Benzenesulfonic acid, 4-hydroxy-, 2,6-dimethylphenyl ester

Endpoint summary

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:

experimental study

Adequacy of study:

supporting study

Study period:

10 January to 6 February 2013

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

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

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

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 10 weeks old at starting.
Body weight: 346-380g, males and 210-250g, females at onset of treatment
Acclimation period: 7 days
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: 22 ± 3 °C
Relative humidity: 30 - 70 %
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.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

Vehicle
Name: Polyethylene glycol 400 (abbreviation: PEG 400)
Lot No.: BCBH5068V
Manufacturer: Sigma-Aldrich
Expiry Date: January 2014
Storage: Room temperature
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 weekly according to stability assessment results. The formulations were stored at 2 – 8 oC in the dark. Stability of the test item in the vehicle was assessed under CiToxLAB study code 12/095-316AN. The test item solution in Polyethylene glycol 400 was stable at concentration range of 1-250 mg/mL when stored for 1 day at room temperature or 7 days refrigerated (at 2-8°C).

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

Remarks:

Doses / Concentrations:
62.5 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 per sez per dose group

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.

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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

High dose animals

Mortality:

mortality observed, treatment-related

Description (incidence):

High dose animals

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):

no effects observed

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:

effects observed, treatment-related

Description (incidence and severity):

Kideneys; determined to be due to species specific effects

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

species specific effects; tubular eosinophilic droplets

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Critical effects observed:

not specified

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. Effects were noted to the kidney organ weights of male high dose animals and microscopic effects (ubular eosinophilic droplets) noted in these dose group animals following pathology. Further examination of these ffects was undertaken. By immuno-staining of the kidney tissues (by the Chromotrope-Aniline-Blue method) the eosinophilic droplets were confirmed to be hyaline droplets. In male rats hyaline indicates the presence of alpha-2 mu globulin, which is specific to male rats and has no known relevance for human health. These effects are therefore deemed to be species specific effects to the test organism with no relevance to effects on humans. Under the conditions of the test the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

in vivo 28-day oral toxicity. K1 quality. Data generated in compliance with GLP

Additional information

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The dose levels for the study were set by the Sponsor in consultation with the Study Director based on results of a preliminary dose range finding.

Justification for classification or non-classification

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. Effects were noted to the kidney organ weights of male high dose animals and microscopic effects (ubular eosinophilic droplets) noted in these dose group animals following pathology. Further examination of these ffects was undertaken. By immuno-staining of the kidney tissues (by the Chromotrope-Aniline-Blue method) the eosinophilic droplets were confirmed to be hyaline droplets. In male rats hyaline indicates the presence of alpha-2 mu globulin, which is specific to male rats and has no known relevance for human health. These effects are therefore deemed to be species specific effects to the test organism with no relevance to effects on humans. Under the conditions of the test the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg bw/day.