Registration Dossier

Toxicological information

Toxicity to reproduction: other studies

Currently viewing:

Administrative data

toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
27. July to 1. October 2004
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed and documented GLP study.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
no guideline available
other: n.a.
not applicable
Principles of method if other than guideline:
Male rats were fed with a diet containing different dosages of Methylparaben for 56 days. Viabilities, clinical observations, body weights and food consumptions as well as all relevant reproductive parameters were determined.
GLP compliance:
Type of method:
in vivo

Test material

Details on test material:
White chrystalline powder
purity: 99.9 %
Melting point: 127°C
Lot No. M3160

Test animals

other: Crl.(WI)BR
Details on test animals or test system and environmental conditions:
Male rats were exposed to Methylparaben. F0 generation female rats were only used as breeders to produce the F1 generation and were not considered part of the study.

F0 Generation Female Rats/F1 Generation Litters:
Female rats were naturally bred at the Suppier´s facility (Charles River Laboratories, North Carolina, USA) by breeder male rats of the same source and strain. The day of delivery was designated day 1 of lactation (postpartum). The female rats were allowed to deliver their litters at the Supplier and shipped to arrive at the Testing Facility on days 12, 13 or 14 postpartum. Upon arrival, dams were placed into nesting boxes in consecutive order, by day postpartum. Day 1 was the day of birth.

Test System Data:
No. of Male Rats Assigned: 64
Date of Birth: 14. to 16. July 2004
Weight on First Day of Exposure: 27.1-40.4 g

F1 Generation Male Rats:
8 litters were selected out of 10 possible litters. From these litters all male rats were assigned to dosage groups as follows: the first and fifth male rats were assigned to Group I, the second abd sixth male rats were assigned to Group II, the third and seventh male rats were assigned to Group III and the fourth and eighth male rats were assigned to Group IV. Six male rats from each dosage group were selected for possible histopathological evaluation of the liver, and adrenal, thyriod and pituitatry glands.

- min. 10 airchanges/hour
- room temperature: 18 - 26°C
- humidity: 30 - 70%
- light: 12 hours light, 12 hours dark
- diet: either CE-2 diet (CLEA Japan Inc., Tokyo, Japan) only (carrier control group) or CE-2 + Methylparaben diet ad libitum
- water: processed by passage through a revers osmosis membrane, ad libitum
- bedding material: Bed-o´cobs (The Andersons Industrial Products Group, Ohio, USA)
After weaninig , F1 generation male rats were individually housed in stainless wire-bottomed cages.

Administration / exposure

Route of administration:
oral: feed
other: CE-2 diet (CLEA Japan, Inc.)
Details on exposure:
A constant concentration of the test article in the diet was offered to the male in each group, and the mg/kg bw/d dosage consumed were calculated and presented for periods corresponding to body weight and feed consumption observations.

A carrier and 3 test diet concentrations were given to the rats. Rats were given continual access to either the carrier control diet (Group I) or Methylparaben in the carrier control diet (Groups II to IV) for at least 56 days beginning on day 21 postpartum.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Analytical procedures were developed for the analysis of Methylparaben in CE-2 Rodent Diet formulations. They involved analysis of the compound by Gas Chromatography with Mass Spectrometry detection. The procedures were applicable for the analysis of dose formulations at the following concentrations: 100, 1000 and 10,000 µg/g of Methylparaben in CE-2 diet. The assay pocedures were sufficienttly linear, reproducible and accurate to support dose formulation analyses.
Samples for this analysis were analysed for concentration, homogeneity and stability verification.
Duration of treatment / exposure:
56 days
Frequency of treatment:
Daily via diet
Duration of test:
56 days
Doses / concentrationsopen allclose all
Doses / Concentrations:
100 ppm
nominal in diet
(equivalent to 11.2 mg/kg bw/d consumed)
Doses / Concentrations:
1000 ppm
nominal in diet
(equivalent to 110.0 mg/kg bw/d consumed)
Doses / Concentrations:
10000 ppm
nominal conc.
(equivalent to 1141.1 mg/kg bw/d consumed)
No. of animals per sex per dose:
16 male rats per dose
Control animals:
yes, plain diet
Details on study design:
Prepared diets containing Methylparaben at constant concentrations of 0, 100, 1000 and 10,000 ppm were available ad libitum to groups of 16 male rats each for a minimun of 56 days. Viabilities, clinical observations, body weights and feed consumption values were recorded. Beginning at the start of week 3 of the exposure period, blood samples were collected every other week from each male and retained frozen for possible analysis of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone.
On the day of sacrifice, all surviving animals were killed and blood samples were collected for possible future analysis for Methylparaben and metabolite levels. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Reproductive organs from all rats, as well as the liver, glands, thyroid and pituitary gland were weighed. Sperm evaluations were conducted to determine sperm concentration, motility and morphology. The left testis from each rat was collected for evaluation of Daily Sperm Production (DSP) determinations. The liver, adrenal, thyroid and pituitary gland from 10 male rats/group were quick-frozen and retained for possible hormone measurements. Histological examination was performed on the reproductive organs from all rats assigned to the control and high concentration group. Additionally, the liver, adrenals, thyroid and pituitary glands from 6 rats/group were retained for possible histopathology. A detailed quantitative examination of the testes was conducted, taking into account the tubular stages of the spermatogenic cycle. A gross necropsy was conducted on the rat found dead.
- Bartlett´s Test (parametric)
- Fisher´s Exact Test
- Dunnett´s Test
- Kruskal-Wallis Test (non-parametric)
- Dunn´s Test

Clinical observations and other proportional data were analysed using the Variance Test for Homogeneity of the Binomial Distribution.
Continuous data (e.g. body weights, organs weights etc.) were analysed using Bartlett´s Test of Homogeneity of Variances and the Analysis of Variance, when appropriate. If the Analysis of Variance was significant, Dunnett´s Test was used to identify the statistical significance of the indivitual groups. If the Analysis of Variance was not appropriate, the Kruskal-Wallis Test was used. In cases where the Kruskal-Wallis Test was statistically significant, Dunn´s Methods of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75%ies, Fisher´s Exact Test was used to analyse the data. Count data were evaluated using the procedures described above for the Kruskal-Wallis Test.

