N-butylphosphorothioic triamide

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 4 February 1998 and 2 November 1999 (F0 animals arrived: 4 February 1998, F0 treatment commenced: 10 February 1998, F0 generation completed: 19 June 1998, F1 treatment commenced: 12 June 1998, F1 generation completed: 23 October 1998)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Ministry of Agriculture, Forestry and Fisheries of Japan (1985)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material: N-(n-butyI) phosphorothioic triamide (NBPT)
- Substance type: Organophosphorus
- Physical state: White powder
- Analytical purity: 88.67%
- Lot/batch No.: C98-1
- Expiration date of the lot/batch: January 2003
- Storage condition of test material: Approximately 4 °C in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England to form the F0 generation.
- Age at study initiation: (F0) 6 weeks; (F1) 4 weeks
- Weight at study initiation: (F0) Males: 143 to 243g; Females: 122 to 191g;

- Fasting period before study: Not stated

- Housing:
Depending upon the phase of the study, rats were housed in TRl8 from Arrowmight Biosciences, Hereford, England and RB3 or RB3 modified cages from North Kent Plastic Cages Limited, Erith, Kent, England. The cages consisted of stainless steel (TR18) or high density polypropylene (RB3 and RB3 modified) bodies with lids of stainless steel grid. TR18 and RB3 modified cages had stainless steel floors and were suspended in batteries over trays covered with absorbent paper which was replaced twice weekly or, daily during pairing or, on each day that food scatter was apparent on the paper. RB3 cages had solid polypropylene floors and autoclaved wood shavings were provided as bedding, renewed at least twice weekly.

- Use of restrainers for preventing ingestion (if dermal): no

- Diet (e.g. ad libitum): The animals were allowed free access to a commercially available laboratory animal diet (LAD 2 SQC) manufactured by Special Diet Services Limited, Witham, Essex, England. This powdered diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

- Water (e.g. ad libitum): Water was taken from the public supply (Essex and Suffolk Water pic, Chelmsford, Essex, England) was freely available via polyethylene or polycarbonate bottles fitted with sipper tubes

- Acclimatisation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): target values was 21°C (Range 19-23°C)
- Humidity (%): target value was 55% (Range40-70% )
- Air changes (per hr): 15 room air changes per hour.
- Photoperiod (hrs dark / hrs light): 12-hour light: 12-hour dark cycle with the lights on at 6.00 GMT.

Administration / exposure

Route of administration:

oral: feed

Type of inhalation exposure (if applicable):

other: Not applicable

Vehicle:

other: basal laboratory diet

Details on exposure:

PREPARATION OF DIETARY ADMIXTURES:

DIET PREPARATION
Fresh diet was prepared at various intervals during the study ranging from three times a week to once every two weeks. Diets were prepared at Huntingdon Life Sciences, Huntingdon.

The particle size of the test material was reduced prior to weighing by use of a coffee grinder. Accurately weighed amounts ofthe test material were ground with plain diet.

For the 3200 and 800 ppm concentrations, a further amount of plain diet was added and the mixture diluted to the required premix weight with further quantities of plain diet. Mixing of each premix was achieved using a turbula mixer for a minimum of 5 minutes.

For the 200 ppm concentration, a further amount of plain diet was added and the grinding repeated. The mixture was then diluted to the premix weight with a further quantity of plain diet. Mixing of the premix was achieved using a turbula mixer for a minimum of 5 minutes.

The dietary concentrations required for feeding were made by diluting the appropriate premix with plain diet, mixing being achieved using a turbula mixer. A separate premix was used to prepare each dietary concentration.

All diets (including the control diet) were deep frozen (approximately -20°C) as soon as possible after mixing.

The frozen diets were then transferred to the Pharmacy, Huntingdon Life Sciences, Eye. Dicts for all phases of the study except weaning were subsequently removed from the freezer, subdivided into weighed daily aliquots and then returned to a freezer prior to use.

During weaning phases of the study when larger amounts of diet were required for feeding, diet mixes to cover a day of feeding were prepared, frozen and transferred to Eye and used directly without aliquotting.

The diet mix size was approximately 25 kg except during the periods of the study between weaning and terminal sacrifice of the FI and F2 offspring when the mix size was 18.5, 14 or 11 kg.

Details on mating procedure:

Following the scheduled period of treatment (10 weeks), males and females from within the same treatment groups were paired on a one male to one female basis for a period of up to 3 oestrous periods or a maximum of 2 weeks with no exchange of partners. Care was taken to avoid pairing siblings. Each morning following pairing, the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each female and examined for the presence of spermatozoa and the stage of the oestrous cycle. The day on which evidence of mating was found was designated Day 0 of gestation.

Once mating occurred, the males and females were generally separated and smearing was discontinued. However after inconclusive mating, smearingwas continued for up to 5 days to confirm positive mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Quality control of dosage form
Specimen formulations were analysed to assess the homogeneity and stability of the test substance in the diet matrix at concentrations of 200 and 5000 ppm.

The homogeneity of NBPT in specimen batches of LAD2 diet formulations was confirmed for the freshly-prepared formulations and following frozen transportation between the Huntingdon and Eye Research Centres.

The stability ofNBPT in LAD2 diet formulations was confirmed during freezer storage for 33 days and during ambient temperature storage for 1 day following both fresh preparation and freezer storage, during which time the diet concentrations remained within 10% of nominal values. The concentration of NBPT in the diets decreased by approximately 20% at 200 ppm and 7% at 5000 ppm during ambient temperature storage for 4 days. It was accordingly recommended that ambient temperature storage of diets should be limited to a maximum of 1 day i.e. diets should be fed freshly each day.

The Sponsor requested that an additional stability assay was performed as part of this study following storage of diets containing 200 and 3200 ppm NBPT for 55 hours at 21°C Three 200 g samples were taken from each of the Control, 200 ppm and 3200 ppm diets prepared for use during the first week of treatment of the Fo generation. These samples were initially deep frozen and were then placed at ambient temperature and one sample per group analysed after 0, 31 or 55 hours storage. The mean analysed concentrations of NBPT remained within -10% of nominal following 31 hours storage at ambient temperature but decreased to -18% at the low concentration of 200 ppm following 55 hours storage at ambient temperature, therefore a maximum ambient temperature storage period of 31 hours was recommended. These results confirmed that the practice used on this study of removing frozen diets from the freezer at 09.00 hours for feeding between 15.00 and 16.00 hours on the same day and removal on the following day was acceptable.

