Chlorpyrifos

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

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 OPP 83-4 (Reproduction and Fertility Effects)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302/EEC: Two-generation Reproduction Toxicity Test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: MAFF Toxicity Testing Guidelines for Reproduction Studies

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Chlorpyrifos

EC Number:

220-864-4

EC Name:

Chlorpyrifos

Cas Number:

2921-88-2

Molecular formula:

C9H11Cl3NO3PS

IUPAC Name:

O,O-diethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate

Test material form:

solid

Specific details on test material used for the study:

AGR 273801
Purity: 98.5 or 97.8%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

This strain of rat was selected because of its general acceptance and suitability for toxicity testing, and the availability of a reliable commercial source.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Portage, Michigan
- Age at study initiation: Approximately 6 weeks of age
- Fasting period before study: No
- Housing: Rats were housed singly in wire-mesh, stainless steel cages in racks provided with deotized cage board to minimize odor and aid in maintaining a clean environment. During the gestation/lactation phases of the study, females were housed in plastic cages provided with ground corn cob nesting material.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Atleast one week

ENVIRONMENTAL CONDITIONS
- The animal rooms of the facility were designed to maintain adequate environmental conditions concerning temperature, relative humidity, airflow and lighting, and were regulated for the species on test.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: feed

Details on exposure:

DIET PREPARATION: Diets were prepared by serially diluting a test material/feed concentrate (premix). The mixing procedures used to prepare the diets produced a homogeneous distribution of the test substance in the rodent chow.

Details on mating procedure:

Breeding of the F0 and F1 adults commenced after approximately 10 and 12 weeks of treatment, respectively. Each breeding program consisted of three 7-day cohabitation periods with one female and one male of the respective treatment group. For the F1 mating, cohabitation of male and female litter mates was avoided. During each breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating.
The day on which sperm were detected was considered Day 0 of gestation. Sperm positive females were separated and placed in nesting cages. Females which failed to mate during the first 7-day mating period were placed with an alternate male from the same treatment group for the second 7-day period; the same procedure was followed for the third 7-day mating period. The adult females continued to receive the respective test diet throughout gestation, lactation and until all litters on study were weaned as previously outlined. After the mating periods, males continued to receive the test diet until necropsied.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the test diets to determine the concentration of the test material were performed at least three times per generation. The average concentration of the test substance in the diets during the course of the study ranged from 93 to 100% of the targeted concentrations.

Duration of treatment / exposure:

Treatment started for F0 animals approximately 6 weeks of age for 2 generations until the F2 litters are weaned on day 21 postpartum.

Frequency of treatment:

7 days per week

Details on study schedule:

The treatment of the F0 rats began at approximately 6 weeks of age. After approximately 10 weeks on test diets, F0 rats were mated (one male to one female of the respective treatment group) to produce the F1 litters. Following weaning (3 weeks of age) of the F1 litters, 30 males and 30 females from each treatment group were randomly selected and assigned to the respective treatment group to become the parents for the next generation. After approximately 12 weeks of treatment following weaning of the last F1 litter, the F1 adults were bred to produce the F2 litters.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.1 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

5 mg/kg bw/day

No. of animals per sex per dose:

30 (F0 adults)
30 (F1 adults)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of the previous dietary toxicity studies in rats. The high dose level in both the males and females was expected to produce depressions in brain, plasma and RBC cholinesterase.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT AND FOOD CONSUMPTION: All F0 animals had body weights and feed consumption recorded weekly, during the 10-week pre-breeding treatment period, beginning on or before the first week of the study. Body weights for males were recorded weekly throughout the course of the study. Sperm positive females were weighed on Days 0, 7, 14 and 21 of gestation. Females that delivered litters were weighed on Days 1, 4, 7, 14, and 21 of lactation. During breeding, feed consumption was not measured in males or females due to cohousing. Following completion of the breeding periods, weekly feed consumption was again measured in males, and dietary concentrations adjusted accordingly. During gestation, feed consumption was measured at weekly intervals in sperm positive females. After parturition, feed consumption was measured once during the first week of lactation, twice during the second week of lactation and at 2 - 3 day intervals during the last week of lactation. A similar schedule was followed for the F1 generation.

