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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
three-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2nd May 1977 to 2nd January 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted with methods similar to recognised OECD guidelines although was conducted for 3 generations rather than 2. Although no data is available on the study's GLP compliance, equivalent quality assurance methods were in place at the testing laboratory. No analysis of diet formulations were made in this study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Principles of method if other than guideline:
The study carried out was a 3-generation reproduction study which is longer than a 2-generation study, however the methods used were similar to that of an OECD guideline for a 2-generation study just with more information on further generations of the species.
No analysis of diet formulations were made in this study.
GLP compliance:
no
Remarks:
Study pre-dates GLP however, Quality Assurance audits/inspections were performed during the study and included as a QA Audit record within the report
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test article, a brown crystalline solid (batch number MM 200025), was supplied in a Winchester by the study sponsor. The test article was melted by allowing hot water to run over the container. When the contents had melted, the Winchester was agitated to ensure homogeneity and the test article was decanted into a number of wide-necked containers, which were stored in a domestic refrigerator when not in use. The study was carried out using this one sample of test article.
Purity was determined to be 98%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Sprague Dawley derived rats of the CD strain obtained from a specific pathogen-free colony were used for the study. They were obtained from Charles River (UK) Ltd., Manston Road, Margate, Kent.
- Age at study initiation: (P) = ~40 days old at the start of the study
- Weight at study initiation: (P) Mean bw = Males: 191 g, Females: 158 g; (F1) Males: 86 g; Females: 80 g; (F2) Males: 67 g; Females: 64 g
- Housing: The animals were housed in solid floor polypropylene cages with stainless steel lids (North Kent Plastics, Dartford, Kent: dimensions 56 cm x 38 cm x 18 cm). Autoclaved soft wood chips were provided for bedding (supplied by the Sawdust Marketing Company, Standon, Herts.). Before parturition, mated females were provided with shredded tissue paper for nesting material.
Number of animals per cage:
Males - Females
pre-mating periods 5 - 5
mating periods (1 x 1) paired together
gestation periods 5 - 1
lactation periods 5 - 1 + litter
behavioural animals 6 - 6

- Diet (e.g. ad libitum): The basic diet used was Rat and Mouse Diet No. 1 Expanded and Reground (B.P. Nutrition, Stepfield, Witham, Essex) obtained in powdered form and offered in non-spill hoppers. After commencement of the study, the manufacturer modified the diet to increase the vitamin K level from 1ppm to 10ppm. This modified diet was used from the 35th week of the study until termination.
- Water (e.g. ad libitum): The animals had free access to mains water available from glass bottles attached to each cage. The water was changed daily and the bottles were replaced at weekly intervals with clean sterilised bottles.
- Acclimation period: The animals were acclimatised to the laboratories for a period of 4 days prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 10%
- Air changes (per hr): approximately 18 air changes per hour
- Photoperiod (hrs dark / hrs light): The animals were exposed to an artificial fluorescent photoperiod of 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): Batches of test diets were prepared at weekly intervals, although this schedule was altered infrequently to accommodate holidays or other unforeseen factors and unused test diet was discarded at the end of each week.
With the exception of the mating periods when male and female animals were housed together, separate diets were prepared for each dose level and sex, and a separate aliquot of test article was weighed for each dosage level.
- Mixing appropriate amounts with (Type of food): The required amount of test article was ground in a mortar with a small amount of powdered diet to form a mixture of high test article concentration. This concentrate was made up to the final weight with the remaining powdered diet and mixed in either a Morton '50E' batch mixer or a Gardner 3C double cone blender for approximately 15 minutes.
- Storage temperature of food: The formulated test diets were stored in galvanised metal bins with tightly fitting lids. This storage condition was necessary in view of the known property of the test article to migrate into plastic.
The stability of the test article in the powdered diet was carried out by HLE.



Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Vaginal smears were taken daily. Mating was confirmed by the presence of sperm in the vaginal smear. The day on which sperm was observed was designated day 0 of gestation. Smearing of individual animals was discontinued when mating was confirmed or the end of the 14 day mating period.

