Dimethylammonium 2,4-dichlorophenoxyacetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic 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:

publication

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: EPA-Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation, Human and Domestic Animals, Series 82-1, 83-1, and 82, revised ed., November, 1984, US Department of Commerce, National Technical Information Service PB86-108958.

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dimethylammonium 2,4-dichlorophenoxyacetate

EC Number:

217-915-8

EC Name:

Dimethylammonium 2,4-dichlorophenoxyacetate

Cas Number:

2008-39-1

Molecular formula:

C8H6Cl2O3.C2H7N

IUPAC Name:

dimethylammonium 2,4-dichlorophenoxyacetate

Details on test material:

- Name of test material (as cited in study report):2,4-D dimethylamine salt, DMA
- Physical state: technical grade aqueous solution
- Analytical purity: 66.2% active ingredient
- Source of the test substance. Industry Task Force II on 2,4-D Research Data

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: 4 weeks
- Housing: individually in elevated stainless steel, wire-mesh cages
- Diet: Purina certified rodent chow 5 002, ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 /12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

acetone

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Purina certified rodent chow 5 002

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of DMA in the diets were analytically verified by gas chromatography.
Concentration analyses of the dietary formulations for each group were determined weekly till the end of the study.
They indicated that all diets were homogeneous and within ±10% of targeted concentrations. DMA intakes were generally within ±10% of targeted doses, as calculated from the diet concentration analyses, the actual food consumption, and body weight measurements.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Feed mixed with the test substance was available ad libitum

Doses / concentrations

Remarks:

Doses / Concentrations:
1, 15, 100 and 300 mg/kg bw/day (acid equivalent)
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose selection was based on the results of a previous short-time testing (Gorzinski et al. 1987)
- Rationale for animal assignment (if not random): Animals were randomly assigned by a weight randomization computer program designed to ensure homogenicity of body weights
- Animal selection. Animals were selected based on the examination of a veterinarian.

Examinations

Observations and examinations performed and frequency:

Toxicity, moribundity, and mortality were observed at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examinations were conducted prior to treatment and at the end of the study (week 13).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: hematology was performed during weeks 6 and 13
- Anaesthetic used for blood collection: Yes, blood samples were collected by orbital sinus venipuncture under ketamine hydrochloride anesthesia
- Animals fasted: Yes, over night
- Parameters checked: cell morphology, corrected leukocyte count, erythrocyte count, hematocrit, hemoglobin, leukocyte count,
leukocyte differential, and platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: clinical chemistry was performed during weeks 6 and 13
- Animals fasted: Yes, over night
- Parameters checked: alanine aminotransferase (ALT), albumin, aspartate aminotransferase (AST), blood urea nitrogen (BUN),
lactate dehydrogenase (LDH), alkaline phosphatase, calcium, chloride, creatinine, globulin, glucose, inorganic phosphorus, potassium, sodium,
total bilirubin, total cholesterol, total protein, triiodothyronine (T3), and thyroxine (T4)

URINALYSIS: Yes
- Time schedule for collection of urine: Collection of urine was performed during weeks 6 and 13
- Metabolism cages used for collection of urine: Yes, urine samples were collected over night in urine collection cages
- Animals fasted: Yes
- Parameters checked: bilirubin, glucose, ketones, pH, protein, specific gravity, and urobilinogen


OTHER:
Clinical pathology was performed during weeks 6 and 13

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals sacrificed at study termination (week 13) were anesthetized by sodium pentobarbital, exsanguinated, and subjected
to gross and microscopic examinations. A complete necropsy was performed on all animals.
Following organs were weight: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, testes (with epididymides), thymus, and thyroid/parathyroids

HISTOPATHOLOGY: Yes
The following tissues from each animal were preserved in 10% neutral-buffered Formalin:
adrenals, aorta, bone marrow (stemum), brain with brain stem, cecum, esophagus, eyes (fixed in Bouin's and stored in 70% alcohol), femur, heart, kidneys, duodenum, jejunum, ileum, colon, rectum, lacrimal gland, liver, lung, mammary gland, mesenteric lymph nodes, ovaries, pancreas, pituitary, salivary gland, sciatic nerve, skeletal muscle, spinal cord (three levels), spleen, stomach, testes with epididymides, thymus,
thyroid/parathyroids, trachea, urinary bladder, uterus, and any other tissues with gross lesions.
Preserved tissues were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically.

Statistics:

Different tests were performed (e.g. Bartlett, Levene, Dunnett).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

adverse

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

non adverse

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

non adverse

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

non adverse

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
The test substance was relatively well tolerated and no treatment related deaths or illnesses were stated.

BODY WEIGHT AND WEIGHT GAIN AND FOOD CONSUMPTION:
Severe body weight gain depressions were observed at the dose level of 300 mg acid equivalents/kg/day.
The rates of body weight gain and food consumption were retarded in certain groups of treated animals of both sexes when compared with controls.
For males this depression ranged from 37 to 80% of controls; for females the range was 57 to 88%.
The no-observed-effect level (NOEL) for body weight and food consumption effects for DMA was considered to be 100 mg/kg bw/day (acid equivalent).

HAEMATOLOGY AND CLINICAL CHEMISTRY:
Clinical chemistry, hematology, and urine-analysis were performed during weeks 6 and 13. Changes in red cell mass, platelet count, T3, and T4 were observed. Blood urea nitrogen (BUN) was increased in the high dose animals (females) whereas the haemoglobin concentration was reduced at this concentration. 100 mg/kg bw/day (acid equivalent) significantly reduced the T3 and T4 levels (females), the red blood cell count (females), and platelet counts (females).
The NOEL for hematology and clinical chemistry effects was set to 15 mg/kg bw/day (acid equivalent).

