Pirimiphos-methyl

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Jun 1993 to 07 Mar 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Pirimiphos-methyl

EC Number:

249-528-5

EC Name:

Pirimiphos-methyl

Cas Number:

29232-93-7

Molecular formula:

C11H20N3O3PS

IUPAC Name:

O-2-(diethylamino)-6-methylpyrimidin-4-yl O,O-dimethyl phosphorothioate

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (F0) 6 weeks
- Weight at study initiation: (F0) Males and females: ca 100 g
- Housing: The F0 animals were initially housed 2 per cage in polypropylene cages with stainless steel grid bottoms, mesh tops and food hoppers. The cages measured 42 x 27 x 20 cm and were suspended on racks containing 6 rows of 4 cages. Males and females were racked separately. Excreta were collected on a tray lined with absorbent paper, suspended beneath each cage. Three days prior to the initiation of mating, the males were transferred to individual cages. For mating, the females were transferred to the cage of the appropriate co-group male. Mated females were transferred to individual solid bottomed cages of the same dimensions, in which sterilised white wood shavings were provided as bedding. White paper tissue was supplied to each mother for incorporation in the nest; this was replaced when it became soiled. Dams and their litters retained this type of cage until termination. F1 animals selected as parents fo the next generation were housed 2 per grid-bottom cage, sexes separate, and then followed the same caging regime as described for the F0 animals.
- Diet: Rat and Mouse Breeder Diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2
- Humidity (%): 50 ± 15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 Jun 1993 To: 7 Mar 1994

Administration / exposure

Route of administration:

oral: feed

Vehicle:

ethanol

Details on exposure:

DIET PREPARATION
Fresh batches of treated diets were prepared weekly during the study, and used within one week of preparation. An appropriate quantity of the test material was dissolved in a suitable volume of ethanol. This solution was added to a suitable quantity of untreated diet, then mixed for at least 2 h using a Hobart mixer with fan assisted venting to form a premix. A control premix was prepared using the same proportion of ethanol and untreated diet. The formulated diets for the Control, Intermediate and High doses were prepared by dilution of the appropriate premix with untreated diet to give the desired concentrations. The Low dose diet was prepared by dilution of the High dose diet with untreated diet. The formulations were mixed on a Winkworth tumblemixer for at least 20 min

Details on mating procedure:

- M/F ratio per cage: Animals were generally paired on a one male to one female basis, sibling matings being avoided. On one occasion the number of males in a group was reduced by mortality, and so one male was paired on a one male to 2 females basis, such that all females were paired.
- Length of cohabitation: until mating was detected or 7 nights had elapsed
- Proof of pregnancy: A vaginal lavage was examined early each morning, commencing on the morning of pairing, until a mating sign was detected; the day of detection of sperm in the lavage, or of a copulatory plug in situ was considered to be Day 0 of gestation
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility; If no mating sign was detected during that time, a rest period of 2 days was allowed before the female was placed with a second co-group male, which had already mated. After 7 nights with the second male, if no mating sign had been detected, attempts to breed from the female ceased and she was transferred to an individual, solid bottom cage
- Further matings after two unsuccessful attempts: no
- Any other information: The stage of the oestrous cycle in each lavage was recorded to assist with interpretation of mating performance and to indicate possible adverse effects on the oestrus cycle

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On 7 occasions triplicate samples were taken from each formulated diet: comencement of treatment (week 1), F0 pre-mating period (week 2), F0 pre-mating period (week 7), F0 gestation period (week 12), F1 pre-mating period (week 19), F1 pre-mating period (week 26), F1 lactation period (week 34)

The method may be summarized as follows: Ten gram aliquots of treated diet were weighed into 250 mL glass centrifuge bottles. Toluene was added to the sample, which was allowed to soak for four hours, then homogenized using a high speed blender. Following centrifugation to separate the solid and liquid phases, an aliquot of the toluene supernatant was collected and dried over granular anhydrous sodium sulfate. A portion of the dried aliquot was placed into an autosampler vial for analysis using a gas chromatograph equipped with a flame photometric detector (FPD). The samples were chromatographed on a 15 meter, DB-1 column.