Litter observations:

All litters were examined as soon as possible after delivery. The following parameters were recorded on each litter: the date of parturition, litter size on the day of parturition (Day 0), the number of live and dead pups on Days 0, 1, 4, 7, 14, and 21 postpartum, and the sex and weight of each pup on Days 1, 4 (before and after culling), 7, 14, and 21 of lactation. Visible physical abnormalities or demeanor changes in the neonates were recorded during the lactation period.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after the last litters in each generation were weaned
- Maternal animals: All surviving animals after the last litter of each generation was weaned

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Tissues routinely collected (Table 1) were preserved in neutral, phosphate-buffered 10% formalin. The testes of 10 F1 control and high dose males were also preserved in Bouin's Fixative. The lungs were infused with formalin to their approximate normal inspiratory volume. The nasal cavity was flushed with formalin via the pharyngeal duct to ensure rapid fixation of the tissue. Histologic examination of potential target organs and reproductive tissues (Table 1) was performed on the control and high-dose groups. In addition, the livers of 10 control and high-dose F1 adult males were examined microscopically. Examination of tissues from the low and middle groups was limited to the adrenals which demonstrated treatment-related histologic changes in the high-dose group, or gross lesions.

Postmortem examinations (offspring):

SACRIFICE
- At the time of weaning, 10 pups/sex/dose level from the F1 and F2 litters were randomly selected for a complete necropsy.

GROSS NECROPSY
- Grosspathologic examination was performed as described above for adults, except that terminal body weights were not recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS
- Histologic examination of tissues was not performed.

Statistics:

Body weights, body weight gains and plasma, RBC and brain cholinesterase levels were evaluated by Bartlett's test for equality of variances. Based upon the outcome of Bartlett's test, either a parametric or nonparametric analysis of variance (ANOVA) was performed. If the ANOVA was significant, a Dunnett's test or the Wilcoxon Rank-Sum test with Bonferroni's correction was performed.
Descriptive statistics (means and standard deviations) were reported for feed consumption. Statistical outliers were identified by the method of Grubbs and excluded from these calculations. Gestation length, average time to mating and litter size were analyzed using a non parametric ANOVA. If the ANOVA was significant, the Wilcoxon Rank-Sum test with Bonferroni's correction was performed. The fertility indices were analyzed by the Fisher exact probability test. Evaluation of the neonatal sex ratio was performed by the binomial distribution test. Survival indices and other incidence data among neonates were analyzed using the litter as the experimental unit by the Wilcoxon test as modified by Haseman and Hoel.

The nominal alpha levels to be used are as follows:
Bartlett's Test (Winer, 1971): α=0.01
Parametric ANOVA (Steel and Torrie, 1960): α=0.10
Nonparametric ANOVA (Hollander and Wolfe, 1973): α=0.10
Dunnett's Test (Winer, 1971): α=0.05 two-sided
Wilcoxon Rank-Sum Test (Hollander and Wolfe, 1973): α=0.05, two-sided with Bonferroni correction (Miller, 1966)
Fisher's Test (Siegel, 1956): α=0.05 one-sided
Censored Wilcoxon Test (Haseman and Hoel, 1974): α=0.05 one-sided
Outlier Test (Grubbs, 1969): α=0.02 two-sided
Binomial Distribution Test (Steel and Torrie, 1960): α=0.05 two-sided

