Dichlobenil

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 July 1987 to 21 April 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1988

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1992

Reference 3

Reference Type:

study report

Title:

Unnamed

Year:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Deviations:

yes

Remarks:

(actual relative humidity was 20-94%; three incorrect pups were selected for the F1 generation; in addition, the following protocol deviation was also reported; on three occasions test diets were prepared in advance and stored frozen)

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

Details on test material:

- Physical state: solid
- Storage condition of test material: in the dark at ambient room temperature

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (P) approxaimtely 6 wks; (F1) nominally 4 wks
- Weight at study initiation: (P) Males: 139.7-217.4 g; Females: 107.7-149.9 g;
- Housing: stainless steel wireless cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 40-70 %
- Air changes: minimum 15 per hour
- Photoperiod: 12 hours light/12 hours dark

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly (excpet on three occassions when the diet was prepared in advance and frozen)

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 15 days
- Proof of pregnancy: sperm in vaginal smear

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken from the diet formulations prepared in weeks 1, 13, 26 and 36 and analysed by shaking samples with hexane and water (low dose groups) or hexane only (high dose groups). Hexane solutions are further diluted and analysed using GC.

Duration of treatment / exposure:

Two generations

Frequency of treatment:

Daily

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

60 ppm

Doses / concentrations 2

Dose / conc.:

350 ppm

Doses / concentrations 3

Dose / conc.:

2 000 ppm

No. of animals per sex per dose:

Thirty (P); twenty-five (F1 generation)

Control animals:

yes, plain diet

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly except for mated females which were weighed on days 0, 6, 12, 15 and 20 of gestation and days 1, 4, 7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes (weekly)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (sex distribution as equal as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
stillbirths, live births, postnatal mortality, litter weights, and clinical condition of pups

GROSS EXAMINATION OF DEAD PUPS:
yes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals (at time of weaning of F1 generation).
- Maternal animals: All surviving animals (at time of weaning of F1 generation).

GROSS NECROPSY
- Gross necropsy was performed.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were removed: ovaries, uterus, cervix, vagina, lesions, pituitary, testes, epididymides, seminal vesicles, prostate and coagulating gland.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at time of weaning.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy was performed.
- Number of implantation sites were counted and recorded for F1 females.

Statistics:

Using nested analysis of variance for normally distributed errors or by non-parametric techniques for non-normally distributed errors.

The standard deviation obtained from analysis of variance was used for 't' tests between control and treatment groups. Where necessary, data were transformed before analysis.

Non-parametric testing was performed using the Kruskal-Wallis test for a treatment-related response.

Significant differences between control and treatment groups were determined using the Wilcoxon rank sum test.

Reproductive indices:

Median pre-coital time = time by which half the females had mated

Fertility index = (no. of pregnant females / no. of paired females) x 100

Fecundity index = (no. of pregnant females / no. of mated females) x 100

Gestation index = (no. of females with live pups / no. of pregnant females) x 100

Offspring viability indices:

Post-implantation survival index (F1 only) = (no. of pups born / no. of implantation sites) x 100

Live birth index = (no. of pups alive on day 1 / no. of pups born) x 100

Viability index 1 = (no. of pups alive on day 4 before culling / no. of pups alive on day 1) x 100

Viability index 2 = (no. of pups alive on day 7 / no. of pups alive on day 4 after culling) x 100

Viability index 3 = (no. of pups alive on day 14 / no. of pups alive on day 7) x 100

Viability index 4 = (no. of pups alive on day 21 / no. of pups alive on day 14 x 100)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical observations of P generation adults were restricted to those commonly seen in this strain of rat in these laboratories e.g. occasional instances of rough hair coat, slight hair loss, fur staining and chronnodacryorrhoea. Incidence of fur staining and slight hair loss was slightly increased at 2000 ppm in P generation females during late gestation.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Treatment did not adversely affect mortality. One P generation male was found dead during week 14; necropsy revealed a liver mass. This death was not attributable to treatment. There were no other adult deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gains were lower in males and females at 2000 ppm; body weight gain in females during gestation was also lower at 2000 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was lower than controls in males and females at 2000 ppm. Initial lowering of food consumption in P generation animals was so marked as to suggest adverse palatability of the test material in the diet.

Food efficiency:

no effects observed

Description (incidence and severity):

Food conversion efficiency was similar in all groups, with the exception of week 1 for the highest dose level.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no significant histopathological effects on either the pituitary or reproductive tract attributable to test material administration.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance, fertility, fecundity, pregnancy rate and duration of gestation were not adversely affected by treatment.