Duration of treatment / exposure:

Commencing at ca 6 weeks of age, F0 animals were treated for 10 weeks prior to mating for the production of F1 litters. Treatment continued throughout the mating, gestation and lactation periods, until termination after weaning of these litters. F1 animals were weaned onto the same diets as were fed to their respective parents. The selected F1 animals were treated for ca 12 weeks after weaning, prior to mating. Treatment then continued for both sexes throughout the mating, gestation and lactation periods, until termination after weaning of the F2 litters.

Frequency of treatment:

Continuous

Details on study schedule:

- F1 parental animals not mated until 9 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 12 weeks

F1 WEANING
For the selected pups, weaning occured on Day 24 of lactation, when they were removed from their mother and re-housed 2 per grid bottomed cage, sexes on seperate racks. The remaining F1 pups and all F2 pups remained with their mother until termination

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 ppm

Remarks:

Low dose group - Equivalent to 1 mg/kg bw/day (F0 and F1 generations)

Doses / concentrations 2

Dose / conc.:

40 ppm

Remarks:

Middle dose group - Equivalent to 3 and 4 mg/kg bw/day (F0 an F1 generations, respectively)

Doses / concentrations 3

Dose / conc.:

160 ppm

Remarks:

High dose group - Equivalent to 12 and 15 mg/kg bw/day (F0 an F1 generations, respectively)

No. of animals per sex per dose:

- F0: 28
- F1: 24

Control animals:

yes, plain diet

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: morning and evening every day

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily
- Clinical observations included behaviour, appearance, respiration, skin and coat condition.

BODY WEIGHT:
- Weights of F0 animals were recorded one week prior to the first day of test material administration, then weekly thereafter until the start of the mating period. Males continued weekly weighing until termination. Weights for females were also recorded on Day 0 of gestation (the day of detection of a positive mating sign) and Days 7, 14 and 20 of gestation, then on Days 1, 7, 14 and 21 of lactation (where the day of birth of the litter was designated Day 0 of lactation).
- Post-weaning F1, animals were weighed weekly from selection until the start of their mating period, after which they followed the same regime of weighing as described for F0 animals.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined: yes
- Time schedule: For F0 animals, food consumption was measured over the week prior to the first administration of test material and then weekly thereafter, until the start of the mating period. For males, consumption continued to be measured over complete one week periods, except during periods of cohabitation with females. For females, weekly food consumption measurements resumed over the 3 weeks of gestation (Days 0-7, 7-14, 14-20) and of lactation (Days 0-7, 7-14, 7-21). For F1 animals, measurement began shortly after selection and then followed the same regime as for the F0 animals.

OBSERVATIONS ON FEMALES DURING THE LACTATION PERIOD
- The females were allowed to litter normally. The day on which parturition commenced was designated Day 0 of lactation.
- The duration of gestation days was calculated.
- Any deficiencies in maternal care were recorded: points looked for included inadequate construction and cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding

CLINICAL CHEMISTRY
- Plasma and red blood cell cholinesterase activity were assessed

Oestrous cyclicity (parental animals):

The stage of the oestrous cycle in each vaginal lavage (during mating) was recorded to assist with interpretation of mating performance and to indicate possible adverse effects on the oestrus cycle.

Sperm parameters (parental animals):

Parameters examined in all male generations: From the High dose and Control groups, the testes and epididymides were examined for qualitative and quantitative effects on spermatogenesis (except for premature decedents).

Following a short (less than 24 h) fixation in bouin’s fluid, a single transversesection disc was cut from each testis. These discs were then transferred to absolute alcohol, followed by processing and embedding in paraffin wax. Sections were cut and stained with haematoxylin and PAS (periodic acid-Schiff), and examined by light microscopy.
For each pair of testes, the number of tubule cross-sections per mm2 was recorded and averaged from 5 microscope fields (each of 2.54 mm2), the calculation of the relative tubule length was performed according to the method of Lennox et al (1970), using the product of testis volume and the average number of tubule crosssections per mm2. From each pair of testes, 10 cross-sections of tubules in Stage VII of the cycle of the seminiferous epithelium, staged according to Leblond and Clermont (1952), were examined for the numbers of preleptotene spermatocytes,
pachytene spermatocytes, and Sertoli cells. The ratio of pachytene spermatocytes to Sertoli cells was calculated. The diameters of 20 cross-sections in Stage VII were also recorded using an eyepiece graticule (with 86 units equalling 1 mm). In cases where one of the testes was grossly abnormal, all assessments were made from the unaffected organs.
After an initial preservation in Hams medium, an Eosin-stained smear of sperm from one cauda epididymis from each male was prepared and then examined for morphological abnormalities using criteria described by Wyrobek and Bruce (1975)