Results and discussion

Effect levels

Dose descriptor:
Effect level:
10 000 ppm
Basis for effect level:
other: No effects

Observed effects


Any other information on results incl. tables

- Test Substance

The test diets used for exposure were within +/-15% of theoretical concentrations. Diets were homogeneous with an RSD of +/-5% and stability was established for up to 8 weeks.

- Mortality (Table 2 of attachment)

No substance-related mortality occured. One rat in the 10000 ppm exposure group was found dead on day 31. No cause of death could be determined. This single event although it occured in the highest dosage group was not considered related to Methylparaben because the rat was essentially normal until being found dead. The only adverse clinical observation was microphthalmia on days 27 and 31, which was considered secondary to retro-orbital bleeding. The rat was gaining weight and had normal feed consumption. All tissues appeared normal at necropsy.

- Clinical Observations (Table 2 of attachment)

Urine-stained abdominal fur occured in 3 of the 16 rats in the 10000 ppm exposure group. These clinical observations were not considered related to Methylparaben even though they occured in the highest exposure group because the observations did not persist and these observations occur sporadically in rats at the performing Testing Facility. No other clinical observations related to Methylparaben occured at exposure levels as high as 10000 ppm. The clinical observations that did occur were not considered related to the test item because:

1. the incidence was not dosage dependent and/or

2. the observation was secondary to retro-orbital bleeding.

These observations included chromorhinorrhea, chromodacryorrhea, a scab or abrasion on the nose, mouth, right axilla, right forelimb, tip of tail or neck, localised alopecia, excess salivation, microphthalmia, swollen right hindpaw, lacrimation, ulceration on the mouth, missing tip of tail, neck abrasion and soft or liquid feces.

- Body weights and Body weight gain (Tables 3 and 4 of attachment)

Body weights and body weight gains were not affected by exposure to Methylparaben as high as 10000 ppm.

- Absolute (g/day) and Relative (g/kg bw/d) Feed Consumption Values (Tables 5 and 6 of attachment)

These values were not affected by the exposure to Methylparaben as high as 10000 ppm.

- Necropsy Observations (Table 7 of attachment)

No necropsy observations related to Methylparaben occured. The number of rats that appeared normal in the 100 ppm exposure group was significantly reduced (p</=0.01). The reduction was the result of 4 of 16 rats in the 100 ppm exposure group with gross lesions. One rat had small ventral and dorsal prostate and seminal vesicles; two rats had a diaphragmatic hernia and one rat had small testes and epidymides. The only other gross lesion occured in one 10000 ppm rat (small seminal vesicles). None of these lesions were considered related to Methylparaben because this incidence was not dosage-dependent and/or these lesions are common in this strain of rat.

- Terminal Body Weights, Organ Weights and Ratios (%) of Organ Weight to Terminal Body Weight (Tables 8 and 9 of attachment)

Terminal body weights, organ weights and ratio of these organ weights to the terminal body weight were not affected by exposure to Methylparaben as high as 10000 ppm. No statistically significant differences occured among the groups, except for a statistically significant increase (p</=0.05) in the ratio of the liver weight to the terminal body weight in the10000 ppm exposure group. This increase was not considered related to Methylparaben because the absolute weight was not significantly increased.

- Sperm Evaluation (Tables 10 and 11 of attachment)

Exposure to Methylparaben at levels as hight as 10000 ppm did not affect sperm motility, sperm count, morphology or daily sperm production.The number of normal sperm was significantly reduced (p</=0.05) and corresponding values for percent abnormal were significantly increased in the 1000 ppm (p</=0.01) and 10000 ppm (p</=0.05) exposure groups. The percent abnormal sperm in these groups (mostly composed of sperm with no head) was significantly increased compared to the control group values. These differences wer not related to Methylparaben because the values were not dosage-dependent.

- Histopathology

Exposure to Methylparaben at levels as high as 10000 ppm did not produce any adverse findings in the reproductive organs.

Applicant's summary and conclusion

On the basis of these data, the NOEL for general toxicity for Methylparaben including histopathology of reproductive organs and sperm analysis is 1000 ppm (equivalent to 1141.1 mg/kg bw/d). No effects occured at the highest dose tested.
Executive summary:

Methylparaben was administered daily by diet to Crl:(WI) BR male rats at dose levels of 100, 1000 and 10000 ppm (equivalent to 11.2, 110.0 and 1141.1 mg/kg bw/d) for a period of 56 days. A control group was treated similary with plain diet only.

The groups comprised 16 animals which were sacrificed at the end of treatment. With the exception of 1 spontaneous death (10000 ppm), all rats survived until scheduled necropsy. No treatment-related clinical signs were noted at any dose level. There were no effects upon daily food consumption. The mean body weights and the mean body weight gain values of the test item-treated rats were similar to those of the controls. The absolute and relative organ weights of the treated rats compared favourably with those of the controls.

No macroscopical changes were evident in the males. The histopathological exmination did not reveal changes. There were no influences on the daily sperm production and sperm morphology.