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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
Study period:
1988-03-22 to 1988-11-08
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate
EC Number:
403-080-9
EC Name:
Sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate
Cas Number:
92484-48-5
Molecular formula:
C16 H17 N3 O4 S .Na
IUPAC Name:
sodium 3-(2H-1,2,3-benzotriazol-2-yl)-5-(butan-2-yl)-4-hydroxybenzene-1-sulfonate
Constituent 2
Reference substance name:
sodium 3(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate
IUPAC Name:
sodium 3(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate
Details on test material:
- Name of test material: sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate (FAT 75309/A)
- Physical state: Powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf/Switzerland
- Age at study initiation: males: 7 weeks; females: 8 weeks
- Weight at study initiation: males: 161 - 181 g; females: 141 - 166 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: Seven days under laboratory conditions, after veterinary examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 degree C
- Humidity (%): 40 - 70 %
- Air changes: 10-15 air changes per hour
- Photoperiod: 12 hours artificial fluorescent light/12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
4 % in distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was weighed into a glass beaker on a tarred Mettler PK 300 balance and the vehicle, carboxymethyl cellulose 4 % (CMC), was added. The mixture was prepared daily prior to administration using a homogenizer and kept stable during application with a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability of the test article/vehicle mixtures was determined once during pretest. Intercurrent sampling for analyses were additionally performed during treatment period in the RCC Analytical Laboratory, according to a method supplied by the sponsor.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days per week.
Doses / concentrations
Remarks:
Doses / Concentrations:
20, 50, 200, 800 mg/kg; dose volume: 10 mL/ kg b.w.
Basis:
other: nominal concentrations by gavage
No. of animals per sex per dose:
5 males, 5 females (groups 2, 3 and 4 );
10 males, 10 females (groups 1 and 5 )
Allocation see below at "Any other information on materials and methods incl. tables"
Control animals:
yes, concurrent vehicle
Positive control:
No positive control

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during the acclimatization and treatment period

FOOD CONSUMPTION AND COMPOUND INTAKE:
The food consumption was recorded once during the acclimatization period and weekly thereafter using an on-line electronic recording system consisting of a
Mettler PK 4800 balance connected to the RCC computer.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examinations were performed at termination of treatment.

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

URINALYSIS: Yes

ORGAN WEIGHTS: The following organ weights were taken from all animals necropsied at termination of treatment: Adrenal glands. Kidneys, Liver, Testes.

NECROPSY: All animals were necropsied and descriptions of all macroscopic abnormalities were recorded. The animals were fasted for approximately 18 hrs before necropsy but water was provided. Necropsies were performed by experienced prosectors supervised by a pathologist. All animals surviving to the end of the observation period and all moribund animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.