One thousand sperm were evaluated per animal for the following abnormalities:
A: Sperm hood upturned or elongated
B: Banana-shaped sperm head
C: Amorphous sperm head
D: Sperm tail tightly coiled
E: Sperm tail acutely folded

Litter observations:

PARAMETERS EXAMINED
The number of live pups born and the number found dead in each litter was recorded as soon as possible after completion of parturition. The live pups were sexed, counted, examined for the presence of milk in the stomach and for any externally visible abnormalities daily up to Day 4 of lactation; they were counted and examined for abnormalities again on Days 7, 14 and 21. Pre-weaning F1 and F2 pups were weighed en masse by litter (sexes separate) on Days 1, 4, 7 and 14 of lactation. The pups were weighed individually on Day 21 of lactation, and the total litter weight was also recorded. Where practical, any pups found dead or killed during lactation were sexed and examined as above.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
Males were killed around the time of weaning of the first litters. Females were killed at or shortly after the time of weaning of their litter.

GROSS NECROPSY
All F0 and F1 animals were subjected to a necropsy, consisting of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities. Any gross lesions were described and representative samples taken and fixed in neutral buffered 10 % formalin.

ORGAN WEIGHTS / FIXATION
- The following organs were weighed (where indicated) and fixed: ovaries, uterus, cervix and vagina, testes (weight and volume recorded individually), epididymides (weighed individually), seminal vesicles, coagulating gland (weighed), prostate gland (weighed), pituitary gland, adrenal glands, brain (weighed).
- The female reproductive tract was examined for signs of pregnancy and the number of visible implantation sites was recorded.
- The testes were fixed in Bouin’s fluid, and one cauda epididymis was preserved in Ham’s F10 medium. The remaining tissues and organs were fixed in 10% formalin.
- Carcasses were discarded following these procedures.
- 0.1 g sample of brain (from the frontal cortex) was removed, snap frozen in liquid nitrogen and stored at -20°C until analysed.

HISTOPATHOLOGY
- From the High dose and Control groups, the testes and epididymides were examined for qualitative and quantitative effects on spermatogenesis
- Following a short fixation in bouin’s fluid, a single transverse sections was cut from each testis. These discs where then transferred to absolute alcohol, followed by processing and embedding in paraffin wax. Section were cut and stained with haematoxylin and PAS and examined by light microscopy.
- After an initial preservation in Hams medium, an Eosin-stained smear of sperm form one cauda epididymis form each male was prepared and then examined for morphological abnormalities.
- One thousand sperm were evaluated per animal for abnormalities
- The remaining epididymis, together with the seminal vesicles, coagulating gland, prostate gland and pituitary gland form each male, and the ovaries, uterus, cervix, vagina and pituitary gland from male were processed, and a haematoxylin and eosin stained section from each organ was examined histologically
- Brain cholinesterase (Brain ChE) was measured in the sample of the brain

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were subjected to postmortem examinations (macroscopic and/or microscopic examination)
- Pre-weaning: offspring found dead or killed before Day 14 of lactation were sexed, examined for externally visible abnormalities and for the presence of milk in the stomach. Offspring dying on or after Day 14 were subjected to a gross necropsy
- At Weaning: From each litter, 2 male and 2 female pups were necropsied. The remaining pups (except F1 weanlings selected for rearing to produce the next generation) were killed after external examination and the carcasses discarded without necropsy.

GROSS NECROPSY
- Pre-weaning: the cranial, thoracic and abdominal contents were examined macroscopically; any findings were recorded and abnormal tissues sampled, as appropriate.
- At weaning: The necropsy consisted of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities. Any gross lesions were described and representative samples taken and fixed in neutral buffered 10% formalin. Carcasses were discarded following these procedures. The remaining pups were killed after external examination and the carcasses discarded without necropsy.

