Dichlobenil

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 February 1987 to 17 June 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dichlobenil

EC Number:

214-787-5

EC Name:

Dichlobenil

Cas Number:

1194-65-6

Molecular formula:

C7H3Cl2N

IUPAC Name:

2,6-dichlorobenzonitrile

Test material form:

solid: particulate/powder

Test animals

Species:

hamster

Strain:

other: Bio F1 Alexander

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 55-75 %
- Photoperiod: 12 hours light/12 hours dark

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Spratt's LAD 2 Standard Rodent Diet
- Storage temperature of food: -20 °C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

GC analysis on samples taken from all dose groups from residues left in the foodhopper at the end of weeks 3, 6, 9 and 12 and in the fresh untouched diets of week 12 (used as reference).

Duration of treatment / exposure:

90 days (up to 4pm on the day before terminal kill in week 14 for main group)

Frequency of treatment:

Feed was available ad libitum and fresh food was given once a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

41 mg/kg diet

Doses / concentrations 2

Dose / conc.:

209 mg/kg diet

Doses / concentrations 3

Dose / conc.:

1 289 mg/kg diet

Doses / concentrations 4

Dose / conc.:

4 648 mg/kg diet

Remarks:

reduced after two weeks from 7500 mg/kg diet

No. of animals per sex per dose:

Ten (main group and recovery group for controls and 4648 mg/kg diet). Twenty for main group controls.

Control animals:

yes, plain diet

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: weekly
- At autopsy animals were weighed after overnight fasting to determine autopsy body weights to be used for calculating organ weights relative to body weight.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: individual food intake calculated for periods of seven days

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before treatment started and in 11th week of treatment
- Dose groups that were examined: control and highest dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 13 weeks of treatment and from the recovery animals, after 4 weeks recovery
- Anesthetic used for blood collection: Yes (light ether anaesthesia)
- Animals fasted: Yes (18 hours)
- How many animals: all
- Parameters checked: RBC, Hb, PCV, MCV, MCH, MCHC, platelet count, WBC and differential WBC, reticulocytes, PTT and APTT. At autopsy 1.8 mL of blood was taken for clotting observations.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 13 weeks of treatment and from the recovery animals, after 4 weeks recovery
- Animals fasted: Yes (18 hours)
- How many animals: all
- Parameters checked: AST, ALT, ALP, GCT, glucose, total protein, albumin, urea, creatinine, cholesterol, phospholipids, calcium, sodium and potassium

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- a full macroscopic examination was done on all animals and any abnormalities found were recorded.
- the following organs were weighed: brain, heart, liver, spleen, kidneys, thymus, prostate, seminal vesicles, testes, uterus and ovaries.
- for the recovery animals, the liver and kidney weights were recorded.

HISTOPATHOLOGY: Yes
- the following tissues were taken, fixed, sectioned and stained where appropriate: aorta, heart, lungs, trachea, salivary glands, liver, gall-bladder, pancreas, oesphagus, stomach, duodenum, ileum, jejunum, caecum, colon, rectum, urinary bladder, kidneys, testes, prostate, epididymides, seminal vesicle, ovaries, uterus, vagina, mesenteric lymph node, spleen, thymus, adrenals, pituitary gland, thyroid with parathyroid, skin with pelvia mammary gland, muscle (quadriceps femoris), sciatic never, brain, spinal cord, eyes and optic nerve

Statistics:

The basic analysis consists of linear regression analysis with treatment and block effects as parameters assuming constant error variance. The regression analysis was followed by Williams's test.

With some variables, one or two animals showed outlying responses. In such cases the Williams's test was replaced by its nonparametric version and the data were summarised by medians instead of means.

The incidence of macroscopic findings was subjected to a test for trend against dose level in a 5 x 2 contingency table. The statistical significance level for the trend statistic was obtained by enumeration of all possible permutations (permutation test).

Microscopic findings obtained on an ordinal scale were subjected to Shirley's test (as modified by Williams for comparing several dose levels with a zero-dose control).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed.

Mortality:

no mortality observed

Description (incidence):

No treatment-related mortality was observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The initial diet concentration of 7,500 mg/kg diet was not tolerated, after two weeks of treatment the animals had lost weight and were only 75 % and 80 % of the weight of concurrent controls (males and females respectively). Reducing the diet concentration to 4,648 mg/ kg resulted in an immediate return to weight gain although at a slower rate than that of the controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption of the males was reduced at the 1,289 and 4,648 mg/kg diet level during the entire treatment period and up to week 6 in the females of the 4,648 mg/kg diet group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related ophthalmoscopic abnormalities were observed.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematology effects indicated a very slight, reversible, macrocytic tendency in the highest dose group. Withdrawal of treatment for 4 weeks resulted in an increased number of platelets, reticulocytes and neutrophils, in the males at this dose level, indicating an active bone marrow.