Single samples (nominally 200g) were taken from all groups/sex (2 assays from each sample) to check for the content of NBPT at approximately 10 week intervals, equivalent to the start of treatment, first week of pairing, week of selection for F1 generation, second week of pairing of F1 generation and, the third week of lactation of the F1 generation.

The results for the test diet formulations analysed during the study were within + 6.5% / -10% of the nominal concentrations confirming the accuracy of formulation.

Duration of treatment / exposure:

The test material was administered to the animals in their diet which was available on an ad libitum basis.

F0 generation:
Males and females of the F0 generation were treated for 10 weeks before pairing and throughout the study until termination.

F1 generation:
Animals of the F1 generation had access to the same diet as their parents throughout, but the F1 generation was deemed to formally start at approximately 4 weeks of age, and were treated from weaning for 10 weeks before pairing, and until termination when litters were weaned.

Frequency of treatment:

The test material was administered to the animals in their diet which was available on an ad libitum basis.

For both generations, on each day of treatment fresh diet was made available to the animals in the afternoon between 15.00 and 16.00, approximately one hourafter diet from the previous day had been removed between 13.30 and 14.30. In order to ensure that diets had warmed up prior to feeding, diets to be fed on a given day were removed from frozen storage in the animal facility at approximately 09.00 hours on that day. Records were maintained oftime of removal of food from the animals, time of feeding fresh food and time of removal of diet from frozen storage.

Details on study schedule:

-Males and females of the F0 generation were treated for 10 weeks before pairing and throughout the study until termination.

-Animals of the F1 generation had access to the same diet as their parents throughout, but the F1 generation was deemed to formally start at approximately 4 weeks of age, and were treated from weaning for 10 weeks before pairing, and until termination when litters were weaned.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
200 ppm
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
800 ppm
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
3200 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

32 males per sex/dose group/generation
32 females per sex/dose group/generation

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
NBPT was administered in the diet to simulate the conditions of possible human exposure.

Dietary concentrations of 200, 800 and 3200 ppm were selected in collaboration with the Sponsor based on results from a preliminary dietary study performed at the testing facility (Report No. ICN001/97l744) and a 13 week ToxicitylNeurotoxicity study in rats by dietary administration (Study No. IMFI7). On ICN/001, treatment at 5000 ppm NBPT resulted in all five females which produced a litter being killed for reasons of animal welfare between Days 15 and 20 of lactation. Effects at 1000 ppm were confined to a possible marginal reduction of bodyweight of the offspring from Day 7 of lactation.Accordingly, 3200 ppm was chosen as the highest dietary concentration. 200 ppm was used at the lowest dietary concentration as this had not been shown to produce any adverse effects in the preliminary study and was considered by the Sponsor to provide an adequate margin of safety. The intermediate level of NBPT was chosen to provide a geometric progression of concentrations.

- Allocation to treatment groups and animal identification:

F0 generation
On receipt of animals, each litter was identified and allocated to cages, a maximum of 4 of one sex being placed in each cage. Before commencement of treatment, the animals within each sex were weighed and the three litters per sex showing the greatest within-litter variation in bodyweight, were discarded to leave 32 litters of males and 32 litters of females. One animal from each of these litters was randomly allocated to each study group, assigned a number and identified by tail tattoo. Records of the litter relationships were retained. This procedure ensured that for each sex, all groups contained populations of rats with the same genetic background and similar initial mean bodyweight and bodyweight range. No group contained more than one male and one female from the same litter of origin.

F1 generation
32 males and 32 females per group were selected to form the F1 generation when the F1 offspring were approximately four weeks of age. A minimum of 1 male and 1 female per litter were selected from as many litters as possible by using a random number procedure within each available litter. Where appropriate, additional offspring (to a maximum of 2 males and 2 females per litter) were selected from randomly selected litters until the required number of animals were selected for the F1 generation. Each animal was assigned a number and identified by tail tattoo. For reasons of animal welfare, it was considered that tail tattooing should not be performed until the animals reached Day 28 of age. Prior to Day 28, selected animals were identified with their unique F1 animal number marked on the side or underside of the tail with a permanent marker.

Cage labels, identifying the occupants by experiment, treatment group, sex and animal number were colour coded.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
All animals were observed at least twice daily throughout the study and any visible signs of reaction to treatment were recorded, with details of type, severity, time of onset and duration.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
A more detailed weekly examination was performed throughout the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males were weighed on the day that treatment commenced (F0) or the formal start of the generation (F1), then weekly thereafter. F0 and F1 Females were weighed on the same schedule until mating was detected and then on Days 0, 7, 14, 20 and 21 after mating, and on Days 1, 4, 7, 14, and 21 of
lactation.

Non-selected F1 and F2 offspring were weighed on Days 28 and 35 of age.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was recorded on a cage basis (four animals per cage unless reduced by mortality) for F0 and F1 males and females daily before pairing for mating. Food consumption for females after mating was recorded daily on an individual basis for Days 0-20 of gestation and Days I to 13 of
lactation. After Day 13 ofiactation, food intake is increasingly influenced by the pups and is no longer an accurate reflection of maternal intake.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Mating procedure
Following the scheduled period of treatment (10 weeks), males and females from within the same treatment groups were paired on a one-to-one basisfor a period of up to 3 oestrous periods or a maximum of 2 weeks with no exchange of partners. Care was taken to avoid pairing siblings. Each
morning following pairing, the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each femaleand examined for the presence of spermatozoa and the stage of the oestrous cycle. The day on which evidence of mating was found was designated Day 0 of gestation.

Once mating occurred, the males and females were generally separated and smearing was discontinued. However after inconclusive mating, smearingwas continued for up to 5 days to confirm positive mating.

Pre-coital interval (females only)
The time elapsing between initial pairing and detection of mating was recorded.

Parturition and duration of gestation
From Day 20 after mating, animals were checked three times daily for evidence of onset, progress and completion of parturition All females from eachgroup were permitted to deliver their young naturally and rear their own offspring until Day 21 of lactation.

The time elapsing between the detection of mating and commencement of parturition was reported as the duration ofgestation.