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No analysis of diet formulations were made in this study. Stability of Dinoseb in the diet when stored at room temperature for 7 days was demonstrated.
Duration of treatment / exposure:
The test diets were continuously available over 3 generations (87 weeks).
Frequency of treatment:
ad libitum in diet
Details on study schedule:
F0 generation:
Groups of 25 male and 25 female rats were fed the appropriate diet for a 13 week pre-mating period. The females were then paired on a 1:1 basis with males from the same dosage group for a 14 day period to produce the F1a litters. Vaginal smears were taken daily. Mating was confirmed by the presence of sperm in the vaginal smear. The day on which sperm was observed was designated day 0 of gestation. Smearing of individual animals was discontinued when mating was confirmed or the end of the 14 day mating period.

The females were allowed to litter and to rear their young to weaning. At the end of the weaning period, all the F1a pups were killed and necropsied. The parent (F0) animals were then re-mated on a 1:1 basis, normally with the same mate as for the first mating period. Ten (F0) females from each group were selected at random and killed on day 21 for teratological examination. Five (F0) females from each group were selected at random and designated 'behavioural animals'; these animals were allowed to litter and rear their young to weaning.
During the pre-weaning period a range of developmental parameters were examined in the offspring. At weaning, 2 male and 2 female pups were randomly selected from each 'behavioural' litter and the remaining pups were included in the random selection of the parents for the next generation.
The selected 'behavioural' pups were maintained untreated for a 3 month period during which time a range of behavioural functions were assessed. After 3 months, the 'behavioural' animals were killed and necropsied.
The remaining F0 females in each group were allowed to litter and to rear their young to weaning (F1b). At the end of the weaning period, 25 male and 15 female pups from each treatment group were selected at random from all the available F1b pups to form the F1 generation. The surplus pups and the F0 parents were then killed and necropsied.
Administration of the appropriate test diets to the F0 animals continued throughout the mating, gestation and lactation periods of both the F1a and F1b generation phases.

F1 generation:
The selected pups continued to be fed the experimental diets for a 13 week period and were then paired as for the F0 generation (avoiding sibling matings) to produce the F2a and F2b generations. These were examined as described for the F1a and F1b generations.

Administration of the appropriate test diets to the F1 animals continued throughout the mating, gestation and lactation periods of both the F2a and F2b generation phases.


F2 generation
The selected pups continued on the respective diets for a 13 week pre-mating period and then paired as for the F0 and F1 generations (avoiding sibling matings) to produce the F3a generation. The F3a pups were examined as described for the F1a generation. The F2 animals were then re-mated to produce the F3b generation. The F3b generation was examined as follows.

Twelve (F2) females from each group were selected at random and designated 'caesarean animals'. Of these females, those with known mating records were killed on day 21 for teratological examination. The remaining (F2) females in each group were allowed to litter and to rear their young to weaning. At weaning, 25 male and 25 female pups were selected at random from each group to form the breeding nucleus of an additional 2 generation reproductive study which will be reported separately. From the remaining pups, 5 male and 5 female pups in each group were randomly selected, killed, and a complete necropsy with histopathology was performed. The surplus pups were killed and necropsied.

At this time, 10 male and up to 10 female F2 animals were randomly selected, killed, and a complete necropsy with histopathology was performed. The remaining F2 adults were killed and necropsied.

