2-(2-butoxyethoxy)ethyl 6-propylpiperonyl ether

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

Corresponding to OECD guideline 416

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-butoxyethoxy)ethyl 6-propylpiperonyl ether

EC Number:

200-076-7

EC Name:

2-(2-butoxyethoxy)ethyl 6-propylpiperonyl ether

Cas Number:

51-03-6

Molecular formula:

C19H30O5

IUPAC Name:

2-(2-butoxyethoxy)ethyl 6-propylpiperonyl ether

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sprague Dawley CD-Crl COBS CD BR
- Age at study initiation: Six weeks
- Weight at study initiation: 124 to 181 g (F0 males) 115 to 151 g (F0 females).
- Number of animals per group: 26 males and 26 females for each dose group both in F0 and F1 generation.
- Duration of mating: 3 weeks
- Fasting period before study:
- Housing: The rats were housed individually in wire mesh-bottomed stainless steel cages except during the mating period when 1 female was placed with each male.
The females in the littering phase were placed in clear plastic shoebox-type cages, with Beta Chip bedding from day 15 of gestation to the end of the lactation period. The unmated females were housed the same way at the end of the mating period.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 3/4 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 55 ± 15
- Photoperiod: 12 hrs dark / 12 hrs light.

IN-LIFE DATES: From July 17 1984 (arrive in the laboratory): To October 13, 1985 (the last day of necroscopy):

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Diets containing PBO were mixed weekly by preparing a premix of basal diet (Purina Rodent Chow - No. 5002) and PBO in 10 kg capacity Hobart blender. The final concentrations were achieved by the addition of basal diet to aliquots of the premix and mixing in 60 kg capacity Hobart blender.

DIET PREPARATION
- Rate of preparation of diet (frequency): all rats were fed with certified Purina Rodent Chow (No. 5002) ad libitum with PBO incorporated into the diet where applicable.
- Mixing appropriate amounts with: Purina Rodent Chow - No. 5002

Details on mating procedure:

The estrous cycle of the female was assessed by examination of the vaginal lavage for 10 days prior to placement for the first mating period. Following 85 day of treatment 1 female was placed with a single male with the same dosage group, for a maximum of 7 days. After 7 day the females of those unmated pairs were placed with male of an other unmated pairs within the group. This was repeated after a further 7 days, allowing a total of 21 days for mating to take place. If only 1 pair of rats in any given group had failed to mate after 7 or 14 day no exchange was made.
the female was examined by examination of the vaginal lavage of spermatozoa. The day of positive identification of spermatozoa was termed day 0 of gestation. Female mated were separated from the males and were returned to their stainless-steel mesh bottom cages.
Subsequently mated females were placed in clear plastic shoebox-type cages. The procedure was repeated for the second mating period with the initial pairings being those which mated successfully during the first mating period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Route of administration: Oral via food.
- Concentration: 0, 300, 1000, or 5000 ppm. Achieved intakes were 30, 100 and 500 mg/kg bw/d. For males: 41 – 14 mg /kg bw/d, 135 – 47 mg/kg bw/d and 706 – 251 mg/kg bw/d. For females: 41 – 17 mg/kg bw/d, 133-56 mg/kg bw/d and 738-316 mg/kg bw/d
- Food consumption per day ad libitum. See Table 2 in "Any other information on materials and methods incl. tables"
- Analysis of Test Article Diets: Prior to initiation of the study, stability and homogeneity of mixing were assessed. During the study, sample were taken from the test diets prepared throughout the study and analysis was performed, prior to feeding of the animals where possible, on weeks 1 to 4 and at least every 4 weeks thereafter to assess the concentration of the test article in the diet.

Duration of treatment / exposure:

The F0 generation (26 male and 26 female CD - Sprague-Dawley - rats), were treated with PBO incorporated into the diet at dosages of 0, 300, 1000, 5000 ppm. The animals were treated for 85 days prior to placement for mating, throughout the two mating periods until necroscopy approximately 3 weeks after the end of the second mating period. The females were also treated throughout the mating, gestation and lactation periods. The F1b generation litters were weaned on day 21 post-partum, and groups of 26 males and 26 females were selected to form the F1b adult generation. These animals were treated at the same dosages as their parents, for at least 83 days prior to placement for mating to provide the F2 generation. The treatment of the Fb1 generation adult females was continued throughout the mating, gestation and lactation periods. The F1b generation adult males were treated until necroscopy approximately 3 weeks after the end of the second mating period. The F2 generation pups were killed following weaning.