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on behavior or demeanor were observed in males or females at any dose level during the exposure period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control male was found dead on test Day 125, seven days prior to the scheduled necropsy. No remarkable clinical observations were recorded for this rat prior to its death. Upon gross pathologic examination this animal was found to have perineal soiling, blood around the muzzle and general visceral congestion; however, the cause of death could not be determined.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significant treatment-related effects on body weights were observed at any dose level in males throughout the entire study or in females during the premating, gestation or lactation periods. Similarly, the body weight gains of females during gestation were unaffected by treatment. However, the total body weight gain of females from the 5.0 mg/kg/ day dose group was slightly but statistically decreased during the lactation day interval 1 through 21. This decrease in body weight gain was attributed to the combined stress of the test substance exposure and lactation, and was consistent with the slight decreases in feed consumption observed in high-dose dams during the last two weeks of lactation. The body weight gains of females in the 0.1 or 1.0 mg/kg/day dose groups were not affected by treatment during lactation.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No significant treatment-related effects on feed consumption were observed at any dose level in males throughout the entire study or in females during the premating, gestation or lactation periods. However, the feed consumption of dams given 5.0 mg/kg/day was slightly decreased during the last two weeks of lactation.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

An increased incidence of histopathologic alterations in the adrenal gland was observed in males and females in the 5.0 mg/kg/ day dose group. The histologic alterations were limited to cells of the zona fasciculata and were characterized as very slight to slight vacuolation (consistent with fatty change) in males, and very slight vacuolation and altered tinctorial properties in females.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Dose-related statistically significant decreases in plasma and RBC cholinesterase were observed in males and females in the 1.0 and 5.0 mg/kg/ day dose groups. Statistically significant decreases in brain cholinesterase were also observed in males and females in the 5.0 mg/kg/day dose group. Brain cholinesterase values in males and females from the 5.0 mg/kg/ day dose groups were decreased to 52 and 51%, respectively, of the control values. No significant effect on brain cholinesterase was observed in males or females in the 0.1 or 1.0 mg/kg/day dose groups.

Reproductive function / performance (P0)

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects were observed on the male or female fertility indices, length of gestation, time to mating, pup sex ratio or litter size in any dose group. The statistically identified decreases in the female conception and fertility index observed in the 0.1 mg/kg/day dose group were considered spurious, in view of the lack of effects on these parameters at 1.0 or 5.0 mg/kg/day.

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Effect level:

0.1 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of serum cholinesterase depression

Remarks on result:

other: No effects on fertility were observed at any dose level tested.

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on behavior or demeanor were observed in males or females at any dose level during the exposure period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male from the 0.1 mg/kg/day dose group was found dead on test Day 66. No remarkable clinical observations were recorded for this rat prior to its death. Upon gross pathologic examination this animal was found to have bilateral renal and urinary calculi. The kidneys were enlarged with a thin cortex and contained foul-smelling urine. The cause of death of this animal was attributed to the renal and bladder calculi.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weights of adult males in the 5.0 mg/kg/ day dose group were slightly decreased throughout the test period when compared with the control group; however, the decreases were not statistically significant. The lower body weights of males in the high dose group were consistent with the lower weights of these animals during lactation. Conversely, no effects on body weights were observed at any dose level in females during the premating, gestation or lactation periods. Similarly, the body weight gains of females during gestation and lactation were not affected by treatment. Since the body weight of females in the 5.0 mg/kg/day group recovered from the decreases observed as pups during lactation, the neonatal weight affects in females F0 pups were considered to be of no toxicologic significance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant treatment-related effects on feed consumption were observed at any dose level in males throughout the entire study or in females during the premating, gestation or lactation periods.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related gross pathologic alterations were observed in male or female F1 adults at any dose level.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

An increased incidence of histopathologic alterations in the adrenal gland was observed in males and females in the 5.0 mg/kg/ day dose group. The histologic alterations were limited to cells of the zona fasciculata and were characterized as very slight to slight vacuolation, consistent with fatty change in males and very slight altered tinctorial properties in females.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Dose-related statistically significant decreases in plasma and RBC cholinesterase were observed in males and females in the 1.0 and 5.0 mg/kg/ day dose groups. The statistically significant decrease in RBC cholinesterase observed in males at 0.1 mg/kg/day was not considered biologically significant due to the lack of effect on this parameter in either males or females given 0.1 mg/kg/day in the Fl generation or in females given 0.1 mg/kg/day in the F2 generation. Statistically significant decreases in brain cholinesterase were also observed in males and females in the 5.0 mg/kg/day dose group. Brain cholinesterase values in males and females from the 5.0 mg/kg/ day dose groups were decreased to 47 and 42%, respectively, of the control values. No significant effects on brain cholinesterase were observed at 0.1 or 1.0 mg/kg/day.