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The prostate showed a dose-related weight reduction. Uterus and spleen weights, absolute and relative, were reduced at the 4,648 mg/kg diet level of the females; however, histopathology examination did not show any abnormality.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were indications of reversible morphological and functional effects on the liver. Following the withdrawal of treatment it was evident that the liver had recovered normal function and morphology
An increase in the incidence of mineralisation in the prostate at 209 - 4,648 mg/kg diet levels was noted. Tubular degeneration in the testes and decreased numbers of spermatocytes in the epididymis were seen in the 4,648 mg/kg diet group. Gall-bladder calculi occurred in both sexes at the highest diet level, this effect remained after the recovery period.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No histopathological abnormalities were seen in uterus and spleen in the female group at the 4,648 mg/kg diet level .

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

41 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

gross pathology

haematology

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Body weight gain (g)

		Dose rate (mg/kg diet, actual)
Time period (weeks)	Sex	0	41	209	1289	7500/4648	7500 /4648 (R)
-2 - 0	M	19.4	19.2	20.9	21.6	19.3
	F	20.2	21.7	21.1	20.0	17.8
1 - 3	M	16.4	18.0	17.7	15.4	-12.5**
	F	15.2	14.2	16.9	15.2	-10.3**
4 - 6	M	15.8	17.6	14.5	13.8	7.4**
	F	14.5	14.6	15.6	15.6	18.0*
7 - 9	M	5.8	6.7	4.7	5.0	2.9**
	F	8.5	8.7	10.6	9.3	12.6**
10 - 13	M	1.0	0.4	0.0	-1.1	5.2**
	F	-1.6	0.3	2.7	-1.3	8.8**
14 - 17	M	2.0 (R)					15.6**
	F	-0.7 (R)					6.5*

*statistical significant P≤0.05; **statistical significant P≤0.01

Table 2: Food consumption (g)

		Dose rate (mg/kg diet, actual)
Time period (weeks)	Sex	0	41	209	1289	7500/4648	7500/4648 (R)
-2 - 0	M	116	114	118	113	117
	F	130	128	130	123	125
1 - 3	M	175	171	176	163*	130**
	F	189	184	190	183	155**
4 - 6	M	183	177	177	171**	150**
	F	193	189	198	197	173**
7 - 9	M	161	159	152	151*	133**
	F	187	177	182	176	169
10 - 13	M	193	185	178*	179*	171**
	F	214	212	222	207	212
14 - 17	M	194 (R)					199
	F	226 (R)					216

Table 3: Selected clinical chemistry values

		Dose rate (mg/kg diet, actual)
		After treatment period (13 weeks)					After recovery period (4 weeks)
Parameter	Sex	0	41	209	1289	4648	0	4648
AST (U/L)	M	34	39	34	33	37	40	31**
	F	43	47	41	36	33*	42	35
ALT (U/L)	M	53	56	54	65**	64**	62	56
	F	73	84*	101*	85*	100**	94	84
ALP (U/L)	M	161	149	161	163	168	138	180**
	F	213	202	200	194	200	228	223
Glucose (mmol/L)	M	4.64	4.05	4.62	4.08	4.66	4.07	4.73
	F	5.16	5.21	5.05	5.74	4.25*	4.71	4.98
T. prot (g/L)	M	70.1	69.6	69.1	69.3	71.3	70.9	72.6*
	F	72.4	72.2	73.1	72.4	75.3*	75.7	73.2
Albumin (g/L)	M	55.4	55.6	55.7	57.2	59.8**	45.3	46.8*
	F	53.4	53.3	54.2	55.2	60.1*	43.2	41.7*
Urea (mmol/L)	M	7.00	6.76	7.30	7.31	8.33**	7.98	7.73
	F	8.10	7.88	8.26	8.71	9.39*	9.02	8.46
Creat (µmol/L)	M	28.5	27.0	27.0	26.0*	25.0**	27.2	26.8
	F	31.0	29.0	29.4	28.6	26.0**	29.5	27.3**
Chol. (mmol/L)	M	3.45	2.99+	2.78 ++	2.94+	4.27 ++	3.64	3.59
	F	4.08	4.18	4.56	4.88**	6.31**	4.76	4.75
Ph. Lip. (mmol/L)	M	2.91	2.70	2.61	2.74	3.65**	2.95	3.06
	F	3.10	3.24	3.62**	3.67**	4.82**	3.19	3.40
Sodium (mmol/L)	M	147.2	147.7	145.4	146.9	147.1	147.0	145.0
	F	145.8	146.3	145.5	146.4	145.3	146.3	143.8*

*statistical significant, P≤0.05; **statistical significant, P≤0.01; results of Student's test since the mean response is not a monotonic function of dose: + P≤0.05, ++ P≤0.01