Administration of the appropriate test diets to the F2 animals continued throughout the mating, gestation and lactation periods of both the F3a and F3b generation phases.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1, 3 & 10 mg/kg/day
Basis:
nominal in diet
No. of animals per sex per dose:
25 males & 25 females
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The levels of the test material were selected by the sponsor.
Positive control:
No data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined at least once daily to determine their general health and behaviour. Any changes observed were recorded on an individual clinical record.
Any animal that appeared to be ill was isolated to permit further observation. If its condition improved, the animal was returned to the group cage; if the death of an animal appeared probable, it was killed. These animals together with any found dead (with the exception of pre-weaned pups) were subjected to a macroscopic necropsy.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of non-pregnant, weaned animals were recorded at weekly intervals throughout the study (although this schedule was altered infrequently to accommodate holidays or other unforeseen factors).
The body weights of pregnant animals were recorded on days 0, 3, 6, 9, 12, 15, 18 and 21 of gestation. The body weights of lactating animals were recorded on days 1, 7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The group food consumption of the selected F0, F1 and F2 generation animals was recorded weekly throughout the study (although this schedule was altered infrequently to accommodate holidays or other unforeseen factors).
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OTHER:
In the females designated as 'behavioural' dams, the duration of gestation was noted and as soon as possible after birth, the number of live and dead pups was counted. Live pups were sexed and weighed individually on days 1, 4 and 21 post-partum. Where possible, pups dying during the pre-weaning period were examined to investigate the cause of death.

During lactation, the pups in each litter were examined daily. The following developmental parameters/functional tests were measured for each litter.
1. Pinna unfolding
2. Righting reflex
3. Hair growth
4. Ears open
5. Incisor eruption
6. Eye opening
7. Auditory startle

For each parameter, the day on which pups in each litter first showed the observation was recorded. Recording continued until all the pups in the litter showed the observation. At day 14 post-partum all the pups in each litter were tested for their ability to balance on a rotating rod.
The animal was placed on a horizontal metal bar with a non-slip surface (dimensions 25 cm length: 2.5 cm diameter) which was suspended 50 cm above a padded surface. The bar was rotated mechanically at a constant speed of 12 revolutions per minute. Rotation of the rod continued for a maximum of 60 seconds or until the animal fell off. A record was maintained of the trial time for each pup.
The pups selected for further behavioural/functional observations (2 male and 2 female pups per litter) were tested at approximately 5-6 weeks of age for the following parameters:
1. Auditory startle
2. Visual placing
3. Observation of gait
4. Rotating rod

Between the age of 10 and 12 weeks, the selected animals were re-tested for auditory startle, visual placing response and observation of gait.
Between the age of 13 and 14 weeks the selected animals were tested for their ability to balance on a rotating spiral.
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
No data
Litter observations:
The duration of gestation was noted and as soon as possible after birth, the number of live and dead pups was recorded. Live pups were sexed and weighed individually on days 1, 4 and 21 post-partum. Clinical changes of the offspring were recorded during the pre-weaning period and where possible, pups dying during the pre-weaning period were examined to investigate the cause of death.

The females were killed on day 21 of gestation by cervical dislocation. The females were subjected to gross necropsy, the ovaries and uteri were removed and the following data recorded:
- number of corpora lutea
- number and position of implantations subdivided into:
a) normal foetuses
b) early intra-uterine deaths
c) late intra-uterine deaths
- individual foetal weights
- individual foetal crown/rump lengths
- sex of the foetuses

Intra-uterine deaths were classed as early or late. Late intra-uterine deaths showed embryonic or foetal tissue in addition to placental tissue, whereas early intra-uterine deaths showed decidual or placental tissue only.
Foetuses were individually identified within a litter to enable each foetus to be matched with its weight and position in the uterine horn.

Before being weighed and measured, the foetuses were killed by an intra-cardiac injection of sodium pentobarbitone (200 mg/ml, Euthatal, May & Baker, Dagenham, Essex).

The exterior of all foetuses was examined. Two thirds of the foetuses in each litter (selected by systemic sampling) were dissected and the viscera examined. The foetuses were then eviscerated and the carcasses placed in 1% (w/v) aqueous potassium hydroxide containing 0.005% Alizarin Red S to stain the ossified skeleton.

The specimens were processed- through ascending concentrations of glycerol/distilled water, examined then preserved in 100% glycerol with thymol crystals, to prevent fungal growth.

The remaining foetuses were placed in Bouin's fixative to allow fixation and partial decalcification. Free-hand transverse sections were made with a razor and the sections examined according to Wilson's technique. The sections from each foetus were stored in airtight plastic tubing containing Bouin's fluid.