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated until 12 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: 15 weeks

Number of generation studies: 2 (F1 n F2)

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (control), 300, 1000, 5000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

26/male/0; 26/male/300; 26/male/1000; 26/male/5000;
26/female/0; 26/female/300; 26/female/1000; 26/female/5000;

Control animals:

yes

Details on study design:

The purpose of this study was to assess the reproductive performance and development of rats fed PBO. The male and female rats of the F0 and F1 adult generations were given either a control rodent diet or a diet containing PBO at concentration of either 300, 1000 or5000 ppm of the diet. The reproductive performance of these animals as well as the development of the F1a, F1b, F2a, F2b generation offspring w2as assessed.

Examinations

Parental animals: Observations and examinations:

SERIAL OBSERVATION (for the parental animals F0 and F1b generations)

- CLINICAL EXAMINATIONS: All parental animals (alias F0 and F1b generations) were examined at least twice each day for mortality and once each day for signs of overt toxicity. In addition, a detailed physical examination was performed once each week, and those animals designated as abnormal were examined by a veterinary aide.
- BODY WEIGHT: All parental animals were weighed 10 and 3 days before the initiation of treatment and weekly during the treatment period except for mated females which were weighed on days 0,7,15 and 21 of gestation and on days 0,4,7,14 and 21 postpartum.
- FOOD CONSUMPTION: Individual food consumption was determined once during the acclimation period (up to the start of the mating period) and fir mated females during days 0 to7, 7 to 15 and 15 to 21 of gestation and days 0 to7, 7 to 14 and 14 to 21 postpartum.
The group achieved intake was calculated weekly as follows:
group achieved intake (mg/kg/day)=( [PBO] X food/intake/rat/day(g))/(mid-week body weight (g)) X10

Oestrous cyclicity (parental animals):

The oestrus cycle was assessed by examination of the vaginal lavage for 10 days prior to the first mating period.

Sperm parameters (parental animals):

Testis with epididymis weight were recorded.

Litter observations:

- GENERAL CONDITION
The general condition of the pups (F1a, F2b and F2 generations pups) was evaluated each day during the lactation period. Any pup found dead on or before day 4 postpartum was placed in Boin’s fluid and subsequently was examined internally using a modified Barrow and Taylor Technique. Pups dying after day 4 postpartum were given a full macroscopic examination.

- BODY WEIGHT
In addition to assessment of body weight at birth (day 0 postpartum), the pups were weighed by sex in each litter on days 4, 7, and 14 and individually on day 21 postpartum.

- CULLING
On day 4 postpartum the litters were culled using random procedures to 8 pups, where necessary, to give litters of 4 males and 4 females, where possible. Culled pups were preserved in Boin’s fluid for possible subsequent internal examination.

Postmortem examinations (parental animals):

SACRIFICE & GROSS PATHOLOGY
- All animals (F0 and F1 Adult Generations) dying or sacrificed in poor condition during the study were subjected to detailed external and internal gross examination.
Approximately 3 weeks after the end of the second mating period, males were killed and were given a gross necroscopy.
Females which failed to mate were killed approximately 26 days after the end of the second mating period and were given a detailed macroscopic examination. Females which mated but did not litter by day 26 post coitum were sacrificed and were given a macroscopic examination, including an examination of the reproductive tract for any abnormalities which may have prevented pregnancy. The dams were killed on day 22, 23, 24 postpartum and were given a macroscopic examination.

TISSUE PRESERVATION
- On completion of the gross pathology examination, the following tissues and organs from each rat were retained in 10% neutral buffered formalin for fixation and preservation:
Epididymides (+*),
Liver,
Mammary glands (thoracic and inguinal),
Ovaries(+),
Prostate(*),
Seminal vesicles(+),
Testes(+*),
Uterus(+),
Vagina,
Any abnormalities(++).

*Fixed with Zenker’s fluid for sacrificed rats only.
+Histopathological examination for control and high-dose rats.
++Histopathological examination for all groups

HISTOPATHOLOGY
- Histopathological examination was performed upon the tissues of the control and high-dose groups initially. The reproductive tracts as specified above (see TISSUE PRESERVATION) of those rats in the low- and mid-dose groups failing to made successfully in both mating periods were also evaluated Histopathologically.