Maternal behaviour
Dams were observed daily for evidence of abnormal maternal behaviour.

Oestrous cyclicity (parental animals):

For three weeks before pairing of F0 and F1 generations, daily vaginal smears were taken from all females and examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued until evidence of mating was observed.

Sperm parameters (parental animals):

F0 and F1 males

Sperm motility
Immediately after sacrifice, one epididymis of each male was removed (normally the left side), weighed and a sample obtained from a small cut in the left cauda epididymis. A small droplet of epididymal fluid was exuded by gentle squeezing and the sperm allowed to diffuse into the medium (M199 containing 0.5% bovine serum albumin warmed to 37°C) A sample for assessment was taken into a 100 /-1m depth cannula by capillary action and at least200 sperm per animal analysed using the Hamilton Thome IVOS Computer Assisted Sperm Analyser (CASA).

Sperm count
For count of epididymal sperm a 5µl sample of sperm was taken from the left cauda and added to 995 µl of medium. This mixture was homogenised for one minute then aliquots were frozen (approximately -20°C) and stored until assessment. For assessment an aliquot was thawed, stained with IDENT (a DNA-specific fluorescent dye supplied by Hamilton Thome) before analysis by CASA: 10 fields per animal were normally analysed.

Sperm morphology
For morphology assessment a 50 µl aliquot of the sperm medium mixture prepared for the sperm count (before homogenisation) was added to 950 µl of 4% neutral buffered formaldehyde (NBF) One drop of the mixture was placed on a clean glass microscope slide and one drop of I% nigrosin/eosin stain added. A smear was produced and allowed to dry before a cover slip was applied with mounting medium. The fixed sample was assessed by lighmicroscopy and at least 200 sperm per animal analysed.

Homogenisation-resistant spermatids
For counts of homgenisation-resistant spermatids the whole left testis was taken and frozen (approximately -20°C). To prepare the sample the testis was thawed and homogenised in 25 ml of SMT (0.9% saline; 0.01% merthiolate; 0.05% Triton X-IOO). An aliquot was stained with IDENT and analysed as described for sperm counts.

Litter observations:

Observations at Day 1 of age

All offspring were examined at approximately 24 hours after birth (Day 1 of age) and the following were recorded for each litter:
a) Number of offspring (live and dead);
b) Individual bodyweights of live offspring;
c) Sex ratio;
d) Observations on individual offspring.

Clinical signs
Litters were observed daily for evidence of abnormal appearance or behaviour.

Mortality and litter size
Daily records were maintained of mortality and consequent changes in litter size. Wherever possible, any offspring found dead were examined externally and internally.

Litters containing nine or more offspring were culled by random selection to eight (where possible four males and four females) on Day 4 ofage.

Bodyweight
Individual FI and F2 offspring were weighed on Days 1,4, 7, 14 and 21 of age.

Sex ratio
The offspring were sexed at weighing and sexes reported for Days 1, 4 (before and after culling) and 21 of age.

Offspring development
The timing of physical development of the offspring was assessed on a total litter basis by recording the days on which the onset and completion of the following parameters occurred:

Pinna detachment - detachment of the edge of the pinna - from Day I of age until completion.

Hair growth - macroscopic observation of generalised growth of body hair - from Day 1 of age until completion.

Tooth eruption - eruption of the upper incisors through the gum - from Day 5 of age until completion.

Eye opening - separation of the upper and lower eyelids - from Day 9 of age until completion.

For reported values, a continuity correction of half of one day was subtracted from the age (day of age) of litters born overnight.

Sexual maturation of F1 generation
Selected female F1 offspring were examined daily from Day 28 of age until vaginal opening occurred Bodyweight was recorded on day of vaginal opening for each animal.

Selected male F1 offspring were examined daily from Day 38 of age until balano-preputial separation occurred. Bodyweight was recorded on day of separation for each animal.

Postmortem examinations (parental animals):

All adults and weaned animals were killed by carbon dioxide inhalation.

Males were killed once the majority of litters had weaned and it had been established that further litters were not required Females that gave birth werekilled when their respective litters had been weaned. Females which failed to give birth, or whose litters died before weaning, were killed at approximately the same time as the bulk of the females after litters had been weaned.

All adult animals were subject to a complete gross necropsy.
The following organs were weighed from the F0 and F1 parental animals:

Adrenal glands, brain, epididymides, kidneys, liver, ovaries (and oviduct), prostate, seminal vesicles and coagulating gland, spleen, testes, uterus wirth cervix.

Postmortem examinations (offspring):

SACRIFICE
Offspring culled on Day 4 of age, and any killed in extremis or for humane reasons before Day 10 of age, were killed by an intraperitoneal injection of pentobarbitone sodium B.P. Vet. (Sanofi Animal Health Limited, Watford, Hertfordshire, England).

Offspring (unselected F1 offspring and F2 offspring)

F1 weaning offspring not selected for continuation of the study were killed on Day 35 of age following the selection process for the next generation. The offspring were examined internally and externally for macroscopic abnormalities. All offspring had the following organs weighed and retained for possible future histopathological examination: brain, spleen, thymus, ovaries and testes.

F2 weaning offspring were killed on Day 35 of age. The offspring were examined internally and externally for macroscopic abnormalities. All offspringhad the following organs weighed and retained for possible future histopathological examination: brain, spleen and thymus.

Tissues from Day 35 F1 and F2 offspring were fixed in 10% neutral buffered formalin except for the testes which were initially fixed in Bouins fluid and then transferred to industrial methylated spirit.

Culled F1 offspring and F2 male offspring on Day 4 of age were subjected to formal necropsy examination. The pups were examined internally and externally and abnormal tissues retained.

Offspring dying during early lactation were preserved in industrial methylated spirit prior to macroscopic examination.