Abnormalities were recorded as major (rare and/or probably lethal) or minor (common deviations from normal). Variations in the degree of ossification of the phalanges were also recorded at skeletal examination. Incompletely or non-ossified phalanges were classed as variants.
Postmortem examinations (parental animals):
SACRIFICE
All F1a, F2a and F3a pups, all surplus non-selected pups from the F1b, F2b and F3b litters, all adults from the F0 and F1 generations, all non-selected adults from the F2 generation, all F1 and F2 behavioural animals and all weaned animals killed in extremis or found dead were subjected to gross necropsy.
All animals with the exception of caesarean designated females were killed by an intra-peritoneal injection of sodium pentobarbitone (Euthatal 200mg/ml: May & Baker, Dagenham, Essex).

GROSS NECROPSY
This involved examination of all external surfaces. The major organs were incised and the cut surfaces examined. This included examination of the chambers of the heart, the associated vessels and the renal pelvis. Any abnormalities observed were preserved in 10% neutral buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGHTS
After weaning of the F3b pups, 5 male and 5 female pups were selected at random from each group for detailed necropsy/histopathology. At the same time, 10 adult (F2) male and 10 adult (F2) female animals were selected at random from each group for detailed necropsy/histopathology.

These animals were subjected to detailed necropsy. Examination of the following organs and tissues both macroscopic and microscopic were made:

adrenals
liver
bladder
kidney
brain
lung
caecum
ovaries/testes
colon
pancreas
duodenum
pituitary (adults only)
femur (including bone marrow)
spleen
heart
ileum
stomach
thymus
thyroids
jejunum
epididymides

The tissues listed above were preserved in 10% neutral buffered formalin. Five µm sections of tissues from all animals were examined after staining with haematoxylin and eosin.
All tissue sections were examined by light microscopy.
Postmortem examinations (offspring):
SACRIFICE
All F1a, F2a and F3a pups, all surplus non-selected pups from the F1b, F2b and F3b litters, all adults from the F0 and F1 generations, all non-selected adults from the F2 generation, all F1 and F2 behavioural animals and all weaned animals killed in extremis or found dead were subjected to gross necropsy.
All animals with the exception of caesarean designated females were killed by an intra-peritoneal injection of sodium pentobarbitone (Euthatal 200mg/ml: May & Baker, Dagenham, Essex).

GROSS NECROPSY
This involved examination of all external surfaces. The major organs were incised and the cut surfaces examined. This included examination of the chambers of the heart, the associated vessels and the renal pelvis. Any abnormalities observed were preserved in 10% neutral buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGHTS
After weaning of the F3b pups, 5 male and 5 female pups were selected at random from each group for detailed necropsy/histopathology. At the same time, 10 adult (F2) male and 10 adult (F2) female animals were selected at random from each group for detailed necropsy/histopathology.

These animals were subjected to detailed necropsy. Examination of the following organs and tissues both macroscopic and microscopic were made:

adrenals
liver
bladder
kidney
brain
lung
caecum
ovaries/testes
colon
pancreas
duodenum
pituitary (adults only)
femur (including bone marrow)
spleen
heart
ileum
stomach
thymus
thyroids
jejunum
epididymides

The tissues listed above were preserved in 10% neutral buffered formalin. Five µm sections of tissues from all animals were examined after staining with haematoxylin and eosin.
All tissue sections were examined by light microscopy.

Statistics:
Adult bodyweight gains = analysis of co-variance

Duration of gestation, Numbers of pups born, Pup weights, Sex ratios, Number of corpora lutea, Foetal weights, Foetal crown/rump lengths = Wilcoxon's rank sum test

Number of pups dead at birth, Number of pups dead at days 1, 4 and 21, Early intra-uterine deaths, Late intra-uterine deaths, Total intra-uterine deaths, Defects, Variants = Fisher's two sum randomisation test with a Monte Carlo simulation for computation of significance levels

Pup weights = Students 't' test
The litter was considered the experimental unit with all statistical