Postmortem examinations (offspring):

SACRIFICE & GROSS PATHOLOGY (F1a & F1b, and F2a & F2b Generations pups)
- Pups born dead or malformed and pups dying on before day 4 postpartum were fixed in Bouin’s fluid for subsequent internal examinations. Pups found dead between days 5 and 21 postpartum were given a detailed gross necroscopy, and all tissues were preserved in 10% neutral buffered formalin.

TISSUE PRESERVATION (for Pups and Weanlings)
Adrenals,
Aorta (thoracic aorta),
Brain,
Cecum,
Colon,
Duodenum,
Epididymides(*)
Esophagus,
Eyes(*),
Heart,
Ileum,
Kidneys,
Jejunum,
Liver (sample of 2 lobes),
Lungs (sample of 2 lobes),
Lymph nodes (mandibular and mesenteric),
Mammary gland,
Optic nerve(*)
Ovaries,
Pancreas,
Pituitary,
Prostate,
Rectum,
Salivary gland,
Sciatic nerve,
Seminal vesicles,
Skeletal muscle,
skin (inguinal)
spinal cord (cervical),
spleen,
sternum,
stomach,
testes,
thymus,
thyroid (and parathyroids),,
tongue,
trachea,
urinary bladder,
uterus,
vagina,
any abnormalities.

*Fixed with Zenker’s fluid for sacrificed rats only.

WEANLINGS
- 10 males and 10 females rats (between 21 and 28 days postpartum) for each group of the F1b and F2b generations were randomly selected from 10 separate litters where possible, were killed and were given a gross necroscopy examination. All gross lesions and those tissues listed above in section “TISSUE PRESERVATION (or Pups and Weanlings)” were retained.
Those weanling rats not selected for either breeding or pathological examination were given an external examination, and those which had external abnormalities were given a complete gross pathological examination.

Reproductive indices:

The following indices were calculated for each group:

- Mating Index = (No. of Females mated/No. of females placed for mating) X 100
- Fertility Index = (No. of Pregnant Females/No. of Females placed for mating) X 100
- Conception Rate = (No. of Pregnant Females/No. of Females Mated) X 100
- Gestation Index = (No. of rats with live pups at birth/No. of Pregnant rats) X 100

Offspring viability indices:

The viability, survival and lactation indices were calculated as follows.

- Viability Index = (No. of live pups on day 4 postpartum/ No. of live pups on day 0 postpartum) X 100
- Survival Index = (No. of live pups on day 7 or 14 postpartum/ No. of live pups on day 4 postpartum) X 100
- Lactation Index = (No. of live pups on day 21 postpartum/ No. of live pups on day 4 postpartum) X 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

See "Details on Results"

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See "Details on Results"

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See "Details on Results"

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

See "Details on Results"

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: See "Details on Results"

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

See "Details on Results"

Reproductive performance:

no effects observed

Description (incidence and severity):

See "Details on Results"

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment-related effects were noted with respect to clinical signs, mortality or gross pathological findings in the parental rats of the F0 generation

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- F0 Parental: Body weights and food consumption for the parental rats in the F0 generation were significantly reduced at the 5000 ppm dosage level (males and females). See Table 1 reported in "Any other information on materials and methods incl. tables"
- F1 Parental: Body weights and food consumption for the parental rats in the F1 generation were significantly reduced at the 5000 ppm dosage level. These reductions were considered related to ingestion of the test diets and therefore treatment related

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
- F0 generation (males): The achieved intake between week 1 and week 22 of the treatment period ranged from 41 to 14, 135 to 47 and 706 to 251 mg/kg/day in the 300, 1000 and 5000 ppm treated groups, rispectively.
- F0 generation (females): PBO dosage in the premating and gestation periods ranged from 41 to17, 133 to 56 and 730 to 316 mg/kg/day in the 300, 1000 and 5000 ppm treated groups, rispectively.
See the table "F0 - ACHIEVED INTAKE" attached in backgorund material.
- F1b generation (males): between week 0 and week 22 the achieved intake of males trated with PBO ranged from 43 to 15, 151 to 47, and 856 to 253 mg/kg/day in the 300, 1000 and 5000 ppm treated groups, rispectively.
- F1b generation (females): the achieved intake of PBO varied from 47 to 19, 158 to 66, and 878 to 353 in the 300, 1000 and 5000 ppm treated groups, rispectively.
See the table "F1b - ACHIEVED INTAKE" attached in backgorund material.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- F0 and F1 generations (females): The estrous cycles of the females in the PBO trated groups were unaffected

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- F0 and F1 generations: The parental performance for the first and second reproductivity phase, in terms of the mean day of mating, mating and fertility indices, and conception rates wa unaffected.