Reproductive function / performance (P1)

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects were observed on the male or female fertility indices, length of gestation, time to mating, pup sex ratio, pup survival or litter size in any dose group.

Effect levels (P1)

Key result

Dose descriptor:

NOEL

Effect level:

0.1 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of serum cholinesterase depression

Remarks on result:

other: No effects on fertility were observed at any dose level tested.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related, clinical observations or physical alterations were observed in pups from any dose group

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Pup survival, however, was statistically decreased on days 14 and 21 of lactation in the 5.0 mg/kg/ day dose group. No significant effects on pup survival were observed at earlier time points in the 5.0 mg/kg/day dose group or in the 0.1 or 1.0 mg/kg/day dose groups at any time during lactation. Importantly, the decrease in pup survival observed at 5.0 mg/kg/ day occurred only at a dose level that produced parental toxicity (significant decreases in brain cholinesterase and histopathologic alterations of the adrenal zona fasciculata).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weights of male and female pups from the 5.0 mg/kg/day dose group were slightly decreased on day 1 of lactation and statistically decreased on days 4, 7, 14 and 21 of lactation. The decreased pup weights at the high dose were consistent throughout the lactation period, were accompanied by decreased survival and lower body weight gain of dams in this dose group during lactation, and therefore, appeared to be treatment-related. The decreased pup weights occurred only at a dose level that produced parental toxicity (significantly decreased brain cholinesterase and histopathologic alterations of the adrenal zona fasciculata). No treatment related effects were observed at dose levels of 0.1 or 1.0 mg/kg/day.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related gross pathologic alterations were observed in weanlings at any dose level

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

Results: F2 generation

General toxicity (F2)

Description (incidence and severity):

No significant treatment-related effects on clinical observations were observed in any dose group. However, the incidence of weak, thin, or cold pups and pups with no milk in their stomach was increased in both the control and the 5.0 mg/kg/ day dose groups. Most of these observations occurred early in the lactation period, preceded pup death and were associated with the death of entire litter; and therefore, were considered secondary to maternal neglect. The apparent increased incidence of these observations in the 5.0 mg/kg/day dose group, relative to the control, were not considered treatment-related, as most of the observations were considered secondary to maternal neglect and occurred in an equal number (five) of litters in both the control and 5.0 mg/kg/day dose groups.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Pup survival, was lower than expected during lactation in both the control and the 5.0 mg/kg/ day dose group. The decreased pup survival in these groups was due to the death of three entire litters (25 pups) in the control group and five entire litters (48 pups) in the 5.0 mg/kg/ day dose group, was associated with an increased incidence of weak, thin, or cold pups or pups with no milk in their stomach, and was considered secondary to maternal neglect. Although the survival of pups in the 5.0 mg/kg/ day dose group was also lower than the control group, the decrease was due to a greater number (five) of dams that neglected their litters relative to the number of control dams that neglected their litters (three). Since the decreased pup survival in the 5.0 mg/kg/ day dose group was due to a limited number of litters and was not a general finding across most litters, the lower pup survival observed at 5.0 mg/kg/ day was not statistically significant and was not considered to be treatment-related. Analyses of pup survival, excluding entire litters that were lost due to dam neglect (three in the control, one at 1.0 mg/kg/ day and five at 5.0 mg/kg/ day), clearly show that no treatment-related effects on pup survival were observed at any dose level.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences in male or female pup weights were observed between the treated groups and the control group.