Reproductive indices:
Reproductive function was assessed by the following indices:
- mating index
- fecundity index
- male fertility index
- female fertility index
Offspring viability indices:
- gestation index
- live birth index
- viability index
- lactation index
- % pre-weaning loss
- % pre-implantation loss
- sex ratios

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

- Bodyweights - males:
Treatment with Dinoseb at 10 mg/kg bw/day (high dose) over 3 generations elicited a marked retardation of body weight gain in comparison with controls. In each generation, when the animals in all dose groups had attained a mean body weight of approximately 300 g, the onset of body weight retardation of the high dose males occurred. The difference in body weight between the control and high dose males at the end of each generation was approximately 20% in the F0 and F1 phases and 15% in the F2 phase.
In the F0 generation, males treated at 3 mg/kg bw/day (intermediate dose) showed a similar rate of weight gain to the controls. F1 generation males exhibited a slightly lower weight gain and F2 generation males showed a superior rate of weight gain than in the control group.
Animals treated at 1 mg/kg bw/day (low dose) exhibited a lower rate of weight gain than the intermediate and control groups in all generations. This observation was most marked in the F1 and F2 generations.

- Body weights - females:
Treatment with Dinoseb at 10 mg/kg bw/day (high dose), elicited a retardation of body weight gain in comparison with controls.
The effect was less marked in each of the succeeding generations.
Females treated at 3 mg/kg bw/day (intermediate dose) showed a similar rate of weight gain to the controls throughout all 3 generations.
Females treated at 1 mg/kg bw/day (low dose) showed a similar rate of weight gain to the controls in the F0 and F2 generations, but a lower rate of weight gain to the controls in the F1 generation.

- Food consumption:
Food consumption data were available for the first 13 weeks of F0 and last 22 weeks of F2. There was no evidence of treatment related intergroup differences in group mean food intake for the males during the periods reported.
For the first 13 weeks of the F0 generation there was no evidence of treatment related intergroup differences in group mean food intake for the females. Food intake of the high dose females during gestation and lactation of both the F2-F3a and F2-F3b litters was generally slightly lower than in the control group.

- Clinical observations/mortalities - males:
There were 2 deaths in the high dose group at the end of the F2 generation. These deaths were considered to be associated with the extremes of temperature and humidity at that time. Discounting these deaths, the intergroup distribution of mortalities during the study was 4/300, 2/300, 1/300 and 4/300 in the control, low, intermediate and high dose groups, respectively. Necropsy of all animals revealed varying degrees of pulmonary disorders. All the deaths were considered incidental and not related to treatment with Dinoseb. Clinical changes occurred in the surviving animals in all dose groups, including the controls, and were mainly lesions caused by fighting or nasal and ocular discharges. Males in the high dose group in all generations generally developed a yellow tinge to the coat.

- Clinical observations/mortalities - females:
At the end of the F2 generation, 5 animals in the control group, 2 in the low dose group, 5 in the intermediate dose group and 3 in the high dose group died. These deaths were considered to be associated with the extremes of temperature and humidity at that time. Discounting these deaths, the intergroup distribution of mortalities during the study was 0/300 in the control group and 3/300 in the low, intermediate and high dose groups. Necropsy of all animals revealed varying degrees of pulmonary disorders. All the deaths were considered incidental and not related to treatment with Dinoseb. Clinical changes in the surviving animals were mainly minor in nature and occurred in all dose groups including the controls. Females in the high dose group in all generations generally developed a yellow tinge to the coat.