GROSS PATHOLOGY & HISTOPATHOLOGY (PARENTAL ANIMALS)
- F0 and F1 generations: there were no gross or histopathogical findings among males and females in the PBO dosage groups which were attributed to the treatment. See the "ADULTS PATHOLOGIST’S REPORT" attached in backgorund material.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

1 000 ppm

Based on:

test mat.

Remarks:

Administered orally with food

Sex:

male/female

Basis for effect level:

other: With regard to parental and neonatal decrease in body weight gain, equivalent to 100 mg/kg bw/day With regard to reproductive parameters, equivalent to 100 mg actual Piperonyl Butoxide /kg bw/day

2

Dose descriptor:

LOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Remarks:

Administered orally with food

Sex:

male/female

Basis for effect level:

other: Body weights and food consumption were significantly reduced at 5000 ppm

Results: F1 generation

General toxicity (F1)

Mortality / viability:

no mortality observed

Description (incidence and severity):

See "Details on Results"

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See "Details on Results"

Gross pathological findings:

no effects observed

Description (incidence and severity):

See "Details on Results"

Histopathological findings:

no effects observed

Description (incidence and severity):

See "Details on Results"

Details on results (F1)

VIABILITY (OFFSPRING)
- F1a, F1b, F2a and F2b generations: The viability, survival and lactation indices in the PBO treated groups were not significantly different form control values.

CLINICAL SIGNS (OFFSPRING)
- F1a, F1b, F2a and F2b generations: There were no findings considered to be related to treatment.

BODY WEIGHT (OFFSPRING)
- F1 pups: Body weights for the F1a and F1b pups were significantly reduced at the 5000 ppm dosage level. See Table 3 reported in "Any other information on materials and methods incl. tables"
- F2 pups: Body weights for the F2a and F2b pups were significantly reduced at the 5000 ppm dosage level. See Table 3 reported in "Any other information on materials and methods incl. tables"

GROSS PATHOLOGY & HISTOPATHOLOGY (OFFSPRING)
- F1a generation: 1, 4, 0 and 1 pups were born with external malformations in the control, 300, 1000 and 5000, respectively.
Among pups born dead or dying, absence/reduction of the renal papilla(e) was the most common finding.
- F1b generation: 2 pups in the 100o ppm groups from separate litters which died had ectopic/pelvis kidney(s). One control group weanling had unilateral anophthalmia, and one weanling in the 5000 ppm treated group and an inguinal hernia and hydrocephalus.
Reduction of the renal papilla(e) /dilatation of the renal papilla(e) was common finding among males and females found death at birth, dying during the lactation period or sacrificed following weaning.
- F2a generation: 0, 1, 2 and 0 pups were born with malformations in the control, 300, 1000 and 5000, respectively.
- F2b generation: one pup in the control group was found with a major malformation (anophthalmia). A second control pup had a pelvic kidney.
See the "PUPS PATHOLOGIST’S REPORT" attached in backgorund material.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 ppm

Based on:

test mat.

Remarks:

Administered orally with food

Sex:

male/female

Basis for effect level:

other: With regard to parental and neonatal decrease in body weight gain, equivalent to 100 mg/kg bw/day With regard to reproductive parameters, equivalent to 100 mg actual Piperonyl Butoxide /kg bw/day

2

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

5 000 ppm

Based on:

test mat.

Remarks:

Administered orally with food

Sex:

male/female

Basis for effect level:

other: Body weights and food consumption were significantly reduced at 5000 ppm

Results: F2 generation

Effect levels (F2)

1

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

1 000 ppm

Based on:

test mat.

Remarks:

Administered orally with food

Sex:

male/female

Basis for effect level:

other: With regard to parental and neonatal decrease in body weight gain, equivalent to 100 mg/kg bw/day With regard to reproductive parameters, equivalent to 100 mg actual Piperonyl Butoxide /kg bw/day

2

Dose descriptor:

LOAEL

Generation:

F2

Effect level:

5 000 ppm

Based on:

test mat.

Remarks:

Administered orally with food

Sex:

male/female

Basis for effect level:

other: Body weights and food consumption were significantly reduced at 5000 ppm

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

RS-Freetext:
F0 and F1b parental generationNo treatment-related effects were noted with 

respect to clinical signs, mortality or gross pathological findings in the 

parental rats of the F0 generation.Body weight gains were  significantly reduced 

for the 5000 ppm group and also food intake was  reduced.Body weights for the 

1000 and 300 ppm groups were  unaffected.Parental performance in terms of the 

mean day of mating,  mating and fertility indices, and conception rates were 

unaffected.  Estrous cycles, gestation index, length of gestation, duration of  

parturition, number of live and dead pups at birth and sex ratio of pups,  all 

parameters of maternal performances were not significantly different  from 

controls.F1 generation pupsViability, survival and lactation indices  were 

unaffected, there were no treatment-related clinical signs and no  gross 

pathological findings. The F1a and the F1b generation pup weights  were 

significantly lower in the 5000 ppm group.Pup weights for the 1000  and 300 ppm 

groups were unaffected.F2 generation pupsViability, survival  and lactation 

indices were unaffected, there were no treatment-related  clinical signs and no 

gross pathological findings. The F2a and the F2b  generation pup weights were 

significantly lower in the 5000 ppm group.Pup  weights for the 1000 and 300 ppm 

groups were unaffected.

Applicant's summary and conclusion

Conclusions:

MATERIALS AND METHODS
In a two-generation study, groups of 26 male and 26 female Charles River rats received diets containing Piperonyl Butoxide at concentrations of 0, 300, 1000, or 5000 ppm equivalent to 0, 30, 100, and 500 mg/kg bw per day. The F0 parental generation were treated for a minimum of 85 days before the first of two matings. The same numbers of weanlings from the F1b litters were selected randomly to become F1-parents and were treated for a minimum of 83 days before being mated twice to produce the F2a and F2b litters.

RESULTS AND DISCUSSION
- F0 and F1b parental generation: No treatment-related effects were noted with respect to clinical signs, mortality or gross pathological findings in the parental rats of the F0 generation.
Body weight gains were significantly reduced for the 5000 ppm group and also food intake was reduced.
Body weights for the 1000 and 300 ppm groups were unaffected.
Parental performance in terms of the mean day of mating, mating and fertility indices, and conception rates were unaffected. Estrous cycles, gestation index, length of gestation, duration of parturition, number of live and dead pups at birth and sex ratio of pups, all parameters of maternal performances were not significantly different from controls.
- F1 generation pups
Viability, survival and lactation indices were unaffected, there were no treatment-related clinical signs and no gross pathological findings. The F1a and the F1b generation pup weights were significantly lower in the 5000 ppm group.
Pup weights for the 1000 and 300 ppm groups were unaffected.
- F2 generation pups
Viability, survival and lactation indices were unaffected, there were no treatment-related clinical signs and no gross pathological findings. The F2a and the F2b generation pup weights were significantly lower in the 5000 ppm group.
Pup weights for the 1000 and 300 ppm groups were unaffected

CONCLUSION
The NOEL/NOAEL was 1000 ppm with regard to reproductive parameters, equivalent to 100 mg actual Piperonyl Butoxide /kg bw/day.
The NOEL/NOAEL was 1000 ppm with regard to parental and neonatal decrease in body weight gain, equivalent to 100 mg/kg bw/day

LO(A)EL = 5000 ppm
Parent males: body weights and food consumption were significantly reduced at 5000 ppm
Parent females: body weights and food consumption were significantly reduced at 5000 ppm
F1 males: body weights and food consumption were significantly reduced at 5000 ppm
F1 females: body weights and food consumption were significantly reduced at 5000 ppm
F2 males: body weights were significantly reduced at 5000 ppm
F2 females: body weights were significantly reduced at 5000 ppm

NO(A)EL = 1000 pp
Parent males: 1000 ppm, equivalent to 100 mg actual Piperonyl Butoxide /kg bw/day.
Parent females: 1000 ppm, equivalent to 100 mg actual Piperonyl Butoxide /kg bw/day
F1 males: 1000 ppm equivalent to 100 mg actual Piperonyl Butoxide /kg bw/day
F1 females: 1000 ppm, equivalent to 100 mg actual Piperonyl Butoxide /kg bw/day
F2 males: 1000 ppm equivalent to 100 mg actual Piperonyl Butoxide /kg bw/day
F2 females: 1000 ppm equivalent to 100 mg actual Piperonyl Butoxide /kg bw/day

Reliability: 1
Deficiencies: No

The no significant adverse effect level for parental toxicity and pup development was 1000ppm of PBO in the diet and for reproductive toxicity 5000ppm of PBO in the diet.