Statistics:

Significance tests, employing analysis of variance followed by an inter-group comparison with the control, were performed on the following parameters, and results are presented in relevant tables of this report: Bodyweights and bodyweight change, food consumption, litter data, organ weights and histopathological findings.
For data recorded and/or processed by the Xybion computer system (adult organ weights and weekly bodyweights/changes) for the parental animals, homogeneity of variance was assessed using Bartlett's test. Whenever this was found to be statistically significant a Behrens-Fisher test was used to perform pairwise comparison, otherwise a Dunnett's test was used. Intergroup differences in macroscopic pathology and histopathology were assessed using Fisher's Exact test.
For data that was collected by hand or processed using Excel (bodyweight change during gestation and lactation, food consumption, bodyweight and age at sexual maturation, seminology data, litter data and offspring absolute organ weights) the statistical analysis was performed using the Startox programme
developed by Huntingdon Life Sciences. Dependent on the heterogeneity of variance between treatment groups, parametric tests (analysis of variance, Snedecor and Cochran 1967) followed by Williams' test (Williams' 1971/2) or non-parametric tests, (Kruskal-WaUis, Hollander and Wolfe 1973) followed by
Shirley's test (Shirley 1977) were used to analyse these data, as appropriate. For litter data the basic sample unit was generally the litter and, due to preponderance of non-normal distributions, nonparametric analyses were routinely used. Where 75% or more of the values for a given variable are the same, a Fisher's exact test (Fisher, 1950) was used.All significant (i.e. p<0.05) inter-group differences from the control are reported only where supported by a significant analysis of variance (i.e. p<0.05).

Reproductive indices:

Mating performance and fertility

For each group and sex the following were calculated:

Percentage mating = (Animals mated/Animals paired) x100

Conception rate = (Animals pregnant or siring a pregnancy/Animals mated) x 100

Fertility index = (Animals pregnant or siring a pregnancy/Animals paired) x 100

Gestation index = (Number of live litters born/Number pregnant) x 100

Offspring viability indices:

Survival indices

The following were calculated for each group:
Post-implantation survival index = (Total number ofoffspring born/Total number of implantation sites) x 100

Live birth index = (Total number of live offspring on Day 1/Total number ofoffspring born) x 100

Viability index = (Number of live offspring on Day 4 ofage/Number of live offspring on Day I of age) x 100

Lactation index = (Number of live offspring on Day of examination/Number of live offspring at Day 4 after culling) x 100

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

no effects of toxicological importance

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

F0 GENERATION

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
The general condition of animals remained similar in all groups throughout the generation and clinical signs were largely restricted to areas of hair loss, minor encrustations on the skin and coat staining.

There were three mortalities among females treated at 200 or 800 ppm but these were not considered to be related to treatment: in the 200 ppm group female 1190 was killed prior to Day 1 of lactation due to poor clinical condition and apparent problems with parturition. Necropsy examination did not reveal any obvious cause for the condition. Female 1191 (200 ppm) was killed on Day 18 of gestation due to poor clinical condition. Necropsy examination revealed changes to the right ventricle of the heart, jejenum and kidneys and, the urinary bladder contents were dark. In addition, three of the seven conceptuses present in the uterus had died. At 800 ppm, female 1206 was killed on Day 7 after mating due to poor clinical condition. Necropsy revealed multiple changes including dark and thickened meninges, enlarged lymph nodes, enlarged, swollen and pale liver and, swollen and pale spleen. No implantation sites were detected in the uterus. Microscopic examination of tissues revealed a malignant lymphoma. In addition to the mortalities among treated females, there was one mortality in the control group: female 1146 was killed due to poor clinical condition on Day 3 of lactation. Necropsy revealed yellow fluid in the thorax and duodenum and the stomach contents were yellow. The litter had already died on the day of sacrifice.

BODY WEIGHT OF MALES (PARENTAL ANIMALS)
Bodyweight gain was slightly, but generally statistically significantly, lower than Controls for males receiving 3200 ppm from the commencement of treatment (approximately 91% of Control by the end of the pre-mating period (Week 10) and 90% of Control at termination, after 17 weeks of treatment).
There was no effect on weight gain of animals receiving 800 and 200 ppm.

BODY WEIGHT OF FEMALES (PARENTAL ANIMALS)
Bodyweight gain prior to pairing was slightly lower than Controls for all groups of females receiving NBPT: the effect showed a concentration relationship but only attained statistical significance at 3200 and 800 ppm. At Week 10, weight gain was 83%, 88% and 93% of controls at 3200, 800 and 200 ppm respectively. These differences were reflected in the lower group mean absolute bodyweights on Day 0 of gestation.

Bodyweight gain at 3200 ppm continued to be significantly lower than in Controls during gestation. Although there was no significant effect on weight change during lactation at 3200 ppm, females did not show the usual pattern of slight weight loss during late lactation (as seen in the controls) but
showed marginal weight gain during Days 14-21.

At 800 and 200 ppm, there were no effects of treatment on weight change during gestation and lactation.

FOOD CONSUMPTION OF MALES (PARENTAL ANIMALS)
Food consumption of males receiving 3200 ppm was marginally lower than in Controls; differences attained statistical significance for Weeks 1, 3, 4 and 5. There was no effect On food intake at 800 or 200 ppm.

FOOD CONSUMPTION OF FEMALES (PARENTAL ANIMALS)
There was no significant effect of treatment on food intake offemales prior to pairing.

At 3200 ppm, marginally lower food consumption (statistically significant) was observed during Days 7-13 of gestation.

FOOD CONVERSION EFFICIENCY OF MALES AND FEMALES BEFORE PAIRING (PARENT ANIMALS)
Food conversion efficiency of males receiving 3200 ppm was slightly inferior to that of Controls during the first week of treatment. Thereafter, there was no consistent effect of treatment. For females at 3200 ppm, food conversion efficiency was slightly inferior to Controls during the first two weeksof treatment. Thereafter, there was no consistent difference.

There was no effect of treatment on the food conversion efficiency of males and females at 800 and 200 ppm.

ACHIEVED CHEMICAL INTAKE (PARENT ANIMALS)
At the highest dietary concentration (3200 ppm) chemical intake was approximately 370 mg/kg/day for males and females during the first week of treatment. Intake decreased to 180 mg/kglday for males and 223 mg/kg/day for females in the week immediately prior to pairing (Week 10) reflecting the natural decline associated with changes in bodyweight: food intake ratio as animals mature. Chemical intake by females is inherently greater than males during the pre-mating phase; during gestation it was slightly higher than in the period immediately before pairing and increased further during early lactation in line with the rise in food intake at peak physiological demand.