- Macroscopic findings at necropsy, microscopic findings and organ weights:
In the adult males, the observed lesions were primarily associated with the pulmonary tissue and the incidence did not suggest an effect of treatment with Dinoseb at any of the dose levels employed.
Observed lesions in the adult females were generally of pulmonary origin and the intergroup distribution did not suggest an effect of treatment with Dinoseb at any of the dose levels employed.
The only treatment related observation at necropsy was in the group treated at 10 mg/kg bw/day. The majority of the animals in this group developed a yellow tinge to the fur. This change appeared to have resulted from direct contact of the fur with the test article (which was yellow-brown in colour) in the diet.
Based on the nature and distribution of microscopic findings in both the F2 parent animals and in F3b progeny, there was no evidence of an effect of treatment with Dinoseb at any of the treatment levels employed.
Examination of organ weight data from adult F2 animals revealed decreased absolute organ weights, but not of relative weights, in animals treated at 10 mg/kg bw/day. These changes were considered a reflection of the observed growth retardation and not a pathological change. Organ weight data from selected F3b progeny showed absolute and relative organ weights from treated groups broadly similar to those of the controls. However, the intra and intergroup variations in individual weights were generally more marked than those observed in mature animals. In young animals the major organs will be undergoing a rapid growth phase. Therefore, the intergroup variations in group mean data probably reflected the different ages of these randomly selected progeny.

- Mating performance and fertility:
There was no effect of treatment with Dinoseb on male or female fertility. Fertility of both sexes was similar to the controls in all 3 generations.
Fecundity in the Dinoseb treated groups was similar to the controls in each mating phase in all 3 generations.
There was little intergroup variation in mating performance in either mating phase in each generation.

F0 generation:
In the first mating (F0-F1a), the fecundity indices were 96.0% in the low dose group and 100% in the control, intermediate and high dose groups. Mating indices were 100%, 96.2%, 92.3% and 100% in the control, low, intermediate and high dose groups, respectively.
In the second mating (F0-F1b) the fecundity indices were 100%, 88%, 100% and 96% in the control, low, intermediate and high dose groups, respectively. Mating indices were 100% in each group.
The male fertility index for the F0 generation was 100% in each group and the female fertility index was 96% in the control group and 100% in each of the low, intermediate and high dose groups.

F1 generation:
In the first mating (F1-F2a), the fecundity indices were 100%, 90.9%, 100% and 96% in the control, low, intermediate and high dose groups, respectively. Mating indices were 100%, 90.9%, 100% and 86.2% in the control, low, intermediate and high dose groups, respectively.
In the second mating (F1-F2b), the fecundity indices were 88%, 88%, 96% and 91.3% in the control, low, intermediate and high dose groups respectively. Mating indices were 92%, 76.7%, 86.2% and 88.5%.
The male fertility index for the F1 generation was 100% in each group and the female fertility index was 100%, 92%, 100% and 95.8% in the control, low, intermediate and high dose groups, respectively.

F2 generation:
In the first mating (F2-F3a), the fecundity indices were 95.8%, 96.0%, 100% and 100% in the control, low, intermediate and high dose groups, respectively. The mating indices were 100% in the control, low and intermediate dose groups and 92% in the high dose group.
In the second mating (F2-F3b), the fecundity indices were 96% in the control group and 100% in each of the Dinoseb treated groups. The mating indices were 100% in the control and low dose groups and 95.8% in the intermediate and high dose groups.
The male fertility index for the F2 generation was 100% in each group and the female fertility index was 96% in the control group and 100% in each of the Dinoseb treated groups.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
3 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Body weight gain
Remarks on result:
other: Generation: All (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