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Effect level:

60 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: equivalent to 3 mg/kg bw/day Based on lower bodyweight gain in offspring in both adults and pups, no effects on fertility were noted

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical observations of F1 generation adults were restricted to those commonly seen in this strain of rat in these laboratories e.g. occasional instances of rough hair coat, slight hair loss, fur staining and chronnodacryorrhoea.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Offspring survival was not adversely affected by treatment. One F1 generation male was killed during week 12 following accidental injury to the snout. This death was not attributable to treatment. There were no other adult deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gains were lower in males and females at 2000 ppm; body weight gain in females during gestation was also lower at 2000 ppm. Body weight gain was lower than controls in F1 males at 350 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was lower than controls in males and females at 2000 ppm.

Food efficiency:

no effects observed

Description (incidence and severity):

Food conversion efficiency was similar in all groups.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy findings of weaned offspring were not adversely affected by treatment.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no significant histopathological findings for either the pituitary or reproductive tract attributable to test material administration. Hyaline droplet nephropathy was recorded in the kidneys of three high dose male. This finding was considered related to test material administration.

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

60 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: equivalent to 3 mg/kg bw/day Based on lower bodyweight gain in offspring in both adults and pups, no effects on fertility were noted

2

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No observed adverse effects

Target system / organ toxicity (F1)

Critical effects observed:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Summary of adult performance

	Dose group (ppm)
	0		60		350		2000
	P	F1	P	F1	P	F1	P	F1
Males
In group	30	25	30	25	30	25	30	25
Dead: post pairing	0	0	0	0	0	0	1	1
Failing to induce pregnancy	1	-	2	-	2	-	1	-
Females
In group	30	25	30	25	30	25	30	25
Pregnant/%	29/96.7	25/100	28/93.3	20/80	28/93.3	24/96	29/96.7	25/100
With total resorption	1	-	0	-	0	-	0	-
Total litter loss	4	4	3	4	3	4	5	2
With live pups at day 21 post-partum	24	21	25	16	25	20	24	23

Table 2: Group mean body weight gain (g)

		Dose group (ppm)
		0		60		350		2000
Time interval	Sex	P	F1	P	F1	P	F1	P	F1
0 -5	M	199.9	260.6	192.0	256.7	192.7	242.6	148.1	201.9
	F	90.5	120.5	91.9	122.1	88.0	111.5	67.1	98.7
6 -10	M	88.9	104.6	98.8	113.6	83.5	93.8	68.8	71.0
	F	32.1	40.3	31.0	37.7	34.5	39.4	23.8	33.3
11 -13	M	33.2	44.7	23.3	43.8	29.5	37.6	-6.1	41.7
	F	100	112	109	115	109	108	88	85
14 -16	M	29.8	31.8	29.3	41.8	30.7	48.3	53.7	42.6
	F	29	46	40	36	53	45	65	60

*14-17 weeks for males of the F1 generation

Table 3: Group mating data

	Dose group (ppm)
	0		60		350		2000
Mating indices	P	F1	P	F1	P	F1	P	F1
Number of paired females	30	25	30	25	30	25	30	25
Number of mated males	30	25	30	22	29	24	29	25
Number of pregnant females	29	25	28	20	28	24	29	25
Fertility index (%)	96.7	100	93.3	80	93.3	96	96.7	100
Fecundity index (%)	96.7	100	93.3	90.9	96.6	100	100	100

Table 4: Group mean pup weights (g)

	Dose group (ppm)
	0		60		350		2000
Day	P	F1	P	F1	P	F1	P	F1
1 (combined)	6.1	5.7	5.7	5.9	5.5	5.9	5.6	5.8
4 (combined)	8.6	7.9	7.9	8.4	7.2	7.7	6.9	7.8
7 (combined)	13.8	13.2	13.1	13.7	12.1	12.8	10.4	12.0
14 (combined)	29.8	30.0	29.1	29.8	27.4	27.4	21.9	24.4
21 (combined)	49.8	50.9	49.5	49.7	46.7	45.98	38.2	39.9
21 (males)	51.2	51.8	50.3	51.0	49.1	47.2	39.5	40.6
21 (females)	49.6	49.6	48.7	48.5	44.7	45.9	37.9	38.9
% weight changes	716	793	4768	742	749	678	582	588

