Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication which meets basic scientific principles

Data source

Reference
Reference Type:
publication
Title:
Examination of the in vitro and in vivo estrogenic activities of eight commercial phthalate esters
Author:
Zacharewski TR, Meek MD, Clemons JH, Wu ZF, Fielden MR and Matthews JB
Year:
1998
Bibliographic source:
Toxicological Sciences, 46, 282-293

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Oestrogenic activity was measured in ovariectomised rats, orally dosed with benzylbutylphthalate and evaluated for uterine weight (uterotrophic assay) and vaginal cell cornification. In vitro evaluation included assays for oestrogen receptor competitive ligand-binding and for gene expression in mammalian cells and yeast.
GLP compliance:
not specified
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): butylbenzylphthalate
- Substance type: no data
- Physical state: no data
- Analytical purity: 98.5%
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: no data; supplied by Monsanto
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: no data

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS - uterotrophic (uterine weight) assay
- Source: Experiment 1: Charles River, Montreal, Quebec, Canada. Experiment 2: Charles River, Portage, Michigan, USA
- Age at study initiation: 24-25 days old on receipt (ovariectomised at 19 days of age)
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: 5/cage
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): Purina Prolab RMH 3000, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: dosing began at day 31

TEST ANIMALS - vaginal cell cornification assay
- Source: Charles River
- Age at study initiation: no data (ovariectomised at 51-56 days of age)
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: no data, probably 5/cage
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): no data, probably Purina Prolab RMH 3000, ad libitum
- Water (e.g. ad libitum): no data, probably tap water, ad libitum
- Acclimation period: not specified
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 40-60
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
no data

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): no data
- Lot/batch no. (if required): no data
- Purity: no data
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
not done
Duration of treatment / exposure:
4 days
Frequency of treatment:
once daily
Duration of test:
4 days of treatment; killed on the day after the final dose
Doses / concentrations
Remarks:
Doses / Concentrations:
20, 200 and 2000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
10 females/dose for each endpoint
Control animals:
yes, concurrent vehicle
other: positive control: ethynyl oestradiol, 1 mg/kg bw/day
Details on study design:
Uterotrophic assay: uterine weights were recorded at necropsy on the day after final dosing (expressed as absolute wet weight and wet weight relative to body weight); assay performed twice (experiments 1 and 2).
Vaginal cell cornification assay: vaginal lavages were performed on the day prior to dosing, pre-dosing each day (4 days) and on the day after final dosing; the number of positive smears and the proportion of smears with vaginal cell cornification were recorded.
Statistics:
One-way analysis of variance; if different, Dunnett's test; residuals tested for normality using Shapiro-Wilk test; if not normally distributed, Blom's transformation

Results and discussion

Effect levels

Dose descriptor:
NOAEL
Remarks:
oestrogenic activity
Effect level:
>= 2 000 mg/kg bw/day
Sex:
female
Basis for effect level:
other: No reproducible effect on uterine wet weight or vaginal cell cornification at any dose; 2000 mg/kg bw/day was the highest dose tested.

Observed effects

In vivo uterotrophic assay - see Table 1. In expt 1, all high-dose animals were lethargic after dosing and 2 died; 1 animal in the mid-dose group also died. In expt 2, no lethargy was seen, but 2 animals at the top dose died. [Note: The investigators pointed out that the animals in the two experiments came from different suppliers.]
In vivo vaginal cell cornification assay - see Table 1.
In vitro assays - weak effects reported in ligand binding, gene expression, and yeast growth assays

Any other information on results incl. tables

Table 1: Effect of phthalate esters on uterine wet weight and vaginal cell cornification in ovariectomized Sprague-Dawley rats

 

Treatmenta

Doseb

(mg/kg bw)

Body weight (g)

mean±SD

 

Uterine wet wt (mg)

mean±SD

 

Uterine wet wt

(mg/100 g body wt)

mean±SD

Number of positive smearsc

% Cornification of smearsd

Expt 1

Expt 2

Expt 1

Expt 2

Expt 1

Expt 2

 

 

 

 

 

 

 

 

 

 

 

 

Sesame oil

0

172±5

129±6

 

30±6

24±6

 

17±4

19±5

0/0

0±0

EE

1

162±6**

114±4**

 

153±32**

105±17**

 

94±18**

92±14**

10/10

100±0

BBP

20

169±7

125±5

 

29±7

17±5*

 

17±4

14±4*

2/10

3±6

 

200

171±8

123±5*

 

30±7

19±4

 

18±4

15±4

0/10

0±0

 

2000

167±10

124±2 (9)e

 

36±14

19±4

 

22±9

16±3

0/8

0±0

 

*Statistically significant difference from control atp< 0.05.

**Statistically significant difference from control atp< 0.01.

aTen animals were used per treatment group.

bAnimals were ovariectomized on day 19. Dosing via oral gavage on days 31-34, and the animals were euthanized on day 35.

cMature ovariectomized (OVX) rats were dosed via oral gavage for four consecutive days. Vaginal lavages were performed prior to dosing to ensure that the animals were not cycling, on each day of dosing (days 1 to 4, inclusive) and 24 h after the last treatment (day 5) prior to asphyxiation. The results in the table are from lavages collected on day 4. Results collected on days 1 to 3 and 5 were comparable. Note that some groups had less then 10 animals due to premature death during the experiment.

dPercentage of cornification of smears was calculated as described by Terenius (1971). The results are a semiquantitative estimate of the percentage of cornified cells relative to leucocytes. Two independent evaluators, both blind to the treatment protocols, were used to score the degree of cornification.

eData from animals used in experiment 2 found to possess ovarian stubs were not included in the data set. The number in the parenthesis indicates the number of animals used to determine the mean±standard deviation.

 

Applicant's summary and conclusion

Conclusions:
In two reliable in vivo assays in ovariectomised rats (uterotrophic and vaginal cell cornification assays), no oestrogenic activity was detected after oral dosing with benzylbutylphthalate at up to 2000 mg/kg bw/day. Weak oestrogenic activity was detected in vitro.
Executive summary:

In a uterotrophic assay, groups of 10 ovariectomised immature rats were dosed orally with benzylbutylphthalate at 20, 200 or 2000 mg/kg bw/day for 4 days. Two further groups of 10 animals received sesame oil only (vehicle control) and ethynyl oestradiol at 1 mg/kg bw/day (positive control). Uterine weights were recorded at necropsy on the day after the final dose, and mean absolute wet weight and wet weight relative to body weight were calculated. The assay was performed twice (experiments 1 and 2). Groups of 10 ovariectomised mature rats were dosed similarly, and vaginal lavages performed prior to dosing, on each day of dosing and on the day after dosing. The number of positive smears and the proportion of smears with vaginal cell cornification were recorded.

No reproducible effect on uterine weight or vaginal cell cornification was detected in benzylbutylphthalate-treated rats, whereas ethynyl oestradiol produced a strong positive response in both assays.

Three in vitro assays for oestrogenic activity were also performed. These included an assay for competitive ligand-binding to rat uterine oestrogen receptors, assessment of gene expression in recombinant receptor/reporter gene assays in mammalian cells, and evaluation of viability in an oestrogen-dependent strain of yeast. Weak oestrogenic activity was detected in all three in vitro assays.

In two reliable in vivo assays in ovariectomised rats (uterotrophic and vaginal cell cornification assays), no oestrogenic activity was detected after oral dosing with benzylbutylphthalate at up to 2000 mg/kg bw/day. Weak oestrogenic activity was detected in vitro.