- Litter data:
There was no significant difference in the mean duration of gestation between the control and Dinoseb treated groups for either litter in each generation. The shortest mean duration of gestation in any of the littering phases was 21.0 days and the longest was 22.5 days.
The gestation indices (number of animals with live pups/number of pregnant females x100) were similar in the control and Dinoseb treated groups in each litter in all 3 generations. In the F0 and F1 generations, the mean numbers of pups born was slightly but not significantly lower than the controls in the animals treated at 10 mg/kg bw/day. In the F2 generation, the mean numbers of pups born in the animals treated at 10 mg/kg bw/day was similar to the controls.
The live birth indices (number of pups born alive/number of pups born x 100) were similar to or higher than the controls in the animals treated at 10 mg/kg bw/day in each litter in all 3 generations.
The viability indices (number of pups alive at day 4/number of pups born alive x 100) were variable in each generation, but there was no evidence of an effect of treatment with Dinoseb at any of the levels employed on the viability of the offspring.
The lactation indices (number of pups alive at day 21/number of pups alive at day 4 x 100) were generally similar to or slightly lower than the controls in the animals treated at 10 mg/kg bw/day, but the intergroup variability of this index within each generation suggested that this was not an effect of treatment with Dinoseb at any of the dose levels employed.
The pre-weaning losses [(number of pups born - number of pups weaned)/number of pups born x 100] were variable in each generation. In the F0 and F1 generations and in the F2 generation (F3a litter), there was no significant difference between the controls and animals treated at 10 mg/kg bw/day. However, in the F2- F3b litter, the number of litters with pre-weaning losses in animals treated at 10 mg/kg bw/day was significantly higher than in the controls.
There was little intergroup variation in calculated group sex-ratios in either litter in each generation. The range of means observed in the litter groups in the study was from 1:0.76 to 1:1.19 males:females.
In the F2 generation, mean pup weights at day 1 post-partum in the F2-F3a litter were significantly lower in the Dinoseb treated groups than in the control group however, as mean pup weight of the Dinoseb treated groups in the F2-F3b litter and in each litter in the F0 and F1 generations was not significantly different from the controls, a treatment effect was not suspected.
In the first litter in each generation and also in the F1b litter, mean pup weight at weaning (day 21 post-partum) was lower than the controls in the group treated at 10 mg/kg bw/day. In all but the F2-F3b litter, the percentage weight increase over the pre-weaning period was lower than the controls in the group treated at 10 mg/kg bw/day.
In certain of the littering phases, the trend towards lower pup weights at weaning and lower percentage weight increases in comparison with the controls was also observed in the groups treated at 1 and 3 mg/kg bw/day but was less marked than in the group treated at 10 mg/kg bw/day.
Where possible, pups dying during lactation were necropsied to investigate the cause of death. Many of the deaths were due to maternal neglect as stomachs of the dead pups were often empty. Other deaths were due to cannibalisation. Pups with notable clinical changes included one female pup in the F2a litter from the group treated at 3 mg/kg bw/day with acaudia and paralysis of the hindlimbs. In the F3b litters, also in the group treated at 3 mg/kg bw/day, one male pup showed crooked tail-and one female pup showed short tail, and in the group treated at 1 mg/kg bw/day one female showed acaudia. These changes were considered incidental and not related to treatment with Dinoseb.
It was also noted that a number of pups in the F3a litters showed ocular damage which appeared to have resulted from irritation of the eye membranes with the powdered diet. As this observation was noted in the control group as well as the Dinoseb treated groups it was considered not to be associated with Dinoseb treatment.

OTHER FINDINGS (OFFSPRING)
There was little intergroup variation in either the F1 or F2 generations in the onset and duration of the various developmental parameters observed during the pre-weaning period.
The group mean trial times for performance on the rotating rod on day 14 post-partum in the Dinoseb treated groups were slightly longer than the controls in the F1 generation and slightly shorter than the controls in the F2 generation.
At 5-6 weeks of age and again at 10-11 weeks of age, all animals in the F1 and F2 generations showed a normal response to the tests employed to demonstrate auditory and visual function and gait.
The group mean trial times for performance on the rotating rod at 5-6 weeks of age were shorter than the controls in the F2 males derived from Dinoseb treated groups. F1 males and F1 and F2 females derived from Dinoseb treated groups showed similar or slightly longer mean trial times than the controls.
The group mean trial times for performance on the rotating spiral at weeks 13-14 were shorter than the controls in the F2 males derived from Dinoseb treated groups. F1 males and F1 and F2 females derived from Dinoseb treated groups showed similar or slightly longer mean trial times than the controls.
No animals in the F 1 or F2 generation at 5-6 or 13-14 weeks were unable to balance on the rotating rods.


Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

- Caesarean data

The mean number of corpora lutea per dam was lower than the controls in the group treated at 10 mg/kg bw/day in all 3 generations. The difference between the controls and the group treated at 10 mg/kg bw/day was statistically significant (p<0.05) in the F0 generation but not in the F1 and F2 generations.