Table 5: Group incidence histopathology findings

		P generation				F1 generation
		Dose group* and sex				Dose group* and sex
		0 ppm M	2000 ppm M	0 ppm F	2000 ppm F	0 ppm M	2000 ppm M	0 ppm F	2000 ppm F
Animals examined		30	30	30	30	25**	24**	25**	25**
Animals unremarkable		10	10	19	23	8	9	15	17
Skin	Acanthosis	0	1	3	3	1 91)	1 (2)	9 (9)	7 (7)
	Dermatitis	2	0	3	0	0 (1)	1 (2)	2 (9)	0 (7)
	Adnexal atrophy	-	-	-	-	0 (1)	0 (2)	0 (9)	1 (7)
Liver	Necrosis (lobar)	0	1	0	0	1 (1)	-	-	-
Kidneys	Focal nephropathy	0	10	0	0	-	-	-	-
	Hydroneophrosis	2	0	0	0	1 (1)	1 (3)	-	-
	Nephroblastoma	0	0	0	1	-	-	-	-
	Hyaline droplets	-	-	-	-	0 (1)	3 (3)	-	-
	Basophilic tubules	-	-	-	-	0 (1)	3 (3)	-	-
	Casts	-	-	-	-	0 (1)	3 (3)	-	-
	Cyst	-	-	-	-	0 (1)	1 (3)	-	-
Pituitary	Altered cell focus	0	2	0	0	-	-	-	-
	Cyst	0	3	0	0	1	0	0	0
	No sample	0	0	1	0	-	-	-	-
Oral cavity	Necropsy finding/ no equivalent finding	2	1	2	2	-	1 (1)	-	-
	Stomatitis	-	-	-	-	-	1 (1)	-	-
Nasal cavity	Rhinitis	-	-	-	-	-	1 (1)	-	-
Abdominal cavity	Necropsy finding/ no equivalent finding	0	1	0	0	-	-	-	-
Lungs	Inflammatory cell foci	-	-	-	-	1 (1)	-	-	-
	Congestion/heamorrhage	2	1	0	1	1 (1)	-	-	-
	Foamy histiocytes	2	1	0	0	-	-	-	-
	Lymphoid hyperlasia	1	1	0	0	-	-	-	-
	Pneunomitis	2	1	0	0	-	-	-	-
Skull	Exostoses	1	1	0	0	-	-	-	-
Foot	Arthritis	1	0	0	0	-	-	-	-
	Dermatitis	0	1	0	0	-	-	-	-
Tail	Dermatitis/folliculitis	0	3	0	0	1 (1)	-	-	-
Mandiubular lymph node	Hyperplasia	-	-	-	-
Ear	Chondritis	-	-	-	-
Testis	Atrophy	1	1	-	-	2	2	-	-
	Sperm granuloma	-	-	-	-	0	1	-	-
Epididymis	Inflammatory cell foci	0	1	-	-	1	0	-	-
	Oligospermia	0	1	-	-	0	2	-	-
Seminal vesicle	Not remarkable	30	30	-	-	24	25	-	-
Coagualting gland	One sample	4	3	-	-	0	1	-	-
Protstate	Inflammatory cell foci	-	-	-	-	6	6	-	-
	Prostatitis	10	10	-	-	3	4	-	-
Ovary	Not remarkable	-	-	30	30	-	-	25	25
	One sample	-	-	-	-	-	-	1	0
Uterus	Arteritis	-	-	1	0	-	-	0	0
	Oestrus stage	-	-	1	1	-	-	0	0
Cervix	Not remarkable	-	-	-	-	-	-	0	0
Vagina	Not remakrable	-	-	30	30	-	-	0	0
Cause of death	Liver lesion		1
	Upper respiratory infection/ stomatitis						1

If less animals were examined, the real number examined is presented between brackets.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test, administration of the test material to rats over two generations at dietary concentrations up to 2000 ppm did not adversely affect adult reproduction or offspring survival. Body weight gains were lower in adults and offspring at 2000 ppm and to a lesser extent at 350 ppm. Food consumption was also reduced in adults at 2000 ppm. No adverse effects of treatment were seen at the low level of 60 ppm which is considered to be the NOEL (equivalent to 3 mg/kg bw/day).

Executive summary:

In a GLP compliant study conducted in line with standardised guideline EPA OPP 83-4, the effect of the test material on reproductive toxicity was determined in the rat.

Rats were administered the test material by oral (diet) at 0, 60, 350 and 2000 ppm.

Adult reproduction or offspring survival was not adversely affected. Body weight gains were lower in adults and offspring at 2000 ppm and to a lesser extent at 350 ppm. Food consumption was also reduced in adults at 2000 ppm. No adverse effects of treatment were seen at the low level of 60 ppm which is considered to be the NOEL (equivalent to 3 mg/kg bw/day).