Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1 - 28 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Data have been generated according to current internationally recognised study guidelines and in accordance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
methyl 2-[(4-hydroxybenzenesulfonyl)oxy]benzoate
Cas Number:
1432505-97-9
Molecular formula:
C14H12O6S
IUPAC Name:
methyl 2-[(4-hydroxybenzenesulfonyl)oxy]benzoate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
EXPERIMENTAL ANIMALS
Species and strain: Crl:WI rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D- 97633
Housing conditions: SPF at arrival; standard laboratory conditions during the study
Justification of species/strain: Wistar rat as a rodent is one of the standard strains for repeat-dose toxicity studies
Number of animals: 30 male and 30 female rats (5 rats/sex/group in the 4 Main groups 1, 2, 3 and 4 and 5 rats/sex/group in the 2 Recovery groups 1 and 4); Spare animals were assigned to CiToxLAB Hungary Ltd. Spare colony at the end of the study.
Age of animals: Young adult rats, approximately 6-7 weeks old at starting.
Body weight: 232-281g, males and 164-200g, females at onset of treatment
Acclimation period: 7 days

Husbandry
Animal health: Only healthy animals were used for the test, as certified by the veterinarian. Females were nulliparous and non-pregnant.
Room number: 524
Cage type: Type III polycarbonate
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG
(Holzmühle 1, D-73494 Rosenberg, Germany)
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.0 – 22.6 °C
Relative humidity: 34 - 58 %
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were housed by 5 animals of the same sex and group/cage. Group housing allows social interaction and the deep wood sawdust bedding allows digging and other normal rodent activities.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
Vehicle
Name: Polyethylene glycol 400
Lot No.: BCBH2687V
Manufacturer: Sigma-Aldrich
Expiry Date: 31 December 2013
Storage: Room temperature

Formulation and analysis of formulation
The test item was formulated in the vehicle at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared fresh prior to administration to animals or at the appropriate frequency to allow their use according to stability assessment results. Stability of the test item in the vehicle was assessed under CiToxLAB study code 12/096-316AN. Based on the results, the test item solution in Polyethylene glycol 400 was stable at concentration range of 5-350 mg/mL when stored for 1 day at room temperature or 7 days refrigerated (at 2-8°C).
Analysis of test item formulations for concentration and homogeneity, using a validated HPLC method, was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Top, middle and bottom duplicate samples were taken from test item formulations twice during the treatment period (during the first and last weeks of treatment). Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements.
All formulations were found to be in the range of 96 to 100% of nominal concentration. All formulations were shown to be homogeneous. No test item was detected in the control samples.

Diet and water supply
The animals were provided with ssniff® SM R/M “Autoclavable Complete Diet for Rats and Mice – Breeding and Maintenance” (Ssniff Spezialdiäten GmbH, D-59494 Soest Germany) and tap water as for human consumption, ad libitum.
The quality control analysis of the water is performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). Copies of the relevant Certificates of Analysis are retained in the archive at CiToxLAB Hungary Ltd.
The diet and drinking water are routinely analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HPLC
Column: Inertsil ODS-2 4.6µm x 150mm
Column temperature: 40oC
Detection Photodiode array / 220nm
Mobile phase MeCN : 0.05% H3PO4aq = 60:40
Flow rate 0.8 ml/min
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
30 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 animals er sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
During the acclimation period, the animals were assigned to their respective dose groups by randomization based on body weights. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight; SPSS/PC+ software was used in order to verify homogeneity/variation among/within groups. Males and females were randomized separately.

The dose levels were set by the Sponsor in consultation with the Study Director, based on available data and information from previous experimental work, including the results of a preliminary dose range finding study conducted at CiToxLAB Hungary Ltd. with the test item.
The oral route is a possible route of exposure to the test item in humans.

Examinations

Observations and examinations performed and frequency:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical observations were made daily, after treatment at approximately the same time.
Detailed clinical observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once weekly thereafter. Observation was performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical signs were noted during the study.
During the last week of treatment (Day 26), each animal was subjected to the functional observation battery, including qualitative assessment of the grip strength, and to measurements of the landing foot splay and fore/hind grip strength.
To measure the landing foot splay, the hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured.
Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs are allowed to grip the support bar and gently pulled back until they release the bar; the device measures the maximum grip strength. This was performed at least 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed. The parameters evaluated included the following: body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or
licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation,
palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation.
Sacrifice and pathology:
On Day 28 (Main animals) or 42 (Recovery animals), all animals were euthanized under pentobarbital anaesthesia by exsanguination.
After exsanguination, the external appearance was examined, cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
Other examinations:
Prior to necropsy, the oestrus cycle of all females was determined by taking vaginal smears, which were prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.
Statistics:
Data were collected using the software PROVANTIS v.7 or were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., then tabulated using the Microsoft Office Word and/or Excel, as appropriate.
Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations.
The statistical analysis was performed using SPSS PC+4.0 software. The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normal distributed, the nonparametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg bw/day.
Executive summary:

The repeat dose toxicity by oral (gavage) has been assessed in male and female Wistar Crl:WI rats at 30, 300 and 1000 mg/kg/day in PEG 400 and solvent control in accordance with the OECD 407 test guideline over 28 days with a 14 day recover period for solvent and high dose animals in compliance with GLP. Under the conditions of the test the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg bw/day.