Effect levels (F2)

Key result

Dose descriptor:

NOEL

Generation:

F2

Effect level:

1 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: Tissues collected and preserved at necropsy 

Adrenals*	Kidneys*	Prostate*
Aorta	Lacrimal/harderian glands	Rectum
Auditory sebaceous glands	Larynx	Salivary glands
Bone	Liver	Seminal vesicles*
Bone marrow	Lungs	Skeletal muscle
Brain (cerebrum, brainstem, cerebellum)*	Mammary gland - females only	Skin
Cecum	Mediastinal lymph node	Spinal cord (cervical, thoracic, lumbar)
Cervix*	Mediastinal tissues	Spleen
Coagulating glands*	Mesenteric lymph node	Stomach
Colon	Mesenteric tissues	Testes*
Duodenum	Nasal tissues	Thymus
Epididymides*	Oral tissues	Thyroid gland
Esophagus	Ovaries*	Tongue
Eyes	Oviducts*	Trachea
Gross lesions*	Pancreas	Urinary bladder
Heart	Parathyroid glands	Uterus*
Ileum	Peripheral nerve	Vagina*
Jejunum	Pituitary*
*Indicates tissues from control and high dose adults that were routinely processed for histological examination

 

Applicant's summary and conclusion

Conclusions:

No effects on fertility were observed at any dose level tested.
Prental NOEL: 0.1 mg/kg/day (based upon the absence of serum cholinesterase depression)
Neonatal NOEL: 1.0 mg/kg/day

Executive summary:

The 2-generation study was conducted according to the guidelines OECD 416 and FIFRA 83-4. The objective of this 2-generation dietary reproduction study was to evaluate the effects of the test substance on the reproductive capability and neonatal growth and survival in rats. Groups of 30 male and 30 female Sprague-Dawley rats, approximately 6 weeks of age, were administered diets that provided 0, 0.1, 1.0 or 5.0 mg/kg/ day of test substance, 7 days per week for two generations. Following 10 weeks of dietary exposure, the F0 adults were mated to produce the F1 litters. After weaning, 30 pups/sex/dose were randomly selected to become F1 adults. Following 12 weeks of exposure, the F1 adults were mated to produce the F2 litters.

Dietary exposure of adult male and female rats to 1.0 and 5.0 mg/kg/day resulted in dose-related parental effects. F0 and F1 adult males and females had statistically significant decreases in plasma and RBC cholinesterase levels. In addition, both the F0 and F1 males and females in the 5.0 mg/kg/day dose group had statistically significant decreases in brain cholinesterase (52 to 42% of the control values) and histopathologic alterations in the zona fasciculata of the adrenal gland. The histopathologic alterations in the adrenal gland were characterized as very slight to slight vacuolation (consistent with fatty change) in males, and very slight vacuolation and/or altered tinctorial properties in females. The significant decreases in brain cholinesterase and histopathologic alterations of the adrenal zona fasciculata observed in males and females given 5.0 mg/kg/day were considered to represent significant parental toxicity. Although parental effects were observed at 1.0 and 5.0 mg/kg/ day, no effects on fertility were observed at any dose level and no neonatal effects were observed at 0.1 or 1.0 mg/kg/day in the F1 or F2 litters. However, the parental toxicity at the high dose was accompanied by neonatal effects at 5.0 mg/kg/ day in the F1 litters. F1 pups from dams given 5.0 mg/kg/day had decreased body weight and slight, but statistically increased mortality.

In conclusion, no effects on fertility were observed at any dose level tested. The parental no-observed-effect level (NOEL) was 0.1 mg/kg/day based upon the absence of serum cholinesterase depression. The neonatal NOEL was 1.0 mg/kg/day. Although neonatal effects were observed in the F1 pups at the high dose of 5.0 mg/kg/day, the significance of the neonatal effects were questionable in view of the presence of significant maternal toxicity at this dose level and the lack of reproducible neonatal effects in the F2 litters. The neonatal NOEL in this study was consistent with the results of previously conducted reproduction and fertility studies with the test substance.