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Toxicity to reproduction: other studies

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Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
received: 5. May 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well performed guideline-conform study with restricted reporting.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 440
Deviations:
yes
Remarks:
analysis of phytoestrogens in diet and daily body weight not reported, 5 animals/dose group
Principles of method if other than guideline:
n.a.
GLP compliance:
not specified
Type of method:
in vivo

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl 4-hydroxybenzoate
EC Number:
202-785-7
EC Name:
Methyl 4-hydroxybenzoate
Cas Number:
99-76-3
Molecular formula:
C8H8O3
IUPAC Name:
methyl 4-hydroxybenzoate
Constituent 2
Reference substance name:
Methylparaben
IUPAC Name:
Methylparaben
Details on test material:
Supplier: Aldrich Chemical Company (Gilligham, Dorset, UK)
Puritiy: > 99%

Test animals

Species:
rat
Strain:
other: Alpk:AP
Sex:
female
Details on test animals or test system and environmental conditions:
Immature rats: 21-22 days old, 35-38 g; from Zeneca breeding unit (Alderley Park)
Ovariectomised rats: obtained from Zeneca breeding unit (Alderley Park). The ovariectomy was performed on 6 to 8 week old animals.They were maintained for 2 weeks after the operation, during which time daily vaginal smears were taken, and only noncycling animals were used for the uterotrophic assay.

Husbandry:
All animals were housed in wire mesh cages; solid bottoms were provided for immature animals. Temperature was controlled at 22+/-3°C, humidity was controlled at 30-70%, and a 12 h/12 h light dark cycle was maintained. Animals were weaned on R&M No. 3 (Special Diet Services Ltd., William Essex, UK) at postnatal Days 19-21 and maintained on R&M No. 1 (Special Diet Services Ltd.) from postnatal Day 21 omward. Diet and water were available ad libitum.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Each experiment was accompanied by a vehicle control group (arachis oil) and a positive control group (17ß-Estradiol (0.4 mg/kg bw/d) by oral gavage or subcutaneous injection. Animals were dosed on 3 successive days with Methylparaben/17ß-Estradiol dissolved/suspended in Arachis oil. The maximum doses used were determined by turbidity of the dosing solutions and restriction of dosing volume.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
3 days
Frequency of treatment:
Daily
Duration of test:
Ovariectomised rats:
- Ovariectomy on 6-8 week old rats
- 2 weeks maintainance after operation (only noncycling females were used for assay)
- 3 days treatment, necropsy 24 h after last treatment

Immature rats:
- 24 h acclimatisation
- 3 days treatment, necropsy 24 h after last treatment
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
40 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
400 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
800 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
40 mg/kg bw/d
Basis:
other: subcutaneous
Remarks:
Doses / Concentrations:
80 mg/kg bw/d
Basis:
other: subcutaneous
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
Negative control: Arachis oil
Positive control: 17ß-Estradiol
Animals were killed by an overdose of halothane 24 h after the final dose. Vaginal opening was recorded for immature animals at the time of death. Vaginal smears were also taken from ovariectomised animals at the time of death. Uteri were excised, trimmed free of fat, pierced, and blotted to remove excess fluid. The body of the uterus was cut just above its junction with the cervix and at the junction of the uterine horns with the ovaries. The uterus was then weighed (wet weight). Uterine dry weight was also determined by drying the uteri at 70°C for 24 h before reweighing. A quantitative estimation of vaginal cytology was performed by counting the number of fully cornified cells in at least 2 fields containing a total of at least 100 cells (x200 magnification).
Statistics:
Variation within experiments for uterus wet and dry weights was determined by analysis of variance using the GLM procedure. Percentages of cornified cells were also considered by analysis of variance, following a double arcsine transformation (Freeman and Turkey, 1950). Differences from control values were assessd statistically using a two-sided Student´s t test based on the error mean square from the analysis of variance.

Results and discussion

Effect levels

Dose descriptor:
NOEL
Effect level:
800 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: no increase in uterus weights or premature vaginal opening

Observed effects

No clinical signs
No increases in uterus weight
No premature vaginal opening
No increase in vaginal cornification

Any other information on results incl. tables

Table 1: The Activity of Methylparaben in the Immature Rat Uterotrophic Assay

 

Compound

Dose/

Route

Uterus weight/[mg]

 

[mg/kg bw/d]

 

Wet

Dry

 

 

 

 

 

Estradiol

0.4

oral

117.5±8.9*

22.5±1.8*

Arachis oil

10 mL/kg bw/d

oral

33.8±5.9

6.4±0.9

Methylparaben

40

oral

34.5±3.2

6.8±0.7

Methylparaben

400

oral

33.3±5.3

6.3±0.9

Methylparaben

800

oral

31.0±4.3

6.2±0.7

Methylparaben

40

subcutaneous

33.5±6.6

6.8±1.1

Methylparaben

80

subcutaneous

32.8±1.5

6.6±0.3

 

 

 

 

 

*    p<0.01

Applicant's summary and conclusion

Conclusions:
Methylparaben (orally as well as subcutaneously applied to immature/ovariectomised rats) was inactive in this uterotrophic assay at concentration up to 800 mg/kg bw/d (NOEL).
Executive summary:

Methylparaben was administered to immature and ovariectomised rats assigned to 5 groups of 5 animals each at doses of 40, 400, 800 mg/kg bw/d (orally) and 40, 80 mg/kg bw/d (subcutaneously). 24 h after the last (third) administration, the rats were sacrificed, the weight of the uteri (wet and dry) was recorded and a quantitative estimation of the vaginal cytology was performed. There were no effects in any group. Based on the results of this assay the respective NOEL is > 800 mg/kg bw/d.