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Description of key information

NOAEL (OECD 407), rat, oral: 250 mg/kg bw/d

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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.
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
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
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.
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
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.
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).
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study has Klimisch score 2.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

There are no data available on repeated dose toxicity for sodium methylparaben. However, there are reliable data on methylparaben which is considered suitable for read-across using the analogue approach.

Sodium methylparaben (sodium 4-(methoxycarbonyl)phenolate) is the sodium salt of methylparaben (methyl 4-hydroxybenzoate). The substance is highly water soluble (418 g/L), dissociates in aqueous solutions and is hydrolysed to sodium hydroxide and the source substance methylparaben. Based on the assumption that upon oral and dermal administration, sodium methylparaben dissociates and is hydrolysed to methylparaben and sodium hydroxide, it was predicted that after exposure the primary effect is local irritation/corrosion at the site of contact due to sodium hydroxide.

Target and source substance are of low acute toxicity. No systemic or local effects were found up to single doses of 5000 mg/kg bw for sodium methylparaben and 2100 mg/kg bw for methylparaben. A dermal absorption of 80% was calculated for sodium methylparaben using QSAR and the available physico-chemical properties. For methylparaben, the dermal absorption was estimated in an in vitro test to be 84.7% in human skin (Fasano, 2004).

Therefore, it can reasonably be deduced that no higher amounts than tested in the acute oral toxicity study will be systemically available via the intact skin barrier. Inhalation is of no concern for the target as well as the source substance because of the low vapour pressure.

The results of several repeated dose toxicity studies indicate a low toxicological concern for methylparaben. Methylparaben caused no systemic toxicity and no effects on fertility or developmental toxicity. Moreover, toxicokinetic data have shown that methylparaben is completely absorbed after oral and dermal administration, hydrolysed, conjugated and rapidly excreted in urine. Thus, there is no evidence of accumulation.

As bioavailability and metabolism and therefore mammalian toxicity of sodium methylparaben were considered to be comparable to that of methylparaben, the assessment of systemic toxicity based on analogue approach can be considered as justified.

In a repeated dose oral toxicity study according to OECD 407 and GLP, 5 Wistar rats per sex and dose were treated for 28 d with Methylparaben (Beerens-Heijnen, 2009). The animals were treated with 50, 250 and 1000 mg/kg bw/d of the test substance in propylene glycol by oral gavage.

No spontaneous mortality occurred during the study and no effects on body weight, body weight gain and food consumption were observed.

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. The necropsy of the male killed in extremis revealed that the gastro-intestinal tract was distended with gas. The forestomach and glandular mucosa had an irregular surface and the caecum had a reddish colour. Furthermore, the thymus size was reduced. In the female killed in extremis, the gastro-intestinal tract was also distended with gas and the duodenum was coloured gray white. The histopathologic examination of both animals sacrificed revealed minimal/slight erosions in the forestomach, 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.

In the surviving animals, piloerection and/or hunched posture were noted 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 14 d 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, estrus cycle, spermatogenesis, necropsy and histopathology).

Based on the results of this study, the NOAEL for Methylparaben was considered to be 250 mg/kg bw/day.

 

In order to meet the standard info requirements according to Regulation (EC) 1907/2006, Annex IX, Column I, 8.6.2 a subchronic repeated dose toxicity study is required. For that reason, in the following, subchronic and chronic repeated dose toxicity studies are described. However, those studies followed no guideline and there are no study reports but publications available. The studies contain partially inconsistent data due to limited documentation or lacking data on examinations. Therefore, the subacute repeated dose toxicity study conducted according to OECD 407 and GLP was used as key study since it represented the most sensitive NOAEL. Due to the fact that unnecessary tests should be avoided in terms of animal welfare, no further subchronic study will be conducted. This is in accordance with Regulation (EC) 1907/2006. 

 

In a chronic dietary study, 6 rats per dose were fed diets containing 2 and 8% Methylparaben for 96 weeks (Matthews et al., 1956). Animals receiving diets containing 8% Methylparaben (equivalent to a Methylparaben intake of 5500 - 5900 mg/kg bw/d) showed a slower rate of weight gain than control animals. This difference was much more striking in the early part of the study. At the end of the study, these differences disappeared. This effect was more striking in the case of males than females. On animals receiving 2% of Methylparaben (equivalent to a Methylparaben intake of 900 - 1200 mg/kg bw/d), however, it was impossible to differentiate treated animals from untreated. Food intake and hence Methylparaben intake remained constant throughout the study. Tissues from all rats surviving the chronic study were entirely normal. The only significant findings were seen in the case of the rats dying during the study; in all such animals there was extensive consolidation of the lung and pneumonia, and in this respect there was no difference between the control and treated animals.

 

Groups of 2-3 Mongrel dogs were treated with Methylparaben by daily application of a capsule containing 500 and 1000 mg/kg bw/d for up to 422 d (Matthews et al., 1956). No toxic symptoms were observed in any of the animals. The weights of the test animals before and after treatment showed respectable increases. At termination of the study, all animals appeared to be in excellent condition. One of the females, receiving 500 mg/kg bw/d Methylparaben was mated toward the end of the study and delivered a litter of healthy pups. At study termination complete blood count examination was made in all dogs. No effect was observed. Complete urinalysis at the end of the study showed no changes. Pathology revealed no changes.

 

Rabbits were treated with 2.5 mg Methylparaben/animal/d (equivalent to approximately 0.5 mg/kg bw/d for males and females with an average bodyweight of 4750 g) for 120 d (Cremer, 1935). All animals survived. There were no clinical signs or effects on body weight, body weight gain, temperature or gastro-intestinal tract observed.

 

In another study guinea pigs were treated with up to 100 mg Methylparaben/animal/d (equivalent to approximately 91 mg/kg bw/d for maleswith an average bodyweight of 1100 gand 108 mg/kg bw/d for femaleswith an average bodyweight of 925 g) for 120 d (Cremer, 1935). All animals survived. There were no clinical signs or effects on body weight, food consumption or haematology.

 

Rats treated with 0.5 to 5 mg Methylparaben/animal/d (equivalent to approximately 1.3 to 13 mg/kg bw/d for maleswith an average bodyweight of 375 gand 2.6 to 26 mg/kg bw/d for femaleswith an average bodyweight of 190 g) for 80 d showed no effects on body weight (gain), gastro-intestinal activity and haematology (Cremer, 1935).All animals survived and no clinical signs were noted.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The selected study is the most reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall quality assessment.

Justification for classification or non-classification

The available data on the repeated dose toxicity of the test substance does not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and is therefore conclusive but not sufficient for classification.