Table 4: Selected absolute and relative organ weights

			Dose rate (mg/kg diet, actual)
			After treatment period (13 weeks)					After recovery period (4 weeks)
Organ		Sex	0	41	209	1289	4648	0	4648
Body weight (g)		M	138	140	137	133	101**	139	119**
		F	136	139	147 ++	138	128+	136	131
Brain (g)	abs^1	M	0.980	0.985	0.990	0.995	0.990	-	-
	rel^1	M	0.709	0.690	0.730	0.752*	0.955*	-	-
Heart (g)	abs	M	0.536	0.529	0.528	0.525	0.437**	-	-
	rel^1	M	0.390	0.381	0.385	0.397	0.435**	-	-
	abs	F	0.591	0.569	0.574	0.578	0.547*	-	-
	rel	F	0.436	0.411	0.391	0.419	0.428	-	-
Liver (g)	abs	M	4.39	4.34	4.29	4.97**	6.66**	4.42	4.44
	rel	M	3.18	3.09	3.12	3.73**	6.59**	3.17	3.72**
	abs	F	4.74	4.93	5.52**	6.36**	8.78**	4.93	5.11
	rel	F	3.47	3.54	3.75*	4.59**	6.84**	3.60	3.88
Spleen (g)	abs	M	0.112	0.118	0.107	0.107	0.074**	-	-
	rel	M	0.081	0.085	0.077	0.081	0.074	-	-
	abs	F	0.166	0.151	0.164	0.157	0.126**	-	-
	rel	F	0.122	0.110	0.112	0.114	0.100**	-	-
Kidneys (g)	abs^1	M	1.005	0.975	0.990	0.990	0.900**	0.949	0.903
	rel^1	M	0.735	0.690	0.725	0.755	0.880**	0.683	0.760**
	abs^1	F	1.160	1.190	1.255	1.230	1.205	1.151	1.019*
	rel^1	F	0.840	0.835	0.830	0.925	0.905**	0.848	0.779**
Thymus (g)	abs	M	0.040	0.048	0.043	0.042	0.034*	-	-
	rel	M	0.029	0.034	0.030	0.030	0.031	-	-
Prostate (mg)	abs	M	388	342	313*	297**	182**	-	-
	rel	M	283	247	228*	224**	182**	-	-
Sem. ves. (g)	abs	M	1.41	1.35	1.19	1.19	0.72*	-	-
	rel	M	1.01	0.97	0.84	0.90	0.70**	-	-
Testes (g)	abs	M	3.27	3.26	3.07	3.15	2.20**	-	-
	rel	M	2.38	2.35	2.25	2.39	2.19	-	-
Uterus (g)	abs	F	0.40	0.41	0.37	0.35	0.30**	-	-
	rel	F	0.29	0.29	0.25	0.26	0.23*	-	-

*statistical significant, P≤0.05; **statistical significant, P≤0.01; 1 medians are presented and nonparameteric test was applied because of an outlying observation; results of Student's t-test since the mean response is not a monotonic function of dose: + P≤0.05, ++P≤0.01

Table 5: Selected microscopic observations

		Dose rate (mg/kg diet, actual)
		Sex	After treatment period (13 weeks)					After recovery period (4 weeks)
			0	41	209	1289	4648	0	4648
Numbers examined		M	20	10	10	10	9	10	10
		F	20	10	10	10	10	10	10
Organ	Observation
Epididymis	No abnormalities	M	19			10	4
	Spermatocytes decreased	M	1			0	5*
Gallbladder	No abnormalities	M	20	10	9	9	2**	8	2
	No abnormalities	F	20	10	10	9	0**	9	0**
Prostate	No abnormalities	M	16	9	6	5	5
	Mineralisation (marginal/slight/moderate)	M	0/0/1	0/0/1	0/1/2	1/3/0	0/2/1
	Prostatis (marginal/slight/moderate)	M	0/2/2	0/0/1	0/0/2	1/2/0	0/1/1
Testes	No abnormalities	M	18				2
	Tubules degenerated (marginal/slight/moderate/marked)	M	0/1/0/1				** 2/0/4/1

* incidences yielding statistical significance (P≤0.1) in a two-sided test against trend with dose level; **Shirley's test P≤0.1

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, the NOEL of the test material in a 90 day repeat dose study in the hamster was determined to be 41 mg/kg diet.

Executive summary:

In a GLP compliant repeat dose (oral) study conducted in line with EPA OPP 82-1, the effects of the repeat dose of the test material over 90 days was determined. Hamsters were fed a diet containing 0, 41, 209, 1289 and 4648 mg/kg diet (7500 mg/kg diet for first two weeks) test material.

Under the conditions of the test, the NOEL of the test material in a 90 day repeat dose study in the hamster was determined to be 41 mg/kg diet.