fluroxypyr methyl ester quantified by high performance liquid chromatography using ultra-violet detection.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
11, 105, 1055 mg/kg/day
Basis:
nominal in diet

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Dosage levels were selected on the basis of a 14-day preliminary dose range-finding study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: On Day -14, prior to dosing and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each cage determined weekly: Yes
- Compound intake calculated from group mean bodyweight and food consumption data and the dietary inclusion level of the test substance: Yes

FOOD EFFICIENCY:
- Calculated from bodyweight and food consumption data as weight of food consumed per unit gain in bodyweight: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Day 1-15 by visual inspection; Day 15-sacrifice by gravimetric analysis

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During Week 4.
- Dose groups that were examined: In all rats before dosing and in the control and high-dosage groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to termination
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (overnight)
- How many animals: All
- Parameters checked: Packed cell volume (PVC), haemoglobin (Hb), red blood cell count (RBC), platelet count (Plts), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), total white blood cell count (WBC), neutrophils (N), lymphocytes(L), eosinophils (E), basophils (B), monocytes (M), thrombotest

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to termination
- Animals fasted: Yes
- How many animals: All
- Parameters checked: Glucose, GPT, GOT, AP, total bilirubin, cholesterol (Chol), urea nitrogen (Urea Nitr), total protein, Albumin (Al), Globulin (Glob), Na, K, Ca, Cl, P, Creatinine

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to termination
- Animals fasted: Yes
- Parameters checked: Volume, pH, specific gravity, protein, glucose, ketones, bile pigments, urobilinogen, haem pigments, microscopy

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Adrenals, brain, kidneys, liver, ovaries, testes (epididymides)

HISTOPATHOLOGY: Yes
adrenals, eyes, heart, kidneys, liver, spleen, any other macroscopically abnormal tissue

Statistics:

All statistical analyses were carried out separately for males and females.
Bodyweight data were analysed using weight gains.
The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology data:
(i) If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75%), the proportion of values different from the mode was analysed by appropriate methods. Otherwise:
(ii) Bartlett’s test (1) was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level) heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
(iii) If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one—way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal—Wallis analysis of ranks (2) was used.
(iv) Analyses of variance were followed by Student’s ‘V test and Williams’ test (4) for a dose—related response, although only the one thought more appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the ‘V test and Williams’ test (Shirley’s test, (3)).
Where appropriate, analysis of covariance was used in place of analysis of variance in the above sequence. For organ weight data, analysis initially involved a correlation analysis between organ weights and final bodyweight. For organs where a correlation at the 10% level of significance was established, analysis of organ weight data was performed using adjusted organ weights by analysis of covariance with final bodyweight as covariate (5). Where a correlation between organ weight and bodyweight was not established, the organ weight analysis was carried out using routine analysis of variance on unadjusted organ weights.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No mortalities but clinical signs at 1055 mg/kg/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortalities but clinical signs at 1055 mg/kg/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1055 mg/kg/day: Impaired bodyweight gain.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1055 mg/kg/day: Decrease in consumption.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

1055 mg/kg/day: Higher food conversion ratios.

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

1055 mg/kg/day: Increase in males through Week 3.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

1055 mg/kg/day: Increase in Na, Cl, Ca and cholesterol.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

1055 mg/kg/day: Minor changes in pH and specific gravity.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

1055 mg/kg/day: Increased kidney weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

1055 mg/kg/day: Kidney changes.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1055 mg/kg/day: Kidney changes.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Several clinical signs were observed among rats receiving 1055 mg/kg/day from Day 27 to termination.
These signs, namely pilo-erection, hunched posture, waddling, lethargy or incoordination and pallor of the extremities, were attributed to an increased sensitivity in response to the fasting/blood sampling procedure.

BODY WEIGHT AND WEIGHT GAIN
Significantly lower bodyweight gains and, consequently, lower actual bodyweights, were shown by male rats receiving 1055 mg/kg/day, over the majority of the treatment period.

FOOD CONSUMPTION, COMPOUND INTAKE & FOOD EFFICIENCY
A slightly lower food consumption was seen for male rats receiving 1055 mg/kg/day throughout the treatment period. The food conversion ratio for these animals was increased during Weeks 3 and 4 in comparison with controls, indicating impaired efficiency of food utilisation.

WATER CONSUMPTION
A marked increase in water consumption was recorded over Week 3 for male rats treated at 1055 mg/kg/day.

CLINICAL CHEMISTRY
Significantly higher sodium, chloride and calcium ion levels were recorded for male rats treated at 1055 mg/kg/day and an increased level of cholesterol, in comparison with controls, was also seen among these rats.

URINALYSIS
A significantly greater volume of urine, produced by male rats treated at 1055 mg/kg/day, reflected the increase in water consumption by these animals. Small but significant reductions were also seen in the urine pH and specific gravity for these rats.

ORGAN WEIGHTS
Absolute kidney weights were significantly higher (P<0.01) for male rats receiving 1055 mg/kg/day in comparison with controls.

GROSS PATHOLOGY
Pale kidneys with irregular cortical scarring were observed in four male rats treated at 1055 mg/kg/day. In three animals of this group, the kidneys were enlarged and indurated.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related changes were seen in the kidneys of male and female rats treated at 1055 mg/kg/day. These changes included areas of dilated basophilic tubules some containing cell debris, hyperplasia of the urothelium and collecting tubule epithelium, degeneration or necrosis of the papillary tip and interstitial inflanunatory cell infiltration sometimes accompanied by loss of tubules.
There were no microscopic changes noted for rats receiving 105 or 11 mg/kg/day that were considered to be related to treatment.

Effect levels

1

Dose descriptor:

LOAEL

Effect level:

1 055 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Changes seen at 1055 mg/kg/day indicated a treatment-related effect particularly affecting the kidneys with susceptibility more pronounced among male rats.

2

Dose descriptor:

NOAEL

Effect level:

105 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Bodyweight gain - Group mean values (g)
Week	Group and dosage (mg/kg/day)
	male				female
	0	11	105	1055	0	11	105	1055
1	60	61	65	48**	22	24	26	27
2	50	49	53	33**	18	21	20	18
3	44	44	50	25	24	23	16	29
4	20	16	19	2*	4	2	8	5
* p<0.05; ** p<0.01

Table 2: Changes in biochemistry - Group mean values (g)
Parameter	Group and dosage (mg/kg/day)
	male				female
	0	11	105	1055	0	11	105	1055
Na (mEq/L)	140	141	140	146**	140	140	141*	141*
Cl (mEq/L)	96	97	97	101**	98	99	98	98
Ca (mEq/L)	5.4	5.4	5.4	5.7*	5.5	5.5	5.6	5.5
Chol (mg/dL)	49	50	48	68**	69	73	70	71
* p<0.05; ** p<0.01

Table 3: Kidney weights - Group mean values (g)
Group and dosage (mg/kg/day)
male				female
0	11	105	1055	0	11	105	1055
3.08	3.44	3.33	4.18**	1.87	1.94	1.95	2.03
** p<0.01

Table 4: Urinalysis - Group mean values (g)
Parameter	Group and dosage (mg/kg/day)
	male				female
	0	11	105	1055	0	11	105	1055
Volume (mL)	7.2	6.4	6.4	12.5**	3.6	3.4	4.3	5.8
pH	6.6	6.4	6.5	6.2**	6.4	6.0	6.1	6.3
** p<0.01