Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
12th Nov., 1996 - 12th Sep., 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance 2,4-dichlorophenoxyacetic acid (94-75-7). In accordance with the ECHA guidance document "Practical guide 6. How to report read-across ant categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Not all parameters tested are documented in the report
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,4-D
- Physical state: solid white powder with specific smell
- Analytical purity: 99.2%
- Impurities (identity and concentrations):
Chlorphenols, expressed as 2,4-dichlorphenol = 0.1%
- Purity test date: 30.10.1996

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Institute of occupational medicine, Lodz, Poland
- Age at study initiation: (P) 7 - 8 weeks;
- Weight at study initiation: (P) Males: 163 g (mean); Females: 131 g (mean); (F1) Males: 75-126 g; Females: 63-109 g
- Housing: steel wire mesh units with a plastic bottom (35 x 30 x 20 cm)
- Diet: Murigran (granulated standard food), ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40 - 80
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): according to Kolodziejczyk (1977, in polish)
- Mixing appropriate amounts with (Type of food): Granulated "Murigran" standard food

Details on mating procedure:
- M/F ratio per cage: 1/2
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually during the pregnant and weaning period
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Treatment of the parental generation: 10 weeks prior mating period, throughout the 3-week gestation period and up to the weaning of the offspring. The F1 generation was treated analogously until the end of the experiment (28-day weaning period).
Frequency of treatment:
Food was provided ad libitum
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 28 days of age.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
200, 600, 1800 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
approx. 10, 30, 90 mg/kg bw/d
Basis:
nominal conc.
based on a mean bogy weight of the animals and the mean food consumption.
No. of animals per sex per dose:
16 males and 32 females
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Based on preliminary data of other experiments and publications.
- Rationale for animal assignment (if not random): Group assignment was performed with a randomisation method

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily throughout the test period
- all behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality were recorded.

BODY WEIGHT: Yes
Time schedule for examinations:
- weekly during the pre-mating period
- during pregnancy body weight was measured on days 0, 6, 10, 15 and 20.
- during lactation body weight was measured on days 1, 4, 7, 14, 21 and 28 after parturition.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Time schedule for examinations:
- weekly during the pre-mating period
- during pregnancy food consumption was measured on days 0, 6, 10, 15 and 20.
- during lactation food consumption was measured on days 1, 4, 7, 14, 21 and 28 after parturition.
Litter observations:
Each litter was examined as soon as possible after delivery.

STANDARDISATION OF LITTERS
-Excess pups of the F1 generation were killed and discarded after weaning (28 days old)

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
- Weight of litters was examined on days 1, 4, 7, 14, 21 and 28 after parturition
- number and sex of pups, stillbirths, live births, presence of gross anomalies, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All males were killed and examined at the end of the mating period.
- Maternal animals: All females were killed and examined after the 28-day weaning period.

GROSS NECROPSY
- Gross necropsy consisted of macroscopically examination for any structural abnormalities or pathological changes with special attention to organs of the reproductive system.

HISTOPATHOLOGY
The ovaries, uterus, cervix, vagina, testes, seminal vesicles, prostate, coagulating gland and pituitary gland were preserved for microscopic examination. These organs were formalin- fixed and stained with hematoxylin and eosin. The preparations were examined with a light microscope.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 28 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
Gross necropsy consisted of macroscopically examination for any structural abnormalities or pathological changes with special attention to organs of the reproductive system and defects.

HISTOPATHOLOGY
The ovaries, uterus, cervix, vagina, testes, seminal vesicles, prostate, coagulating gland and pituitary gland were preserved for microscopic examination. These organs were formalin- fixed and stained with hematoxylin and eosin. The preparations were examined with a light microscope.
Statistics:
For statistical evaluation of the results a t-test with p = 0.05 was used. The results are given as mean values with a standard deviation.
Reproductive indices:
fertility index, pregnancy index, live born index, 4-day survival index, 14-day survival index, general survival index, lactation index

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

The parental animals do not differ in outer appearance and behaviour from the control group (generation P and F1)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
Generation P
- in general, there was no difference in outer appearence and behaviour between the treatment groups
- During the study 4 parental females died, 3 in the 200 ppm-group (13 day of pregnancy, at the parturition and 4 days after parturition) and one in the 1800 ppm-group (21 day of pregnancy).
- 7 rats showed bronchial murmur
- 2 rats had otitis media
None of the deaths were attributed to be an direct effect of the treatment with the test substance. The pathomorphologic examination revealed non specific changes and the incidence was comparable with the historical control values.

F1 generation:
- In the 1800 ppm group 1 parental female died at parturition and a male died in the fourth week before mating. Bleeding in the reproductive organs of the female animal was observed and the dead male showed hydronephrosis.
-In single F1-rats spread over all groups bronchial murmur (3), hind limb paresis (1) and enlargement of eyeball and corneal macula (1) was stated.
-In the killed paternal rats of generation F1 there was found megalosplenia, and cardiosymphsis in one control female and another one belonging to the 1800ppm-group showed uterus enlargement. In one control male stagnation of urine in the bladder was observed.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
Body weight of P-male in the exposed group did not differ significantly from the control.
In the females both, P and F1 in the 1800 ppm-group there was noted a statistically significant decrease of body weight in the whole study time including pre-mating, mating, gestation and weaning period.
F1 males showed a decrease of body weight in the 1800 ppm-group during the first 8 weeks.