Chemical intake by animals in the lower treatment groups was in proportion to the dietary concentrations, reducing steadily at the animals became older: in the week preceeding pairing, values were 11 and 43 mg/kg/day for males and 14 and 55 mg/kglday for female groups receiving 200 and 800 ppm, respectively.

OESTROUS CYCLES (PARENTAL ANIMALS)
The regularity and duration of the oestrous cycles were unaffected by the presence of NBPT in the diet at concentrations up to 3200 ppm.

PRE-COITAL INTERVAL AND MATING PERFORMANCE (PARENTAL ANIMALS)
Almost every animal in all groups mated at the first oestrus opportunity following cohabitation (i.e within four days of pairing). Fertility was unaffected by treatment.

GESTATION LENGTH AND PARTURITION (PARENTAL ANIMALS)
There was a slightly higher incidence of females with the longer gestation lengths of 23 or more days at 3200 ppm compared with Controls. The difference did not attain significance and was considered to be of uncertain biological importance. There was no effect on gestation length at 800 or 200 ppm.
There were no indications ofany adverse effects of treatment upon the parturition process.

The gestation index was not adversely affected by treatment.

SPERM EVALUATION OF F0 MALES (PARENTAL ANIMALS)
There was no effect of treatment on the numbers of homogenisation resistant spermatids in the left testis or sperm in the left epididymis.

Treatment at 3200 ppm only was associated with significantly lower percentages of progressively motile and morphologically normal sperm compared with Controls. The difference in sperm morphology largely reflected the fact that male 1100 (female partner had a litter of 14 pups) was found
to have 96.5% decapitated epididymal sperm but the difference remained significant when values for this animal were excluded from assessment.

Analysis of additional sperm motility parameters revealed significant reductions in straight line velocity (VSL) and curvilinear velocity (VCL) for sperm at 3200 ppm only.

NECROPSY (PARENTAL ANIMALS)
There were no findings considered to be related to treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
At necropsy after approximately 17 weeks of treatment the group mean bodyweight of males were 93%, 101% and 104% of the Controls at 3200, 800 and 200 ppm respectively. Bodyweights for females which had reared their young to weaning were 94%, 95% and 97% of Control at 3200, 800 and 200 ppm respectively.

A few of organ weightslbodyweight relative weights were statistically significantly different from the Control but these were confined mostly to 3200 ppm. Among males at 3200 ppm, absolute kidney weights were lower than in Controls while bodyweight relative weights for the brain, epididymides and liver were higher than in Controls.

Among females at this level, absolute adrenal and spleen weights were lower than in Controls while bodyweight relative weight for the liver was higher than in Controls.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Reproductive organs were examined from all Control and 3200 ppm animals. (The right testis and epididymis were examined - tissues from the left side of the body were used for assessment of sperm counts, motility and morphology and were not subjected to microscopic examination unless macroscopic abnormalities were apparent).

Treatment of all males (32/32) at 3200 ppm was associated with a specific change in the epididymis: the epithelial cells lining the epididymal duct in the distal part of the caput and the proximal portion of the corpus epidiymis exhibited macro and microvesiculation which resembled fat vacuoles. In view of this treatment-related change, the right epididymis was subsequently examined for all males at 800 and 200 ppm and the change was detected in the right epididymis of 9/32 males at 800 ppm. No males were affected at 200 ppm.

No treatment-related findings were detected among females at 3200 ppm.


F1 GENERATION

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
The general condition of animals remained similar in all groups throughout the generation and clinical signs were largely restricted to areas of hair loss and coat staining.

There were no mortality in males at any dose.

Five females at 800 ppm were killed for reasons of animal welfare but these mortalities in females were not considered to be related to treatment: female 2199 was killed on Day 19 of gestation and females 2204,2212,2213 and 2222 were killed during early lactation. Four of the five females had been noted as having red staining on the genital area/vagina prior to sacrifice and this may reflect parturition difficulties. Necropsy examination did reveal evidence for internal uterine haemorrhage in females 2199 and 2213.

There were no mortalities in females at 3200 ppm, confirming the coincidental nature of the deaths at 800 ppm.

BODY WEIGHT OF MALES (PARENTAL ANIMALS)
Absolute bodyweights of the selected offspring at the formal start of the FI generation (Week 0) in all groups receiving NBPT were marginally lower than in Controls and mirrored the weights of the remaining (unselected) population prior to sacrifice.

Absolute weight gains at 3200 ppm were significantly lower than in Controls and mean bodyweight at termination (Week 18) expressed as a percentage of Control was lower than the difference at Week a(see next page) indicating persistence of impaired growth.

At 800 ppm, weight gains were also lower than in Controls but the differences were not statistically significant and mean bodyweight at termination expressed as a percentage of Control was comparable to the difference at Week 0 indicating that the overall growth rate of animals at 800 ppm was comparable to that of Controls.

There was no effect on weight gain at 200 ppm.

BODY WEIGHT OF FEMALES (PARENTAL ANIMALS)
Absolute bodyweights at the formal start of the FI generation (Week 0) in all groups receiving NBPT were marginally lower than in Controls, again reflecting intergroup differences in the population from which these offspring were drawn.

Absolute weight gains at 3200 ppm were generally significantly lower than in Controls and mean bodyweight at pairing (Week 10) expressed as a percentage of Control was lower than the difference at Week 0 indicating continued impairment ofgrowth. This resulted in noticeably lower mean bodyweight on Day 0 of gestation.

There was no clear effect on weight gain or growth prior to pairing at 800 and 200 ppm.

Bodyweight at the start of pairing, on the day of mating and bodyweight gain during gestation were all lower than in Controls at 3200 ppm. Although there was no significant effect on weight change during lactation at 3200 ppm, females did not show the usual pattern of slight weight loss during late lactation (as seen in the controls) but showed weight gain during Days 14-21.

At 800 and 200 ppm, there was no effect of treatment on weight change during gestation and lactation.

FOOD CONSUMPTION OF MALES (PARENT ANIMALS)
Food consumption of males receiving 3200 ppm was marginally lower than in Controls; differences attained significance for Weeks 4·8 and 10. There was no effect on food intake at 800 or 200 ppm.

FOOD CONSUMPTION OF FEMALES (PARENT ANIMALS)
There was no significant effect of treatment on food intake of females prior to pairing.