The mean number of corpora lutea per dam in the 1 and 3 mg/kg bw/day treated groups was not significantly different from the controls.

Group mean pre-implantation loss was variable in each group in each generation but there was no indication of an effect of treatment with Dinoseb on pre-implantation loss.

The mean number of implantations per dam was lower than the controls in the Dinoseb treated groups in the F0 generation, but only slightly different from the controls in the F1 and F2 generations.

There was no effect of treatment with Dinoseb on post-implantation loss (the incidence of intra-uterine deaths).

Mean foetal weight was significantly lower (p<0.05) than the controls in the group treated at 10 mg/kg bw/day in the F2 generation but was not significantly lower in the F0 and F1 generations. Mean foetal weight in the other Dinoseb treated groups was generally similar to the controls and none of the values were significantly different from the controls.

Mean crown/rump length in the Dinoseb treated groups was not significantly different from the controls in any generation.

There was little intergroup variation in calculated mean sex ratios in any group in any generation.

In the F0 generation, one major external and visceral defect was observed in a foetus from a dam (that died in parturition) in the group treated at 10 mg/kg bw/day and, in the F1 generation, a low incidence of hydronephrosis was observed in the Dinoseb treated groups. No major external and visceral defects occurred in the control group in any generation or in any Dinoseb treated group in the F2 generation. No major skeletal defects occurred in any group in any generation. There was no effect of treatment with Dinoseb on the incidence of minor external and visceral defects.

The incidence of minor skeletal defects in the group treated at 10 mg/kg bw/day was significantly higher than the controls in the F0 generation, lower than the controls in the F1 generation and higher but not significantly different from the controls in the F2 generation. There was no significant difference in the incidence of minor skeletal defects between the controls and the other Dinoseb treated groups.

In the absence of a consistent high incidence of minor skeletal defects throughout the 3 generations in the group treated at 10 mg/kg bw/day, it was considered unlikely that the high incidence in the F0 and F2 generation was related to treatment with Dinoseb.

The incidence of variants in the group treated at 10 mg/kg bw/day was significantly higher than the controls in the F2 generation but was lower than the controls in the F0 and F1 generations. The high incidence in the F2 generation was most probably related to the smaller foetal size, as the skeletons of small foetuses are generally less ossified than larger foetuses.

Applicant's summary and conclusion

Conclusions:
Treatment with the test material at 10 mg/kg/day elicited a marked retardation in body weight gain of male and female parental animals. Nevertheless, treatment throughout the 3 generations at a level which elicited toxicity did not affect survival, fertility, fecundity or produce any macroscopic changes at necropsy.
Executive summary:

A 3-generation reproduction study of the test material was carried out in the Sprague-Dawley derived rat.

Groups of 25 male and 25 female rats in each of the 3 generations (F0, F1 and F2) received Dinoseb in the diet at concentrations to provide 1, 3 or 10mg/kg daily for 29 weeks (total duration 87 weeks). Similar groups of animals-fed untreated diet over the same period served as the control group.

In conclusion, treatment with the test material at 10 mg/kg/day elicited a marked retardation in body weight gain of male and female parental animals. Nevertheless, treatment throughout the 3 generations at a level which elicited toxicity did not affect survival, fertility, fecundity or produce any macroscopic changes at necropsy.

There was an indication that treatment at 10 mg/kg/day of the test material resulted in lower mean numbers of pups born, lower mean pup weights at weaning, fewer numbers of corpora lutea in the caesarean females and a lower mean foetal weight in the third generation. All these findings may be associated with the lower body weights of the parental females.

There was however no effect on any other reproductive parameter examined or on the functional development of the offspring and there was no evidence of teratogenicity.

Although in the groups treated at 1 and 3 mg/kg/day of the test material there were isolated significant differences from the controls in parental body weight and in the various reproductive parameters examined, it was considered that these findings were incidental and not related to treatment.

The NOAEL of the study was set at 3 mg/kg bw/day.