HISTOPATHOLOGY: Yes

HISTOTECHNOLOGY: All organ and tissue samples, as defined under Histopathology were processed, embedded and cut at a thickness of 2-4 micrometers and stained with hematoxylin and eosin.
Sacrifice and pathology:
HISTOPATHOLOGY: Yes
Statistics:
The following statistical methods were used to analyze the body weights, food consumption, organ weights and clinical laboratory data :
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for
the comparison between the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
Group means were calculated for continuous data and medians were calculated for discrete data (scores).
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given
parameter, yet display different test statistics values. The exact Fisher-test was applied to the ophthalmoscopic examination data.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
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):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY
No death occurred, which could be related to test article treatment. One female rat of group 5 was killed in a moribund state at day eight after an intubation error.
CLINICAL SIGNS
No clinical signs were observed in male and female animals of groups 1, 2 and 4 as well as in females of group 3 during the treatment and recovery (treatmentfree) period. Slight ruffled fur and/or rales were observed in two males of group 3 and three males and one female of group 5 during the treatment period. During the recovery period no clinical signs were observed.
FOOD CONSUMPTION
The food consumption of the male rats of group 5 was statistically significant decreased between days 1 to 8 of the treatment period. All other statistically significant differences were considered to be incidental and not related to test article treatment.
BODY WEIGHTS
The body weight of the male rats of group 5 was statistically significant decreased at day 28 of the treatment period. All other statistically significant differences were considered to be incidental and not related to test article treatment.
BODY WEIGHT GAIN
The body weight gain of the male rats of group 5 was statistically significant decreased at day 28 of the treatment period. All other statistically significant differences were considered to be incidental and not related to test article treatment.
OPHTHALMOSCOPIC EXAMINATIONS
No ophthalmoscopic changes were observed.
HEMATOLOGY
No changes of toxicological significance were noted at the end of the treatment nor at the end of the treatment-free (recovery) period. The only change of note was a slightly prolonged partial thromboplastin time for males and a slightly increased leukocyte count for females of group 5. These findings were reversible at the end of the recovery period.
CLINICAL BIOCHEMISTRY
For biochemical data the following effects were observed at the end of the treatment for group 5: Slightly increased total lipids and triglycerides level for females, slightly decreased total cholesterol level for males, slightly increased aspartate aminotransferase activity for males, slightly decreased iron level for males and females, slightly increased phosphorus level for females, slightly increased sodium and chloride level for males and females, and slight changes in the protein electrophoretic pattern indicated by an increased albumin fraction for males and females (also increased for males of group 4), decreased alpha-1 globulin and increased alpha-2 globulin fraction for males, decreased beta globulin fraction for males and females (also decreased for males of group 4 ), and increased albumin to globulin ratio for males and females (also increased for males of group 4). These findings were reversible at the end of the treatment-free (recovery) period. In summary, the changes noted were considered to be of an adaptive nature and therefore, not significant in toxicological terms.
URINALYSIS
For urinalysis data no changes of toxicological significance were observed. However, a moderately decreased overnight urinary output (Volume/18 hrs) for males and a slightly increased specific gravity for males and females of group 5 was noted at the end of the treatment. These findings were reversible at the end of the treatment-free (recovery) period.
ORGAN WEIGHTS AND ORGAN WEIGHT RATIOS
A statistically significant increase in absolute and relative liver weights were observed in male rats of groups 4 and 5 and in female rats of group 5 at termination of the treatment period. Additionally the absolute weights of the adrenals were statistically significant decreased in males of group 5 at termination of the treatment and treatmentfree (recovery) period.
NECROPSY AND HISTOPATHOLOGY
Apart from the lesions observed in the animal which was killed in a moribund state after incidental intubation error, all pathology findings recorded were of a spontaneous nature common to rats of this age and strain. There was no evidence of abnormal histopathological findings resulting from treatment with the test substance.

Effect levels

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical chemistry, body weight

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Results of chemical analysis:

The mean concentrations found were in the range 96.6 % to 108.5 % of the nominal concentrations. This is within the range 75.0 % to 115.0 %. The homogeneity varied in the range -2.7 % to +2.1 % of the mean concentration and is therefore within the range of + 15.0 % as proposed by the above mentioned guidelines. The test article/vehicle mixtures are stable over a period of 2 hours.

Applicant's summary and conclusion

Conclusions:
Based upon the results obtained in this study, the "no adverse effect level" (NOAEL) of sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate is 200 mg/kg bw for male and females rats when administered orally by gavage for a period of 28 days.
Executive summary:

A 28-day oral (gavage) toxicity study was performed with sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate in male and female Wistar rats according to OECD 407 Guideline and EC Method B.7. Dose levels administered in this study were selected based on data obtained during a 5-day dose-range-finding toxicity study. A control, four dose groups and a high dose and control recovery group (n= 5 animals per group and sex) were involved in the study. The test item was administered at concentrations of 0, 20, 50, 200 and 800 mg/kg bw prepared in carboxymethyl cellulose (4 %), at a 10 mL/kg bw treatment volume. Stability and homogeneity of test item in this vehicle (CMC) were analytically proven. There were no changes in the animal clinical condition, body weight or food consumption. A statistically significant increase in absolute and relative liver weights were observed in male and female rats at termination of the treatment period at 200 mg/kg bw/day. No histopathological correlation in liver were found. Thus, these findings were considered to be adaptive and not adverse. No other effects were observed at 200 mg/kg bw/day. Additionally the absolute weights of the adrenals were statistically significant decreased in males at termination of the treatment and treatmentfree (recovery) period at 800 mg/kg bw/day. There was no evidence of abnormal histopathological findings resulting from treatment with the test substance. No effects were noted at clinical laboratory investigations (hematology, clinical biochemistry, urinanalysis). There were no ophthaloscopic changes observed. In conclusion, based upon the results obtained in this study, the "no adverse effect level" (NOAEL) of sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate is 200 mg/kg bw for male and females rats when administered orally by gavage for a period of 28 days.