At 3200 ppm, marginally lower food consumption (statistically significant) was recorded during Days 7-19 of gestation.

FOOD CONVERSION EFFICIENCY OF MALES AND FEMALES BEFORE PAIRING (PARENT ANIMALS)
There was no effect of treatment on the food conversion efficiency of males or females treated with NBPT.

ACHIEVED CHEMICAL INTAKE (PARENT ANIMALS)
At the highest dietary concentration (3200 ppm) chemical intake was approximately 480 mg/kglday for males and females during the first week of treatment. Intake then decreased to about 200 mg/kglday for males and 250 mg/kglday for females in the week immediately prior to pairing (Week 10) reflecting the natural decline associated with changes in bodyweight: food intake ratio as animals mature. Chemical intake by females during early gestation was slightly higher than in the period immediately before pairing and increased during early lactation in line with the rise in food intake at peak physiological demand. The higher initial intake compared with the F0 generation (approximately 480 vs. 370 mg/kg/day for F1 and F0, respectively) was consistent with the expected pattern related to the younger age of the animals at the same stage of the study.

Chemical intakes by animals in the 800 ppm and 200 ppm groups were pro-rata to the dietary inclusion levels throughout the generation.

OESTROUS CYCLES (PARENTAL ANIMALS)
The regularity and duration of the oestrous cycles were unaffected by the presence ofNBPT in the diet at concentrations up to 3200 ppm.

PRE-COITAL INTERVAL AND MATING PERFORMANCE (PARENTAL ANIMALS)
The majority of animals in all groups mated at the first oestrus after pairing but a number of females in all groups showed acyclic periods before mating resulting in some extension of the pre-coital interval.

As in the Fo generation, fertility was unaffected by treatment; the fertility index at 3200 ppm was 100%.

GESTATION LENGHT AND PARTURITION (PARENTAL ANIMALS)
As in the Fa generation, there was a slightly higher incidence of females with the longer gestation lengths of 23 or more days at 3200 ppm compared with Controls. The difference did not attain significance and was considered to be of uncertain biological importance. There was no effect on gestation length at 800 or 200 ppm.

There were no indications of any adverse effects of treatment upon the parturition process.

The gestation index was unaffected by treatment.


SPERM EVALUATION OF F1 MALES (PARENTAL ANIMALS)
There was no effect of treatment on the numbers of homogenisation resistant spermatids in the left testis or sperm in the left epididymis.

Treatment at 3200 ppm was associated with significantly lower percentages of motile and progressively motile sperm compared with Controls. At 800ppm, the percentage of motile sperm was significantly lower than in Controls.

Analysis of additional sperm motility parameters revealed a significant reduction in the percentage of 'rapid' sperm at 3200 ppm only.

There was no adverse effect on the percentage of sperm wlith normal morphology.

NECROPSY (PARENTAL ANIMALS)
There were no findings considered to be related to treatment

ORGAN WEIGHTS (PARENTAL ANIMALS)
At necropsy after approximately 18 weeks of treatment the group mean bodyweight of males were 82%,95% and 101% of the Controls at 3200,800 and200 ppm respectively. Bodyweights for females which had reared their young to weaning were 92%, 101% and 99% of Control at 3200, 800 and 200 ppm respectively.

A number of organ weightslbodyweight relative weights were statistically significantly different from the Control. Among males at 3200 ppm, absolutekidney, liver and seminal vesicle weights were lower than in Controls and bodyweight relative weights for the brain, adrenals, epididymides, prostate, spleen and testes were higher than in Controls.

Among females at 3200 ppm, absolute adrenal, spleen, ovaries (plus oviducts) and kidney weights were lower than in Controls and, apart from the ovaries, these organs were also lower than Control when expressed as bodyweight-relative values.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Reproductive organs were examined from all Control and 3200 ppm animals. (The right testis and epididymis were examined - tissues from the left side of the body were used for assessment of sperm counts, motility and morphology and were not subjected to microscopic examination).

Treatment of all males at 3200 ppm was associated with a specific change in the right epididymis: the epithelial cells lining the epididymal duct in the distal part of the caput and the proximal portion of the corpus epididymis exhibited macro and microvesiculation which resembled fat vacuoles. In viewof this treatment related change, the right epididymis was subsequently examined for males at 800 and 200 ppm and the change was detected in the right epididymis of 13 of the 31 males examined at 800 ppm. No malcs wcre affected at 200 ppm.

Among females at 3200 ppm, there was a clear increase in the number of animals exhibiting vaginal epithelium which was atrophic and mucified compared with Controls. These are histological characteristics indicative of anoestrus, indicating that the animals had not yet returned to normal oestrus cycling (females with live litters were killed on Day 21 of lactation).

Effect levels (P0)

1

Dose descriptor:

NOEL

Effect level:

200 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: F0 and F1 (migrated information)

2

Dose descriptor:

NOEL

Effect level:

800 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: F0 and F1 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Description (incidence and severity):

No effects of toxicological importance

Histopathological findings:

not examined

Details on results (F1)

F0 GENERATION

LITTER SIZE AND OFFSPRING VIABILITY (OFFSPRING)
At 3200 ppm, mean implantation count and total and live litter size on Day 1 of age were both slightly lower than in Controls; the difference attained statistical significance for live litter size. There was no effect on implantation count and litter size at 800 and 200 ppm.

There was no effect of treatment on the post implantation survival index, indicating that in-utero survival was not impaired. Indices of offspring viability were largely influenced by the incidences of total litter loss and litters killed (1, 2, 0 and 3 litters for Groups I to 4 respectively) and it was considered that there was no evidence of any treatment related effects upon offspring survival.

SEX RATIO (OFFSPRING)
The ratio of male to female offspring was unaffected by treatment.

OFFSPRING BODYWEIGHT
Bodyweight of male and female offspring on Day 1 of age was not affected by treatment, although it might have been expected that mean pup weights at 3200 ppm would be slightly higher than in Controls in view of the lower litter size and this was not the case.

Bodyweight gain of male and female offspring to Day 14 of age was not adversely affected by treatment. Thereafter, weight gain of unselected males and females at 3200 and 800 ppm was lower than in Controls reflecting impaired growth, as the offspring started to feed independently and had exposure to the test material in their diet; this was reflected in significantly lower weight gain during Days 14-35 of age compared with Controls.

Growth of offspring at 200 ppm was marginally lower than in Controls but differences did not attain significance.

OFFSPRING DEVELOPMENT
There was no effect of treatment on the timing of onset or completion of pinna detachment, hair growth, tooth eruption and eye opening.

NECROPSY OF OFFSPRING
Necropsy of offspring that were examined before weaning revealed absence of milk in the stomach as a consistent finding, unrelated to treatment.

Necropsy of offspring not selected for the next generation and killed at 35 days of age revealed no evidence for change or anomaly that could be related to treatment.

Macroscopic abnormalities considered to be worthy of further investigation were examined microscopically but there were no treatment-related findings.

ORGAN WEIGHT OF OFFSPRING ON DAY 35 OF AGE
Mean bodyweight, spleen and thymus weights were significantly lower than in Controls for males at 3200 ppm. However, differences largely disappeared when spleen and thymus weights were expressed as bodyweight relative values suggesting that there was no independent effect on these organs.

Mean bodyweight for females at 3200 and 800 ppm were significantly lower than in Controls. There were significantly lower absolute ovary weights in all treated groups but these most likely reflected differences in bodyweight as there were no clear differences in bodyweight relative values. In addition, there was significantly lower absolute thymus weight at 3200 ppm only.

SEXUAL MATURATION (F0 PARENTAL ANIMALS)
There were no treatment-related effects on time of sexual maturation of males or females, based on the time of balano-preputial
separation or of vaginal opening, respectively.

F1 GENERATION

LITTER SIZE AND OFFSPRING VIABILITY (OFFSPRING)
As in the F0 generation, mean implantation count and total and live litter size on Day 1 of age at 3200 ppm were slightly lower than in Controls; the differences did not attain significance and, there was no indication of in-utero or peri-natal mortality as contributory factors to the lower live litter size.
There was no effect on implantation count and litter size at 800 and 200 ppm.

Indices of offspring viability were largely influenced by the incidences of total litter loss and litters killed (1, 0, 5 and 2 litters for Groups 1 to 4 respectively). There was no clear evidence of any treatment related effects upon offspring survival.

SEX RATIO (OFFSPRING)
At 3200 ppm, there was a slightly lower than expected mean percentage of males compared with Controls; the difference attained significance at Day 4 of age pre cull. Only 3/32 litters at 3200 ppm contained more than 60% male pups compared with 9/27 Controls.

At 800 and 200 ppm, the ratio of male to female offspring was unaffected by treatment and there were approximately 50% male offspring in these groups.

BODY WEIGHT (OFFSPRING)
Bodyweight of male and female offspring on Day 1 of age was not obviously affected by treatment.

Bodyweight gain of male and female offspring to Day 14 of age was not adversely affected by treatment. Thereafter, weight gain of males and females at 3200 ppm was lower than in Controls reflecting impaired growth as the offspring started to feed independently; this was reflected in significantly lower weight gain during Days 14-35 of age compared with Controls.

OFFSPRING DEVELOPMENT
There was no effect of treatment on the timing of onset or completion of pinna detachment, hair growth, tooth eruption and eye opening.

NECROPSY OF OFFSPRING
Necropsy of offspring which died before weaning revealed absence of milk in the stomach as the only consistent finding unrelated to treatment. Therewere no significant anomalies, among the offspring which died or those which survived to necropsy on Day 35 of age, which were considered to be
associated with treatment.

Macroscopic abnormalities considered to be worthy of further investigation were examined microscopically but there were no treatment related findings.

ORGAN WEIGHT OF OFFSPRING ON DAY 35 OF AGE
Mean bodyweight, brain, spleen and thymus weights were significantly lower than in Controls for males at 3200 ppm. However, there were no clear differences in bodyweight relative weights for these organs suggesting that differences were related to the lower bodyweight. Mean absolute thymus weights were also lower than Controls at 800 ppm.

Mean bodyweight for females at 3200 ppm were significantly lower than in Controls. As for the males, there were significantly lower absolute organ weights (spleen and thymus) but these most likely reflected differences in bodyweight as there were no clear differences in bodyweight relative weights.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

200 ppm (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Adult toxicity:
The No-observed-effect-Ievel (NOEL) for toxicity to the F0 and F1 adult animals was 200 ppm (11 and 14 mg/kg/day for F0 males and females respectively based on Week 10 bodyweights or, 12 and 16 mg/kg/day for F1 males and females respectively) based on decreased bodyweight gain at 3200 ppm inmales and females and at 800 ppm in females; decreased sperm motility at 3200 ppm in the F0 males and at 3200 and 800 ppm in the F1 males, and epididymal lesions at 3200 and 800 ppm in males. There were no treatment-related effects on mortality, clinical signs of toxicity, food consumption or food efficiency.

Reproductive toxicity:
The NOEL for reproductive toxicity was 800 ppm (approximately 43-61 mg/kg/day based on Week 10 bodyweights) based on slightly decreased implantation count and litter size for the F0 and F1 females at 3200 ppm, and delayed or non-recovery of oestrus cyclicity postpregnancy of F1 females at 3200 ppm. Neither mating or fertility indices were affected at any dose. There were no adverse effects on testicular or ovarian function at 3200 ppm.

Pup toxicity:
The NOEL for toxicity to pups was 200 ppm (approximately 32 mg/kg/day achieved maternal dosage during Days 7-13 of lactation) based on bodyweight gain decreases occurring after the onset of self-feeding of treated diets in F1 pups at 3200 and 800 ppm and in F2 pups at 3200 ppm.
There were no treatment-related effects on pup survival, development, or sexual maturation.

Executive summary:

Introduction: The objective of this study was to assess the effects of continuous dietary administration of N-(n-butyl phosphorothioic triamide) (NBPT), an organophosphorrus compound, upon the reproductive performance of the male and female rats of the CD strain through two successive generations. The study was conducted in accordance with the regulations or guidelines of the following authorities:

- Draft guidelines of the Environmental Protection Agency of United States of America OPPTS 870.3800 (1984) (N.B: Revisions of the draft guidelines released as a "Public Draft" (February 1996) were taken into account).

- Organisation for Economic Co-operation and Development, Guideline 416 (1983)

- Ministry of Agriculture, Forestry and Fisheries of Japan (1985)

Method:

The NBPT was administered continuously in the diet at concentrations of 200, 800 or 3200 ppm to groups of rats to the two successive generations. A fourth group received the basal diet without the test material and served as the Control.

The F0 generation, which comprised 32 males and 32 females in each group, received the treated diet for 10 weeks before pairing and throughout mating, gestation and lactation. From the Fl litters, 32 male and 32 female offspring were selected to fonn the F1 generation. Both sexes received treatment for a minimum of 10 weeks from selection, throughout pairing, gestation and lactation. Pup survival, body weight, development, and sexual maturation were evaluated.

All F0 and F1 adult animals were subjected to a detailed necropsy and the reproductive organs and the brain, liver, kidneys, adrenals and spleen were weighed and retained. Sperm counts, motility and morphology were determined for the left epididymis and testis for all F0 and F1 males. Histopathological examinations were performed on reproductive organs from Control and high dosage animals. In addition, the retained epididymis was examined for all F0 and F1 males in the 800 ppm and 200 ppm groups.

Non-selected F1 offspring were subjected to a necropsy examination on Day 35 of age and the brain, spleen, thymus, ovaries and testes weighed. All non-selected F2 offspring were subjected to a necropsy examination on Day 35 of age and the brain, spleen and thymus weighed.

Principal findings

Non-reproductive phases

The inclusion of NBPT in the diet at levels up to 3200 ppm did not affect the general condition of the animals and there were no mortalities considered to be related to treatment.

Treatment of F0 males at 3200 ppm and, of F0 females at 3200 and 800 ppm, was associated with lower weight gains prior to pairing. Bodyweight gain of F1 and F2 offspring at 3200 ppm and of F1 offspring at 800 ppm was retarded from Day 14 of age compared with Controls. The overall growth rate of selected F1 animals from selection to termination was also retarded at 3200 ppm.

F0 and F1 females receiving 3200 ppm showed lower weight gains during gestation and did not show the characteristic pattern of late lactation weight loss.

There was no effect of treatment on pup development or sexual maturation of F1 animals.

Achieved intake of NBPT

In the F0 generation at 3200 ppm, chemical intake was approximately 370 mg/kg/day for males and females during the first week of treatment. In the week before pairing, intake had declined naturally to approximately 180 mg/kglday for males and 220 mg/kg/day for females. Chemical intake for females was higher during gestation and increased to a maximum of around 530 mg/kglday during Days 7-13 of lactation at peak physiological demand.

At 3200 ppm during the first week of the F1 generation, chemical intake was approximately 480 mg/kg/day for males and females. In the week before pairing, intake was about 200 mg/kg/day for males and 250 mg/kg/day for females. Chemical intake for females was higher during early gestation and increased to around 520 mg/kg/day during Days 7-13 of lactation at peak physiological demand.

Chemical intake by animals in the lower treatment groups was in proportion to the dietary concentrations but followed the same patterns as at 3200 ppm.

Fertility and reproductive performance

In both the F0 and F1 generations, there were no adverse effects of treatment on mating performance.

At 3200 ppm only, mean implantation count and total and live litter size on Day 1 of age was slightly lower than in Controls on both generations.

There was no effect of treatment on the parturition process or, peri and post-natal survival of the offspring. Pre-weaning developmental landmarks were unaffected and despite a retardation in growth rate among F1 offspring at 3200 ppm, the attainment of sexual maturity was not delayed.

Terminal studies

Treatment of F0 males at 3200 ppm was associated with a significantly lower percentage of progressively motile sperm in the left epididymis compared with Controls and, further analysis demonstrated reductions in straight line and curvilinear velocity of the sperm. Organ weight analysis showed a higher bodyweight relative weight for the epididymides at 3200 ppm. Microscopic examination of the right epididymis detected a specific treatment-related change for all males at 3200 ppm and some males at 800 ppm: cells lining the epididymal duct in the distal part of the caput and the proximal portion of the corpus epididymis exhibited macro and microvesiculation which resembled fat vacuoles.

Treatment of F1 males at 3200 ppm was associated with significantly lower percentages of motile and progressively motile sperm in the left epididymis compared with Controls. At 800 ppm, the percentage of motile sperm was lower than in Controls Further analysis demonstrated a significant reduction in the percentage of 'rapid' sperm at 3200 ppm only. As in the Fa generation, organ weight analysis showed a higher bodyweight relative weight for the epididymides at 3200 ppm and microscopic examination of the right epididymis detected the same vacuolation of a defined area of the epithelium at 3200 and 800 ppm as had been seen in F0 males. 200 ppm was a no-effect-Ievel for the epididymis.

There was a clear increase in the incidence of F1 females in the 3200 ppm group exhibiting vaginal epithelium which was atrophic and mucified.

Conclusion

Adult toxicity:

The No-observed-effect-Ievel (NOEL) for toxicity to the F0 and F1 adult animals was 200 ppm (11 and 14 mg/kg/day for F₀ males and females respectively based on Week 10 bodyweights or, 12 and 16 mg/kg/day for F1 males and females respectively) based on decreased bodyweight gain at 3200 ppm in males and females and at 800 ppm in females; decreased sperm motility at 3200 ppm in the F0 males and at 3200 and 800 ppm in the F1 males, and epididymal lesions at 3200 and 800 ppm in males. There were no treatment-related effects on mortality, clinical signs of toxicity, food consumption or food efficiency.

Reproductive toxicity:

The NOEL for reproductive toxicity was 800 ppm (approximately 43-61 mg/kg/day based on Week 10 bodyweights) based on slightly decreased irnplantation count and litter size for the F₀ and F₁ females at 3200 ppm, and delayed or non-recovery of oestrus cyclicity postpregnancy of F₁ females at 3200 ppm. Neither mating or fertility indices were affected at any dose. There were no adverse effects on testicular or ovarian function at 3200 ppm.

Pup toxicity:

The NOEL for toxicity to pups was 200 ppm (approximately 32 mg/kg/day achieved maternal dosage during Days 7-13 of lactation) based on bodyweight gain decreases occurring after the onset of self-feeding of treated diets in F1 pups at 3200 and 800 ppm and in F2 pups at 3200 ppm. There were no treatment-related effects on pup survival, development, or sexual maturation.