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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
October 20 -December 17, 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance Methylparaben. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3050
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
the certificate is not included in the report
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Methyl 4-hydroxybenzoate
- Substance type: White powder
- Physical state: solid
- Analytical purity: >99%
- Purity test date: September 26, 2008
- Lot/batch No.: GBGA039810
- Expiration date of the lot/batch: December 31, 2010
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: pre-test mean range: males 146-150, females 121-123 grams
- Fasting period before study: not applicable
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type; height 18 cm; during overnight activity monitoring individual housing in MIII type; height 15 cm.) with sterilized sawdust as bedding material
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days before start of treatment under laboratory conditions


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8 – 21.8ºC
- Humidity (%): 33 - 67%
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: October 31, 2008 To:December 17, 2008

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for specific gravity of the vehicle. In order to obtain homogeneity, the formulations were heated in a water bath with a maximum temperature of 49 °C for a maximum of 42 minutes. The formulations were allowed to cool down to a temperature of maximally 40°C prior to dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX and on information from the sponsor.
- Concentration in vehicle: 0, 10, 50, 200mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analyzed on a single occasion during the in-life phase for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The analytical method used was validated under NOTOX project 489458.
Duration of treatment / exposure:
at least 28 days
Frequency of treatment:
Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females. An extra 5 animals per sex in the control and high dose group were allowed 14 days of recovery.
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: dose levels were based on the results of a 7-day dose range finding study with this compound

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pre-test and at week 4
- Dose groups that were examined: All animals at pretest and Groups 1 and 4 (Main and Recovery group animals) at week 4

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination at the end of the treatment and recovery phase
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination at the end of the treatment and recovery phase
- Animals fasted: Yes, overnight
- How many animals: All animals
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate

URINALYSIS: Yes
- Time schedule for collection of urine: 15-24 hours
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Volume, Specific gravity, Clarity, Colour, pH, Blood, White blood cells (WBC), Bilirubin, Urobilinogen, Protein, Ketones, Glucose, Nitrite , Sodium, Potassium, Calcium, Sediment: White blood cells, Red blood cells, Casts, Epithelial cells, Crystals, Bacteria, Other.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:during week 4 of treatment
- Dose groups that were examined: all dose groups
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity.

ESTROUS CYCLE DETERMINATION: YES
All females received a daily lavage from day 15 up to and including day 28 to determine the satge of estrous. Since no treatment-related effects on the estrous cycle were noted, no lavage was conducted during the recovery phase.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Adrenal glands, (Aorta), Brain [cerebellum, mid-brain, cortex], Caecum, Cervix, Clitoral gland, Colon, Duodenum, Epididymides, Eyes with optic nerve [if detectable] Harderian gland (Skin), Female mammary gland area, (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Oesophagus) Ovaries, (Pancreas), Peyer's patches [jejunum, ileum] if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, (Skeletal muscle), (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid [if detectable], (Tongue), Trachea, Urinary bladder, Uterus, Vagina, all gross lesions

Tissues/organs mentioned in parentheses were not examined by the pathologist since there were no changes in macroscopic appearance indicative of (potential) toxicity.

The following organ weights and terminal body weight were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles, Spleen, Testes, Thymus, Uterus

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all Main group 1 and 4 animals,
- all tissues from animals, which were terminated in extremis,
- all gross lesions.
Based on (possible) treatment-related changes in bone marrow and sternum, the histological examination was extended those particular organs of all animals of groups 2 and 3 (males and females) and recovery group animals. All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-toone t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
One male and one female at 1000 mg/kg/day were sacrificed for ethical reasons on Day 14 and 24 respectively, showing several clinical signs indicative of ill health. Microscopic findings examination revealed minimal/slight erosions in the stomach, correlating to the irregular surface recorded at necropsy, slight red pulp atrophy of the spleen and slight/moderate lymphoid atrophy of the thymus, correlating to the reduced size recorded at necropsy. Since these deaths occurred in the highest dose group, a relation to treatment with the test substance cannot be excluded.

Piloerection and/or hunched posture were noted among the surviving animals at 1000 mg/kg/day during the observation period. Laboured respiration, rales and gasping were noted among animals at 1000 mg/kg/day and one female at 250 mg/kg/day during the observation period. These clinical signs were considered related to treatment with the test substance since these signs occurred predominantly in the highest dose group and were absent during the recovery phase.

Protein levels of males at 1000 mg/kg/day were slightly reduced and chloride levels were slightly increased at the end of the treatment period, achieving a level of statistical significance. At the end of the recovery phase protein and chloride levels were similar to control levels. Urinalyses at the end of the treatment period showed increased calcium excretion in females at 1000 mg/kg/day. These changes had resolved at the end of the recovery phase. Also, considering the slight nature of these changes, and absence of any supportive toxicologically significant morphological findings, no toxicological significance was ascribed to these changes.

Spleen to body weight ratio and/or spleen weight were higher for all test substance- treated males at the end of treatment. Since these changes had resolved at the end of the recovery phase and were not confirmed by related histopathological findings, this increase is considered not to be of toxicological significance.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. functional observations, ophthalmoscopy, body weight, food consumption, haematology parameters, estrous cycle and spermatogenesis).

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for Methyl 4-hydroxybenzoate of 250 mg/kg/day was established.
Based on a read-across approach, Sodium Methylparaben is not subjected for labelling and classification requirements according to regulatory requirements.
Executive summary:

A 28 day oral toxicity study was performed in rats. The animals were treated with 50, 250 and 1000 mg/kg bw/d.

No spontaneous mortality occurred during the study. However, one male and one female at 1000 mg/kg bw/d were sacrificed for ethical reasons on Day 14 and 24 respectively, showing several clinical signs indicative of ill health. Microscopic examination revealed minimal/slight erosions in the stomach, correlating to the irregular surface recorded at necropsy, slight red pulp atrophy of the spleen and slight/moderate lymphoid atrophy of the thymus, correlating to the reduced size recorded at necropsy. Piloerection and/or hunched posture were noted among the surviving animals at 1000 mg/kg bw/d during the observation period. Laboured respiration, rales and gasping were noted in most animals at 1000 mg/kg bw/d and one female at 250 mg/kg bw/d during the observation period. These clinical signs were considered related to treatment with the test substance since these signs occurred predominantly in the highest dose group and were absent during the recovery phase.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. hematology, clinical chemistry, urinalysis, functional observations, ophthalmoscopy, body weight, food consumption, estrus cycle, spermatogenesis, macroscopic and microscopic examination).

Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for the test item, Methylparaben, was considered to be 250 mg/kg body weight/day.