ORGAN WEIGHTS:
Effects of DMA were determined for liver, kidney, testes and thyroid. The thyroid/ bw ratio and the liver weight was significantly increased in the high dose animals (male and female). Furthermore, inkreased kidney weight was observed in animal streated with 100 mg/kg bw/day (acid equivalent)(male and female). The weight of the testes of the male rats was significantly reduced in the highest dose group but unaffected in the other treatment groups. The NOEL for organ weight effects was set to 15 mg acid equivalents/kg/day.

GROSS PATHOLOGY AND HISTOPATHOLOGY: NON-NEOPLASTIC:
Histological alterations were observed predominantly at the 300 mg/kg bw/day (acid equivalent) group and consisted of retinal degeneration and
cataract formation (females), centrilobular hepatocellular hypertrophy (both sexes), atrophy of the testes, hypertrophy in the zona glomerulosa
of the adrenal cortex (both sexes), brush border loss in proximal tubular cells in the kidney (both sexes), and vacuolization of kidney tubular cells
(both sexes). The effects noted in the liver, testes, kidney, and adrenal were considered to be of a slight degree and occurred at a dose that clearly exceeded the maximum tolerated dose (MTD).
The overall NOEL following histological evaluation was considered to be 15 mg/kg bw/day (acid equivalent).

Effect levels

1

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: haematology; clinical chemistry; organ weights

2

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: haematology; clinical chemistry; organ weights

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Key results of the 13-week study in F344 rats on DMA

	mg/kg/day (acid equivalents)
	Control	1	15	100	300
male
Survival	10/10	10/10	10/10	10/10	10/10
Body weight gain (g)	173 ± 12	168 ± 18	172 ± 11	173 ± 15	100 ± 15*
RBC counts (millions/µL)	9.6 ± 0.2	9.6 ± 0.1	9.5 ± 0.3	9.5 ± 0.1	8.5 ± 0.1*
Hemoglobin (g/dL)	16.9 ± 0.4	16.9 ± 0.2	16.8 ± 0.4	16.8 ± 0.3	16.4 ± 0.2*
Platelet counts (thousands/µL)	854 ± 37	813 ± 138	833 ± 39	854 ± 90	708 ± 28*
T3 levels (ng/dL)	85.9 ± 21.7	99.9 ± 28.0	97.4 ± 16.4	89.4 ± 18.3	75.3 ± 17.8
T4 levels (µg/dL)	3.9 ± 0.6	4.5 ± 0.5	4.2 ± 0.7	3.4 ± 0.6	1.0 ± 0.1*
Thyroid/body weight ratio	0.005 ± 0.001	0.006 ± 0.001	0.006 ± 0.001	0.006 ± 0	0.008 ± 0.002*
Testes/body weight ratio	1.41 ± 0.05	1.39 ± 0.05	1.42 ± 0.06	1.42 ± 0.11	0.80 ± 0.08*
Kidney/body weight ratio	0.67 ± 0.04	0.67 ± 0.03	0.71 ± 0.02	0.75 ± 0.03*	0.77 ± 0.03*
Liver/body weight ratio	2.49 ± 0.16	2.51 ± 0.14	2.58 ± 0.16	2.58 ± 0.13	3.06 ± 0.25*
Brush border loss of tubular cells	0/10	0/10	0/10	0/10	7/10
Hypertrophy of adrenal cortex	0/10	0/10	0/10	0/10	10/10
Centrilobular hepatocellular hypertrophy	0/10	0/10	0/10	0/10	0/10
Females
Survival	10/10	10/10	10/10	10/10	9/10
Body weight gain (g)	70 ± 9	75 ± 11	70 ± 6	61 ± 6	24 ± 14*
RBC counts (millions/µL)	9.0 ± 0.2	8.8 ± 0.2	9.0 ± 0.2	8.6 ± 0.1*	7.8 ± 0.5*
Hemoglobin (g/dL)	16.7 ± 0.4	16.4 ± 0.4	16.7 ± 0.3	16.3 ± 0.3	15.4 ± 1.1*
Platelet counts (thousands/µL)	838 ± 62	882 ± 33	882 ± 36	705 ± 52*	669 ± 31*
BUN (mg/dL)	17 ± 1.6	17 ± 1.1	17 ± 1.1	16 ± 1.7	21 ± 3.1*
T3 levels (ng/dL)	96.8 ± 14.9	92.9 ± 17.4	94.9 ± 11.7	75.7 ± 12.7*	71.1 ± 19.8*
T4 levels (µg/dL)	2.7 ± 0.6	2.4 ± 0.5	2.5 ± 0.6	0.9 ± 0.1*	0.9 ± 0.0*
Thyroid/body weight ratio	0.006 ± 0.001	0.008 ± 0.001	0.007 ± 0.003	0.008 ± 0.002	0.012 ± 0.003*
Kidney/body weight ratio	0.75 ± 0.05	0.73 ± 0.03	0.74 ± 0.05	0.80 ± 0.03	0.93 ± 0.10*
Liver/body weight ratio	2.72 ± 0.11	2.77 ± 0.10	2.73 ± 0.13	2.80 ± 0.11	4.10 ± 0.69*
Cataracts	0/10	0/10	0/10	0/10	7/10
Retinal degeneration	0/10	0/10	0/10	0/10	6/10
Hypertrophy of adrenal cortex	0/10	0/10	0/10	0/10	9/10
Brush border loss of tubular cells	0/10	0/10	0/10	0/10	8/10
Centrilobular hepatocellular hypertrophy	0/10	0/10	0/10	0/10	8/10

values reported are at the interval before study termination

* significantly different from the control value, p < 0.05