Statistics:

Where considered appropriate to assist with interpretation, statistical analyses were applied to determine the statistical significance of differences from Control. Cholinesterase data were generally analysed by analysis of variance, with pairwise comparisons made using Fisher’s F-protected t-test (Snedecor and Cochran, 1980). Where necessary, a log transformation was used to stabilise the variances. Where a log transformation failed to stabilise the variances, the Kruskal-Wallis test, followed by Dunn’s procedure was used (Hollander and Wolfe, 1973).
Organ weight data were analysed by analysis of variance and by analysis of covariance (Snedecor and Cochran, 1980) using the terminal body weight as the single covariate. Treatment means were compared using an F-protected Least Significant Difference procedure.

Reproductive indices:

- Fertility index = Number of pregnant females or siring males/Number Paired
- Gestation index = Number bearing live pups/Number pregnant

Offspring viability indices:

- Birth index = Total number of pups born (live and dead) / Number of implantation scars
- Live birth index = Number of pups live on Day 0 of lactation / Total number born
- Viability index = Number of pups live on Day 4 of lactation / Number live on Day 0
- Lactation index = Number of pups live on Day 21 of lactation / Number live on Day 4
- Overal survival index = Number of pups live on Day 21 of lactation / Total number of pups born (live and dead)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

None of the observed clinical observations were considered to be associated with treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Among males, body weight performance was similar in all groups.
Among females, mean weight gain at 160 p.p.m. during the premating and gestation periods were marginally lower than Control; slight differences in body weight performance during lactation were not obviously associated with treatment. Body weight performance of females at 10 and 40 p.p.m. was essentially similar to Control.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Slight intergroup differences in food consumption were considered too small to be attributed to treatment.

Food efficiency:

no effects observed

Description (incidence and severity):

At all times, the achieved dosages were proportional to the nominal dietary concentrations.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mean Plasma cholinesterase:
At 40 and 160 ppm, mean plasma cholinesterase activity during treatment was consistently and significantly lower than Control (38-86 % and 16-56 % of Control for 40 and 160 ppm, respectively). At 10 ppm, mean plasma cholinesterase activity in the F0 generation was significantly lower than Control (61-78% of Control).

Red Blood Cell Cholinesterase:
At 160 ppm, mean red blood cell cholinesterase activity during treatment was generally significantly lower than Control (generally 52-67% of Control). At 40 ppm, there was some indication of reduction in red blood cell cholinesterase activity, especially among the F0 generation. At 10 ppm, there were no consistent differences in red blood cell cholinesterase activity compared with Control.

Brain Cholinesterase:
At 160 ppm, mean brain cholinesterase activity was lower than Control (47-87 % of Control), with the differences attaining statistical significance. At 40 ppm, brain cholinesterase activity of females was slightly lower than Control (47-56 % of Control), but with the differences attaining statistical significance. At 10 ppm, and for males at 40 ppm, brain cholinesterase levels were essentially similar to Control.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The histological assessments did not indicate any obvious adverse effect of treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance, fertility of both sexes and the duration of gestation were similar in all groups.
The mean number of implants were essentially similar in all groups.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

10 ppm

Based on:

test mat.

Remarks:

corresponding to 1 mg/kg bw/day

Sex:

male/female

Basis for effect level:

clinical biochemistry

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

160 ppm

Based on:

test mat.

Remarks:

corresponding to 12 mg/kg bw/day

Sex:

male/female

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

None of the observed clinical observations were considered to be associated with treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Among males, body weight performance was similar in all groups.
Among females, mean weight gain at 160 p.p.m. during the premating and gestation periods were marginally lower than Control; slight differences in body weight performance during lactation were not obviously associated with treatment. Body weight performance of females at 10 and 40 p.p.m. was essentially similar to Control.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Slight intergroup differences in food consumption were considered too small to be attributed to treatment.

Food efficiency:

no effects observed

Description (incidence and severity):

At all times, the achieved dosages were proportional to the nominal dietary concentrations.

