Sodium 5-N-butylbenzotriazole

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 July 1989 - 17 August 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

1988

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Short description of test conditions:

Butyl benzotriazole-sodium salt (hereafter abbreviated to BBT) was formulated daily as 5.22%, I.28% and 0.12% v/v solutions in distilled water and administered orally to rats at dosage levels of 5, 55 and 225 mg/kg/day for 28 consecutive days. Control aninals received distilled water alone.

- Parameters analysed / observed:
Clinical observations
Bodyweight
FOOD CONSUMPTION AND COMPOUND INTAKE
HAEMATOLOGY
CAGE SIDE OBSERVATIONS
GROSS PATHOLOGY
HISTOPATHOLOGY

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 5-N-butylbenzotriazole

EC Number:

404-450-2

EC Name:

Sodium 5-N-butylbenzotriazole

Cas Number:

118685-34-0

Molecular formula:

CCCCc1ccc2c(c1)nnn2[Na]

IUPAC Name:

sodium 5-butyl-1H-1,2,3-benzotriazol-1-ide

Test material form:

solid - liquid: aqueous solution

Remarks:

43.1% active ingredient

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 20 March 1989
- Expiration date of the lot/batch: March 1990

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test material was stored in the original container under ambient conditions in non-continuous artificial light.
- Solubility and stability of the test substance in the solvent/vehicle: The chemical stability of solutions in distilled water were assessed by Huntingdon Research department of analytical chemistry prior to the start of treatment.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Final dilution of a dissolved solid, stock liquid or gel: The test substance was formulated freshly each day as a 5.22%, 1.28% and 0.12% v/v solution in distilled water.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

Crl:CD (SD) BR VAF Plus strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France
- Age at study initiation: 28 +- 1 day
- Weight at study initiation: 70 to 93 g
- Housing: caged in groups of five according to sex
- Diet (e.g. ad libitum): Biosure LAD 1 ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY: Analyses were made on all batches of diet used to establish levels of basic nutrients and of specified substannces and micro-organisms likely to be present in feed components and which , if in excess of specified amounts, might have had an undesirable effect on the test system. Although occasional slight deviation may have been permitted, all batches of diet conformed with the acceptable standards agreed by the study director and head, HRC department of quality assurance (see Appendix 7). The analytical data was lodged in HRC Archives.

Results of the routine physical and chemical examination of drinking water at source (Graham Final Water) as conducted usually weekly by the supplier, Anglian Water Authority, were made available to HRC as quarterly summaries. Additionally, leves of specified substances known to be present from time to time in local water and which, if in excess of the maxima recommended (for humans) might have had an undesirable effect on the test system, were determined in the tap water at approximately 6 monthly intervals. A list of the principal determinants is given in appendix 8. The analytical data was lodged in HRC archives.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): mean maximum of 20.7°C and mean minimum of 20.1°C
- Humidity (%): mean maximum iof 56.5% and mean minimum of 48.2%
- Air changes (per hr):20 air changes per hour
- Photoperiod (hrs dark / hrs light):12 hours artificial light and 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

administered by oral gavage using a syringe and rubber catheter at a dosage volume of 10 ml/kg/day.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

The dosage levels of the test substance were selected on the basis of an acute oral toxicity study ( HRC Report No. 881462D/BTZ 9/AC) and a seven day oral preliminary toxicity study (HRC Schedule No. BTZ /18) carried out at HRC.

VEHICLE
- Concentration in vehicle: 5, 55 or 225 mg a.i./kg/day
- Amount of vehicle (if gavage): 10 ml/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see Addenum: The Analysis of butyl benzotriazole-sodium salt in liquid formulations

Duration of treatment / exposure:

28 days

Frequency of treatment:

one dose of 10 ml/kg/day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

55 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

225 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes / No / Not specified
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed prior to dosing and subsequently at weekly intervals throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The quantity of food consumed in each cage was measured at weekly intervals throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Daily monitoring by visual appraisal was maintained throughout the dosing period

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination (week 4)
- Anaesthetic used for blood collection: Yes, under light ether anaesthesia.
- Animals fasted: Yes, food was withdrawn overnight prior to collection of samples.
- How many animals: all

The following parameters were analysed with an Ortho ELT-1500 analyser:
Packed cell volume (PCV) (%)
Haemoglobin (Hb) (g/dl)
Red blood cell count (RBC) (x10^6/mm^3)
Platelet count (Plts) (x10^3/mm^3)
Absolut indices:
Mean corpuscular haemoglobin concentration (MCHC)
Calculated Hb (g/dl) x 10 / PCV (%)
Mean corpuscular volume (MCV) (fl)
calculated: PVC (%) x 10 / RBC (x10^6/mm^3)
Total white blood cell count (WBC) (x10^3/mm^3)


CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No abnormal clinical findings were noted in the control group or in most rats receiving BBT at 5 mg/kg/day. A swollen appearance of the nasal region was observed on Days 22 and 23 in one female treated at 5 mg/kg/day however, this finding was not considered to be a result of treatment with BBT. No abnormal clinical findings were noted in rats treated at 55 ng/kg/day.

Several clinical findings were noted during the treatment period for
rats receiving BBT, 225 ng/kg/day. Persistent or intermittent increased salivation, pilo-erection, red/brown staining around the nasal region and hunched posture were noted for all rats, with noisy respiration and Iethargy also being noted for periods of one to several days. Abnormal gait (waddling) was recorded on occasions for two male and one female rats treated at 225 mg/kg/day and brown staining of the shoulders was noted for three female rats during week 3.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female from the control group (No. 25) died shortly following a blood sampling which was carried out on Day 28 for repeat analysis of certain haematological parameters. No adverse clinical signs were noted for this animal throuhout the study. Macroscopic pathology revealed red staining of the fur in the peri-orbital region, enlargment of the cervical lymph noder and minimal hydronephrosis of the right kidney. Death was considered likely to be due to an anaesthetic accident.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower bodyweight gains and bodyweight losses were recorded amongst male and female rats treated with BBT, 225mg/kg/day when compared with control rats. This change achieved statistical significance (P<0.01 or P<0.05) during Weeks 1 to 4 for male rats and during Week 4 for female rats. Bodyweigth gains for rats in the intermediate and low dosage groups were similar to those of the control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was lower for rats in the high dosage group (BBT 225mg/kg/day) than for control animals during the four-week treatment period. This can be related to the lower bodyweight gains and bodyweight losses recorded for animals in the high dosage group.
Food consumption for animals in the lower dosage groups was comparable with controls.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The neutrophil count was significantly lower (P<0.05) in female rats from the high dosage group than in controls. However, this apparent shift was not observed for male rats and was considered likely to have arisen by chance.

The MCV for females in the high dosage group was also significantly Iower (p<0.05) than for controls. In this instance, the apparent change was very small in magnitude, was not observed in the corresponding males and was not therefore considered to be treatment-related.

The thrombotest time was decreased in males treated at 225 mg/kg/day. This achieved significance (P<0.05) when compared with controls. However, this change was minor and was not considered to be of toxicological importance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Biochemical investigations were also carried out for all rats during week 4 (Day 27).

AIkaline phosphatase and glutamic-pyruvic transaminase leveIs were statistically significantly higher (P<0.01) in male rats fron the high dosage group than in controls. Significantly higher (P<0.05) glutamic-oxaloacetic transaminase leveIs were recorded for both male and female rats from this group when compared with control rats.

Statistically significantly higher (P<0.01) creatinine levels were also recorded for male and female rats treated at 225 mg/kg/day in comparison with controls.

Urea nitrogen levels were also raised for males and females of this high dosage group, achieving significance (P<0.01) for male rats when compared with control.

For animals in the high dosage group, statistically significantly (P<0.01 or P<0.05) higher potassium ion concentration for male rats, lower inorganic phosphorus levels for male rats, higher inorganic phosphorus levels for female rats and lower chloride ion concentration for female rats were recorded when compared with controls.

These changes in ion concentrations were generally low in magnitude and their toxicological importance remains unclear.

The apparent change to significantly higher (P<0.01) bilirubin levels which was recorded for female rats in the high dosage group in comparison with controls was not considered of toxicological importance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Abnormal gait (waddling) was recorded on occasions for two male and one female rats treated at 225 mg/kg/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Testes weights for male rats receiving BBT were lower than controls. This trend appeared to be dosage-related and statistical significance (P<0.05) was achieved for animals in the high dosage group. The change in testes weights for animals in the lower dosage groups was low in magnitude.

In all other instances, organs weights for animals treated with BBT were comparable with that of control animals.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All macroscopic abnormalities were considered to be incidental and revealed no changes considered attributable to treatment with BBT. See table 6 and 7 appendix 6 origina report.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All abnormalities and variations of normality are reported in Appendix 6. There were no changes seen which were reletated to treatment with butyl benzotriazole-sodium salt.

The focal necrosis seen in the liver of 2 male rats receiving 225 mg/kg/day was considered to be sufficient to raise serum GPT and GOT levels but was considered to be unassociated with treatment.

No morphological changes were seen to account for the lower testes weights recoreded for males treated at 225 mg/kg/day.

Similary, no morpholigical changes were seen to account for the raised serum urea nitrogen or creatine levels.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Dose descriptor:

NOEL

Effect level:

ca. 55 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

dermal irritation

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

neuropathology

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

225 mg/kg bw/day (actual dose received)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Following the repeated administration of BBT, 225 mg/kg/day, several changes were noted in the parameters and tissues examined that were considered to be related to treatment.
For rats receiving BBT, 5 or 55 mg/kg/day, no changes were noted that were considered to be of toxicological inportance. The apparent trend to Iower testes weights for male rats in these low dosage groups was very low in magnitude and, in the absence of any histological changes, was considered to be of limited toxicological significance.
Thus, the level of BBT at which no overt signs of toxicity were recorded is considered to be 55 mg/kg/day.

Executive summary:

Butyl benzotriazole-sodium salt (hereafter abbreviated to BBT) was formulated daily as 5.22%, 1.28% and 0.12% v/v solutions in distilled water and administered orally to rats at dosage Levels of 5, 55 and 225 mg/kg/day for 28 consecutive days. Control aninals received distilled water alone. Several changes were noted in the paraneters and tissues examined that were considered to be related to treatment. For rats receiving BBT, 5 or 55 mg/kg/day, no changes were noted that were considered to be of toxicological importance. The apparent trend to lower testes weights for male rats in these low dosage groups was very low in magnitude and, in the absence of any histological changes, was considered to be of limited toxicological significance. Thus, the level of BBT at which no overt signs of toxicity were recorded is considered to be 55 mg/kg/day.