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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:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to OECD test guideline 407 under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
minor changes not impairing the study
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification: SULFANILIC ACID TECHNICAL GRADE
Formula: H2N-C6H4-SO3H
Molecular mass: 173,19 g/mol-1
Purity: 99,12%
Batch No.: M001/09
Batch production: 01 October 2009
Appearance: Grey powder (micronised)
Storage conditions: Conventional, ambient temperature
Expiry date: 01. Oct. 2011

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Species: Rattus norvegicus
Strain: Wistar-WU
Gender: 20 males, 20 females
Source: Charles River, Sandhofer Weg 7, 97633 Sulzfeld, Germany
Age at acclimatisation: 8-10 weeks
Health status: Specific pathogen free (SPF)
Pregnancy status females: Nulliparous, non-pregnant
Acclimatisation: 5-15 days

Housing conditions
Clean conventional housing: airing with approx. 10 air changes per hour, room climate 22 ± 3°C at 30-70% relative humidity, (aimed
limits - due to meteorological circumstances, upper limit might have been above 70% on short term), artificial lighting 12 h light/12 h dark.

Caging
Groups of up to five animals in open macrolon cages type 2000P, TechniPlast (size slightly larger than GV-SOLAS Type IV).

Bedding:
Lignocel hygienic animal bedding (J. Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg)

Diet
Maintenance diet for rats and mice, No. 1324 TPF (Altromin Spezialfutter GmbH & Co. KG, 32791 Lage), ad lib.

Water
Sterilised community tap water, ad lib.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
suspension in water

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Although the test item’s solubility in water is poor, results from a preceding dose range finding study indicated that the micronised (<100 μm) test item could be applied as a colloid suspension in water at the three dosages specified in this study report.
- Amount of vehicle (if gavage): 4 ml/kg bw

Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
63 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: pre-test
Positive control:
not required

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily/once workdays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: once weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: twice weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once (on day 29)
- Anaesthetic used for blood collection: No data
- How many animals: all
- Parameters. see below

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once (on day 29)
- How many animals: all
- Parameters. see below

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before application and once during the last exposure week
- Dose groups that were examined: all
- Battery of functions tested: grip strength and limb placing
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross lesions (P), Adrenal gland (P, W), Oesophagus (P), Urinary bladder (P), Trachea and thyroid (P), Testes (P, W), Stomach (P), Epididymides (P, W), Thymus (P, W), Prostata / uterus / ovary (P), Liver (P, W), Heart (P, W), Spleen (P, W), Lung (P), Duodenum (P), Peripheral nerve (P), Jejunum (P), Bone marrow (P), Ileum (with Peyer’s
patches) (P), Sternum (P), Cecum (P), Spinal cord (P), Colon (P), Whole brain (W), Rectum (P), Cerebrum (P), Lymph nodes (intestinal area) (P), Cerebellum (P), Kidney (P, W), Pons (P)
[P = preservation, W = weight determination]

HISTOPATHOLOGY: Yes (on organs marked abobe with 'P') with animals of highest dose group
Statistics:
In general, one-Way ANOVA (Analysis of variance), followed by a post hoc t-test was used.
In case of interval-scaled data, the One-Way ANOVA was supplemented by Dunnett’s posthoc t-test.
Ordinal-scaled data were analysed by the Kruskal-Wallis test, supplemented by Dunn’s t-test.
The entire deductive statistics were performed using Graph Pad Prism. The significance level was set to 0.05.

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:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
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
Details on results:
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
- highest dose group (males): slightly increased in week 2 and 3

CLINICAL CHEMISTRY
- highest dose group (males): - a few parameters were altered, but not of toxicological relevance
- highest dose group (female): -sodium slightly reduced

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:
In a study according to OECD test guideline 407 in rats under GLP, the NOAEL of the test substance was determined to be 1000 mg/kg bw for both sexes.
Executive summary:

Aim of the study:

The aim of this study was to assess data on the subacute toxicity of the test substance suspended in water, after oral administration to Wistar rats.

Experimental model:

The test substance was administered daily by oral gavage at dose levels of 63, 250 and 1000 mg/kg body mass (BM) / day to 5 male and 5 female Wistar rats each over a period of 28 days. Another 5 male and 5 female rats received the same volume of water as vehicle control. All liquids were administered to each animal at 4 ml / kg bw.

Assessed parameters:

During the in-life phase, all animals were monitored for fatalities, general clinical signs, detailed clinical signs, grip strength and reactivity to sensory stimuli (limb placing test). The bodyweight as well as group food and group water consumption were also recorded. At the end of the in-life phase, blood samples from all animals were collected to provide data on haematology, serum biochemistry and blood coagulation. All animals were sacrificed humanely immediately after bleeding and examined by gross necropsy. Organ mass was recorded according to the study plan, tissues and organs selected therein were preserved and processed histologically. A histopathological examination was conducted on samples from the high dose groups and the vehicle groups.

Results:

Data monitoring of clinical signs, detailed clinical signs, motor activity, reactivity to sensory stimuli as well as food and water consumption showed no abnormalities. The most noticeable among the very few observable effects were haematological changes in the male test animals of the high dose group. The albumin-globulin ratio, total albumin, GOT1, GPT and LDH were found to be significantly raised in the high dose. In contrast, alpha 1-, beta- and total globulin were significantly reduced in that group. Both findings suggested the liver as target organ of mild to moderate effects, although findings of this nature are in favour of an adaptive response rather than a direct toxic effect of the test item. These findings, not present in the control animals, are considered to be related to the treatment with the test item. Although some additional significant changes were observed, none of the average data points were overall extremely out of range for rats of this strain and age. The histomorphological examination of rat organs from the 28-day toxicity study by oral administration of animals treated with the test substance did not reveal morphological lesions that were considered to be related to the test item. There was no morphological difference between the vehicle control groups and the groups subjected to the high dose of the test item.

Conclusion:

A daily oral administration of the test substance to Wistar rats at a dose level of 63, 250 and 1000 mg/kg body mass over a time period of 28 days (acc. to OECD test guideline 407 under GLP) resulted in very few mild systemic and local effects in test animals treated with the high dose (1000 mg/kg), but did not produce any pathological evidence of a local or systemic toxicity of

the test item. Therefore, the NOAEL was considered to be 1000 mg/kg bw.