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Plasma Cholinesterase:
At 40 and 160 ppm, mean plasma cholinesterase activity during treatment was consistently and significantly lower than Control (38-86 % and 16-56 % of Control for 40 and 160 ppm, respectively). At 10 ppm, mean plasma cholinesterase activity for F1 animals, activity was generally lower than Control except for F1 males sampled shortly prior to mating, although only the value for F1 females at termination attained statistical significance.

Red Blood Cell Cholinesterase:
At 160 ppm, mean red blood cell cholinesterase activity during treatment was generally significantly lower than Control (generally 52-67% of Control), although the value for F1 females at termination was similar to Control (93% of Control). At 40 ppm, there was some indication of reduction in red blood cell cholinesterase activity. At 10 ppm, there were no consistent differences in red blood cell cholinesterase activity compared with Control.

Brain Cholinesterase:
At 160 ppm, mean brain cholinesterase activity was lower than Control (47-87 % of Control), with the differences attaining statistical significance except for F1 males. At 40 ppm, brain cholinesterase activity of females was slightly lower than Control (47-56 % of Control), but with the differences attaining statistical significance. At 10 ppm, and for males at 40 ppm, brain cholinesterase levels were essentially similar to Control.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no statistically significant intergroup differences in the weights of any of the organs assessed.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The histological assessments did not indicate any obvious adverse effect of treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Testicular tubular atrophy was seen in 6 F1 males at 160 ppm, together with one Control F1 male. In 4 of these males at 160 ppm, the finding was only very mild, and in the 2 males with moderate atrophy the lesion was only unilateral. Furthermore, only one of the males with moderate atrophy, together with one male with very mild atrophy and a further male with no atrophy, failed to sire a litter. Also, the morphometic measurements and quantitative assessment of cell types for animals with tubular atrophy were not obviously different from those animals without atrophy. These parameters of the detailed examination of the testes were similar in the Control and High dose groups. The incidence of sperm abnormalities was similar in all groups.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance, fertility of both sexes and the duration of gestation were similar in all groups.
The mean number of implants were essentially similar in all groups.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

10 ppm

Based on:

test mat.

Remarks:

corresponding to 1 mg/kg bw/day

Sex:

male/female

Basis for effect level:

clinical biochemistry

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

160 ppm

Based on:

test mat.

Remarks:

corresponding to 15 mg/kg bw/day

Sex:

male/female

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P1)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The values for Birth, Live Birth, Viability, Lactation and Overall Survival Indices at all dose levels in both generations were similar to the corresponding Control values.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Slight intergroup differences in litter and pup weights did not appear to indicate any adverse effect of treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One pup from the Control Litter was killed in early lactation with absent tail, bladder distended and apparent ataxia. One pup from the 10 ppm Litter had a pale area on kidney. One pup from 160 ppm Litter had a bipartite spleen. None of these abnormalities were considered to have been associated with treatment.

Histopathological findings:

no effects observed

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

160 ppm

Based on:

test mat.

Remarks:

corresponding to 12 mg/kg bw/day

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The values for Live Birth, Viability, Lactation and Overall Survival Indices at all dose levels in both generations were similar to the corresponding Control values.
At 160 ppm the mean number of F2 pups born was slighty lower than the Control value, with consquent slight lowering of the Birth Index.
The mean number of F2 pups on Day 21 of lactation at 160 ppm was greated than the Control value, and it was therefore considered that the slightly lower Birth index as of doubtful biological importance.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Slight intergroup differences in litter and pup weights did not appear to indicate any adverse effect of treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One pup from the 10 ppm Litter had one eye absent. One pup from the Control Litter had a pale area on kidney. One pup from the 40 ppm Litter had left eye opaque and stomach distended. One pup from the Control Litter had free blood in cranial cavity. None of these abnormalities were considered to have been associated with treatment.

Histopathological findings:

no effects observed

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

160 ppm

Based on:

test mat.