Relative food consumption and therefore compound intace decreased over the time of the study. This is expected due to the initially rapid growth and the increasing body weight of the animals.
Food consumption in males did not differ in the exposed and control group, except week 8, where there was noted a decrease in all exposed groups.
A statistically significant higher food consumption of P-females was observed in the pre-mating period of the 1800 ppm-group and partly in the 600 ppm-group.
The 1800 ppm-group of F1-females a decrease of food-intake was only stated in the first 4 weeks of the pre-mating period and in the last week of the lactation period.
A higher food consumption of the females in the exposed groups was observed compared to the control. This was statistically significant in F1-females in the 200 ppm-group, partly in the 600 ppm-group and also in the 1800 ppm-group (but only in the last week).


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All females were successfully mated. No differences were observed in the general reproductive behaviour, pregnancy, amount of dead pups and succsess of breeding.


GROSS PATHOLOGY (PARENTAL ANIMALS):
In the females that died dead pups and bleeding in the reproductive organs were found. In two females (200 and 1800 ppm group) pneumonia purulenta was observed.
In one female of the 200 ppm-group the uterus was enlarged. In 4 females of the 1800 ppm-group pneumonia purulenta was found.
No lesions were observed in the examined organs of males of generation P and F1.


HISTOPATHOLOGY (PARENTAL ANIMALS)
In the pathomorphologic examination there were stated a few non-specific lesions like circulatory disturbances, progressive and inflammatory changes in the control and in the exposed rats. The circulatory disturbance occurred as hyperemia in the hypophysis and in few cases in the ovaries.
No lesions were observed in the examined organs of males.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 800 ppm (analytical)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: body weight
Dose descriptor:
NOAEL
Effect level:
600 ppm (analytical)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: body weight
Dose descriptor:
NOAEL
Effect level:
ca. 30 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: body weight
Dose descriptor:
NOAEL
Effect level:
ca. 180 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: body weight

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING):
No differences were stated in number of born pups, live born and dead born pups and the percentage of males and females in the treated groups and the control.
No differences were observed in these indices in generation F1 and partly in F2 in comparison with the control and within F1 and F2.
The 14-days survival index, the general survival index and the lactation index of the 1800 ppm-group of the F2-generation were significantly smaller than in the control.
The mortality of pups was higher in the 1800 ppm-group in both generations, beginning from the 8th day of life. The general mortality in the generation F1 was 5.4%, 8.4%, 5.4%, and 26% respectively in treatment groups with increasing doses, respectively. In the F2 generation, the mortality was 2.6%, 1.6%, 7.5% and 43.5%.


CLINICAL SIGNS (OFFSPRING):
In single animals of generation F1, evenly spread over all groups, bronchial murmur was stated (9 cases). 3 pups in the 600 ppm-group showed diminution of the eye-ball and corneal macula.
In the offspring F1 in the 1800 ppm-group the following lesions were noted: convulsions (64 animals), dejection (54 animals), enlargement of the eye-ball (2 animals), corneal macula (5 animals) and imbalance (1 animal). At 13 pups in one litter there was stated tympanites.
These lesions were also observed in offspring F2 in the 1800 ppm-group (convulsion – 27), (dejection – 62), (diminish of eye-ball and corneal macula – 1).


BODY WEIGHT (OFFSPRING):
No difference in the mean body weight of the pups in both generations was determined in the control and in the first two dosage groups.
This regards also the 1800 ppm-group till day 7 for F1- and day 4 for F2-generation. Later, a statistically significant decrease of body weight in relation to the control was observed. At the end of the weaning the mean body weight in 1800 ppm-group of F1 was 37% smaller than in the control and in the F2, the body weight was reduced by 39%, respectively.


GROSS PATHOLOGY (OFFSPRING)
In the dead offspring (F1 and F2) of the control and exposed groups no pathologic changes were found.
Gravel in the bladder was observed in single animals of the control and the 1800 ppm-group belonging to killed F1-infants. The 1800 ppm-group showed also pneumonia purulent.
In the 1800 ppm-group of the killed infants belonging to F2-generation, catarrhal enteritis was observed in a few cases.
In two rats, a male and a female of the 200 ppm-group of generation F2, hyperplasia of hypophysis was observed.
Single hyperplasia of uterus was noted in few cases and cysts in ovaries. Pyometritis and ovaritis were found in single females of the control and the treated groups.

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
600 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight gain; clinical signs; mortality
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight gain; clinical signs; mortality

Results: F2 generation

Effect levels (F2)

open allclose all
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 14 day survival index; survival index; lactation index
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
ca. 30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 14 day survival index; survival index; lactation index

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion