Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 December 1988 to 23 February 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report which meets basic scientific principles

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Administration of test compound only during organogenesis (days 6-15 of pregnancy) and not from pre-implantation as indicated in guideline; administration of test compound in feed rather than by gavage
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): benzyl butyl phthalate
- Substance type: no data
- Physical state: clear, oily liquid
- Analytical purity: 96%
- Impurities (identity and concentrations): 1 major and 10 minor, 3.4% in total by weight, no single impurity present at >1%; not identified
- Purity test date: no data
- Lot/batch No.: no data (RTI chemical ID = D60; RTI Log no. = 4911-40)
- Expiration date of the lot/batch: no data
- Stability under test conditions: stable throughout the period of use
- Storage condition of test material: At or below room temperature in sealed containers

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 250(+/-2) g
- Fasting period before study: none
- Housing: individually in solid-bottom polycarbonate cages with stainless steel wire lids and Ad-Sorb-Dri (TM) cage litter
- Diet (e.g. ad libitum): Ground Purina Certified Rodent Chow #5002 ad libitum
- Water (e.g. ad libitum): deionized/filtered water ad libitum
- Acclimation period: 7 days before cohabitation with males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (average)
- Humidity (%): 48 (average)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSED FEED: Even mixing of test chemical to required concentration

DIET PREPARATION
- Rate of preparation of diet (frequency): Once only for each dietary concentration; storage in refrigerator, with fresh supplied obtained from refrigerated stock on days 6, 9 and 12 of gestation
- Mixing appropriate amounts with (Type of food): rodent chow
- Storage temperature of food: under refrigeration

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis by HPLC (high performance liquid chromatography) prior to and after use of the dosed feed verified the formulations to be within 91-108% of the theoretical concentration (0.5, 1.25 or 2%), indicating accurate preparation and stability throughout the period of use.
Details on mating procedure:
Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: until detection of vaginal sperm
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
Days 6-15 of gestation (the organogenesis period)
Frequency of treatment:
Continuous (in feed)
Duration of test:
From pre-mating until gestational day 20.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 420, 1100 or 1640 mg/kg bw/day
Basis:
other: average ingested dose
Remarks:
Doses / Concentrations:
0, 0.5, 1.25 or 2%
Basis:
nominal in diet
No. of animals per sex per dose:
30 pregnant females/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Preliminary study at dietary concentrations of 0, 0.25, 0.5, 1, 2.5 and 5% (in 5-8 confirmed-pregnant females per dose group) indicated no maternal or embryo/foetal toxicity at less than or equal to 1%, but excessive maternal and embryo/foetal toxicity at 2.5%.
- Rationale for animal assignment (if not random): stratified randomization so that body weights did not differ among groups

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: every 3 days from gestational day 0; final examination after sacrifice

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes - calculated every 3 days from gestational day 0; final calculation before sacrifice
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: liver (weighed and a random selection from control and high-dose groups were examined microscopically), kidneys (weighed), uterus (weighed intact)

OTHER: water intake calculated every 3 days from gestational day 0; final calculation before sacrifice; pregnancy rate recorded for each group
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
- General Linear Models (GLM) were applied for the analyses of variance (ANOVA) of maternal and foetal parameters; prior arcsine-square root transformation on all litter-derived percentage data and Bartlett's test for homogeneity of variance on all data to be analysed by ANOVA
- When ANOVA revealed a significant dose-effect, Williams' and Dunnett's Multiple Comparison tests were used to compare exposed to control groups
- One-tailed tests were used for pairwise comparisons (except maternal body and organ weights and foetal body weights)
- X2 test for independence, "a test" for linear trend on proportions and Fisher's exact probability test were used on some data
Historical control data:
Included in appendices to report. No major differences, where comparable, between these and control data from present study.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
At 1.25% in the diet there was a 37% reduction in weight gain (during treatment), an increase in relative liver weight, and increases in relative food and water intakes. At 2% in the diet, decreased maternal weight gain persisted throughout the remainder of the gestation period (93% decrease) and increased food and water intakes were more pronounced; liver weight changes were also more pronounced (although there were no microscopic changes). Relative kidney weights were increased and there were clinical signs of toxicity including hair loss, piloerection, coat discolouration, lethargy, excessive urination, muscular weakness and abnormal gait.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 420 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
ca. 420 mg/kg bw/day (nominal)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
At 1.25% in the diet there was a slight increase in the percentage of foetuses/litter with external, visceral and skeletal malformations (5.9 +/- 1.6, compared with 2.0 +/- 0.8 in the controls); at 2% in the diet the percentage (52.8 +/- 6.6) was statistically significantly increased. The urogenital system, eye, heart and axial skeleton were the most frequently affected. The high-dose group also showed increased resorptions (with a 33% reduction in liver foetuses per litter) and a 20% reduction in foetal body weights.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Benzyl butyl phthalate given to pregnant rats in the diet at levels of 0.5, 1.25 or 2% (resulting in intakes of 0, 420, 1100 or 1640 mg/kg bw/day respectively), on days 6-15 of pregnancy, was found to cause developmental and maternal toxicity at the two higher dose levels. There were no apparent effects on mothers or their foetuses at the lowest tested dose of 0.5%, which was considered to be the no-observed-adverse-effect level (NOAEL) for both maternal and developmental endpoints.
Executive summary:

A well-conducted study was performed in rats to assess the potential of benzyl butyl phthalate to cause developmental toxicity.

Using a similar protocol to that outlined by OECD Test Guideline 414, groups of 30 pregnant rats were given diets containing 0, 0.5, 1.25 or 2% benzyl butyl phthalate (providing 0, 420, 1100 or 1640 mg/kg bw/day respectively) on days 6 -15 of pregnancy. Maternal weight gain, clinical signs and food and water intakes were monitored, and the dams were killed on day 20 of pregnancy. The uterus, liver and kidney were weighed, and some livers from the high-dose and control groups were examined microscopically. The uterine contents were examined for implantations and resorptions. Viable foetuses were weighed and evaluated for external, soft tissue and skeletal malformations.

At 1.25% in the diet, the mothers showed reduced weight gain during treatment, increased relative liver weight and increased relative food and water intakes. At this dose there was a slight (but not statistically significant) increase in the numbers of litters with external, visceral and skeletal malformations.

At 2% in the diet, the maternal effects described above were more pronounced and accompanied by increased kidney weights; there were also clinical signs of toxicity including hair loss, piloerection, coat discolouration, lethargy, excessive urination, muscular weakness and abnormal gait. Foetal malformations were statistically significantly increased (53% malformed foetuses per litter compared to 2% in controls), with the urogenital system, eye, heart and axial skeleton most frequently affected. Increased resorptions and reduced foetal body weights were also reported.

In conclusion, the NOAEL for both maternal and foetal effects was 0.5% (420 mg/kg bw/day).