Remarks:

corresponding to 15 mg/kw bw/day

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1. Analysis of formulated diets: means and overall means of corrected percent nominal for test substance treated diets

Study week	Mean corrected percent nominal
	10 ppm	40 ppm	160 ppm
1	77.8	87.1	85.8
2	90.1	81	81.3
7	79.6	83.1	83.4
12	96.8	87.1	81.7
19	93.8	89.3	87
34	78.4	74.5	77.6
Overall means	86.1	83.7	82.8

Values represent analyses of samples from the top, middle and bottom sections of bulk feed batch

Table 2. Cholinesterase activities (mean; absolute and % of control) in Sprague Dawley rats (14/group) fed diets containing the test substance over 2 generations

	F0 to F1 generation			F1 to F2 generation
Timing	pre-dosing	pre-mating	sacrifice	pre-mating	sacrifice
Dose (ppm)
Erythrocyte
Male       0^#	989	1250	1476	945	1272
10 (%)	123	96	90	79	92
40 (%)	102	78*	83**	99	83**
160 (%)	144	64**	67***	64	63**
Females    0^#	1575	1513	1234	1085	439
10 (%)	113	89*	94	108	112^+
40 (%)	110	79***	73***	83	97^+
160 (%)	105	54***	52***	53**	93^+
Brain
Males       0^£	N.P.	N.P.	16113	N.P.	12558
10 (%)	N.P.	N.P.	96	N.P.	103
40 (%)	N.P.	N.P.	96	N.P.	96
160 (%)	N.P.	N.P.	87***	N.P.	82
Females    0^£	N.P.	N.P.	14735	N.P.	14674
10 (%)	N.P.	N.P.	90*^$	N.P.	94
40 (%)	N.P.	N.P.	74***	N.P.	84*
160 (%)	N.P.	N.P.	47***	N.P.	56***

N.P. - not performed    ^$- only 2/14 animals below control range        

⁺ - Control value low  ^£- iU/mg          ^#- iU/l

* P<0.05     ** P<0.01        *** P<0.001

 

 

Table 3. Reproductive Performance for F0 and F1 and Litter parameters for F1 and F2

Observation	Dose Group (mg/kg/day)
	0	10		40	160
F0 Reproductive Performance
Mean precoital interval (days)	2	2		2	2
Males
Males placed with females	28	28		28	28
Mated	27	27		26	27
Male mating index	96	96		93	96
Males with females pregnant	26	25		25	24
Male fertility index	93	89		89	86
Females
Females placed with males	28	28		28	28
Number inseminated	27	25		25	24
Female mating index	96	89		89	86
Pregnant	27	25		26	25
Female fertility index	96	93		100	93
Mean gestation interval (days)	22.0	22.1		22.0	22.2
Number of litters	27	25		25	23
Gestation index	100	100		100	96
F1 Reproductive Performance
Mean precoital interval (days)	3	2		2	2
Males
Males placed with females	23	23		24	24
Mated	19	23		23	23
Male mating index	83	100		96	96
Males with females pregnant	21	21		23	21
Male fertility index	91	91		96	88
Females
Females placed with males	23	24		24	24
Number inseminated	21	22		23	22
Female mating index	91	92		96	92
Pregnant	21	22		23	22
Female fertility index	91	92		96	92
Mean gestation interval (days)	21.9	21.7		21.7	21.9
Number of litters	21	22		23	21
Gestation index	100	100		100	95
Litter Parameters
F1 Generation
Mean Implantation Sites	17.3		16.0	16.5		16.1
Number born live	417		369	375		339
Number born dead	7		11	7		4
# Deaths  Days 1-4 (%)	12.2		21.1	19.5		7.4
# Deaths  Days 5-7 (%)	14.6		22.5	22.1		9.7
# Deaths  Days 8-14 (%)	14.9		24.7	23.2		11.8
# Deaths  Days 15-21 (%)	14.9		24.7	23.5		12.4
Mean litter size  Day 0	15.3		14.4	14.9		14.7
Day 4	14.6		12.6	13.6		13.7
Day 7	14.2		12.4	13.2		13.3
Day 14	14.2		12.1	13.1		13.0
Day 21	14.2		12.1	13.0		12.9
Parturition index	91		92	91		88
Live birth index	98		97	98		99
Viability index	89		82	82		93
Lactation index	96		92	96		95
F2 Generation
Mean Implantation Sites	17.5		17.6	17.5		16.7
Number born live	323		340	361		294
Number born dead	5		5	6		7
# Deaths   Days 1-4 (%)	8.9		12.9	6.1		4.4
# Deaths  Days 5-7 (%)	13.9		20.0	10.5		4.8
# Deaths  Days 8-14 (%)	19.5		24.1	15.2		6.8
# Deaths  Days 15-21 (%)	19.8		24.1	15.8		6.8
Mean litter size  Day 0	15.4		15.6	16.1		14.0
Day 4	14.0		13.9	15.4		13.4
Day 7	13.2		13.0	14.7		13.3
Day 14	12.4		12.3	13.9		13.0
Day 21	12.3		12.3	13.8		13.0
Parturition index	89		90	93		82
Live birth index	98		99	98		98
Viability index	91		87	91		96
Lactation index	89		84	90		98

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, 10 ppm (corresponding to 1 mg/kg bw/day) was identified as a level in which only plasma cholinesterase was affected; this parameter is generally taken to indicate exposure rather than toxicity. Parental toxicity was observed at 40 and 160 ppm There were no effects on the reproductive parameters at any of the levels tested; therefore 160 ppm (corresponding to 12 and 15 mg/kg bw/day in F0 and F1, respectively) is considered a no observed adverse effect level (NOAEL) for reproductive toxicity  

Executive summary:

In a GLP compliant 2 generation reproductive toxicity study, performed according to EPA 83-4, Sprague-Dawley rats received diets containing 0, 10, 40,160 ppm of test substance. F0 animals were randomised into the 4 treatment groups, each containing 28 males and 28 females. From each treatment group, 24 male and 24 female F1 weanlings were selected for rearing to maturity and mating to produce the F2 generation. The F0 animals were treated for 10 weeks prior to mating, and then throughout the mating, gestation and lactation periods until sacrifice at the time of weaning of the F1 animals. The selected F1 animals were weaned onto the same dietary concentrations as their parents, and were maintained on these diets until termination. The F1 animals were mated at approximately 12 weeks of age. The F1 animals and their F2 litters were killed at the time of weaning of these litters.

Animals were observed for clinical signs, mortality, body weight, food consumption, mating behaviour, gestation length and degree of maternal care (including observations of pup condition and feeding). Reproduction and gestation indices were determined together with pup weight and sex ratio. There was no cull at day 4. All F0 and F1 adults received a gross post mortem examination, with reproductive organs being removed and weighed. Reproductive tissues from control and top dose animals were investigated histologically. In a later investigation (6 years later), testes from all F0 and F1 adult males were examined histologically. Sperm analyses were performed on all surviving adult males from the control and top dose groups. Gross examinations were performed on 2 pups/sex/litter at weaning and on any pups found dead or dying. No cholinesterase determinations were performed on pups.

Dietary analyses showed acceptable levels of incorporation and homogeneity, with overall achieved intakes of 0, 1, 3 or 12 mg/kg bw/d in the F0 generation and 0, 1, 4 and 15 mg/kg bw/d in F1 animals. There were no effects on mating, fertility, litter size, pup weight or pup survival in either generation, with a mean of 12 or more pups/litter in all groups at day 21. Body weight gain was reduced (~ 10%) in top dose females during gestation and lactation. Erythrocyte and brain cholinesterase activities were decreased, in a dose related manner at 40 and 160 ppm, and to a similar extent in both generations with females showing a greater sensitivity than males to inhibition of brain acetylcholinesterase. There were considered to be no adverse effects on cholinesterase activities at 10 ppm, as, with the exception of an isolated incidence in the pre-mating F1 males, the erythrocyte activity was inhibited by <20% as was the inhibition of cholinesterase activity in the brain in F0 females.

Under the conditions of this study, 10 ppm (corresponding to 1 mg/kg bw/day) was identified as a level in which only plasma cholinesterase was affected; this parameter is generally taken to indicate exposure rather than toxicity. Parental toxicity was observed at 40 and 160 ppm There were no effects on the reproductive parameters at any of the levels tested; therefore 160 ppm (corresponding to 12 and 15 mg/kg bw/day in F0 and F1, respectively) is considered a no observed adverse effect level (NOAEL) for reproductive toxicity.