Sodium 1H-benzotriazolide

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: study performed by regulatory organisation, but no guideline followed

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1978

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 451

Deviations:

yes

Remarks:

only two dose groups

Principles of method if other than guideline:

Groups of animals are administered to the test substance for 78 weeks and observed for 26 weeks additionally.
Two dose groups and a control group are chosen per sex.
Body weights and clinical signs are collected and killed animals are pathologicaly examined

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1H-1,2,3-Benzotriazol

IUPAC Name:

1H-1,2,3-Benzotriazol

Constituent 2

Reference substance name:

Benzotriazole

EC Number:

202-394-1

EC Name:

Benzotriazole

Cas Number:

95-14-7

Molecular formula:

C6H5N3

IUPAC Name:

1H-Benzotriazole

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): 1H-Benzotriazole
- Molecular formula (if other than submission substance): C6H5N3
- Molecular weight (if other than submission substance): 119.14 g/mol
- Smiles notation (if other than submission substance): c12c(cccc1)N=NN2
- InChl (if other than submission substance): 1S/C6H5N3/c1-2-4-6-5(3-1)7-9-8-6/h1-4H,(H,7,8,9)
- Structural formula attached as image file (if other than submission substance):
- Distributor: Aldrich Chemical Company, Milwaukee, Wisconsin
- Analytical purity: >99 %
- Purity test date:
- Lot/batch No.: 122917 and 030737
- Storage condition of test material: 4 °C

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

second test animals:
Species : mice
Strain: B6C3F1
Sex: male/ female

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Wilmington, Massachusetts
- Age at study initiation: 42 days (rat), 44 days (mice)
- Weight at study initiation: ca. 90 g (rat), ca. 20 - 25 g (mice)
- Fasting period before study: no
- Housing: steel wire-mesh cages, solid polycarbonate cages
- Diet: ground Wayne Lab Blox animal feed, ad libitum
- Water: Tap water (0.75 - 1 ppm chlorine), ad libitum
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-24 °C
- Humidity (%): no data
- Air changes (per hr): 6
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Mixing a weighed amount of chmical with an aliquot of food in a mortar.
When the premix is homogeneous, it is blended with the remaining feed and
mixed for 20 min.

DIET PREPARATION
- Rate of preparation of diet (frequency): once a week
- Mixing appropriate amounts with (Type of food): Ground Wane Lab Blox animal feed
- Storage temperature of food: 4 °C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis by ultraviolet spectroscopy at intervals of 2 - 6 weeks after mixing.
Concentrations were within 25 % of the theoretical value

Duration of treatment / exposure:

78 weeks

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
6700 ppm
Basis:
other: rats, nominal in diet, low-dose group; time-weighted average dose

Doses / concentrations 2

Remarks:

Doses / Concentrations:
12100 ppm
Basis:
other: rats, nominal in diet, high-dose group; time-weighted average dose

Doses / concentrations 3

Remarks:

Doses / Concentrations:
11700 ppm
Basis:
other: mice, nominal in diet, low-dose group; time-weighted average dose

Doses / concentrations 4

Remarks:

Doses / Concentrations:
23500 ppm
Basis:
other: mice, nominal in diet, high-dose group; time-weighted average dose

No. of animals per sex per dose:

50

Control animals:

yes

Details on study design:

- Dose selection rationale: based on subchronic study

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: every 2 weeks in the first 12 weeks, then every month

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Statistics:

Probabilities of survival are estimated by the product-limit procedure of Kaplan and Meier (1958).
Statistical analyses for a possible dose-related effect on survial use the method of Cos (1972) and Tarone's (1975) extensions.
One-tailed P values have been reported for all tests except the departure from linearity test, which is only reported when its two-tailed P value is less than 0.05. The one-tailed Fisher exact test (Cox 1970) is used to compare the tumor incidence of a control group with that of a group of dosed animals at each dose level.
The Cochran-Armitage test for linear trend in proportions, with continuity correction (Armitage 1971) is also used

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

lowered mean body weights

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Rats:
Eye discoloration or inflammation up to two animals per group (also control).
Inflammation of the posterior ventral surface in one high-dose male.
Alopecia in one control male.
Survival ranged from 64 to 86 %, with the lower values observed in the control groups (For details see table 1)

Mice:
Alopecia in 1 low-dose female, 2 high-dose females, 27 control males, 30 control females
distension of the stomach in 1 control male and female
swelling and inflammation in the anal region in 1 low-dose male
abscess in the anal region in 1 high-dose male
poor balance at the end of study in 1 high-dose female
Survival ranged from 60 to94 %, with the lower values observed in the control groups (For details see table 2)

BODY WEIGHT AND WEIGHT GAIN
Rats:
Mean body weights of the dosed male and female rats were lower than those of the corresponding controls throughout most of the bioassay. A dose-related effect was generally indicated by the data for each sex, except for the last 24weeks in the females when the mean body weights of both the low- and high-dose groups gradually rose toward those of the controls.

Mice:
Mean body weights of the dosed male and female mice were lower than those of the corresponding controls throughout most of the bioassay, and a dose-related effect was indicated by the data for each sex.

PATHOLOGY (gross pathology, histopathology)
Rats:
Hepatic lesions observed in male and female rats consisted of neoplastic nodules of the liver in five males and two females from the high-dose groups. No animals in the control or low-dose groups had neoplastic nodules. Morphologic features of the neoplastic nodules were similar to those described in the literature. Neoplastic nodules were spherical lesions up to several liver lobules in size composed of plates of hepatocytes which were sharply demarcated and compressed the adjacent normal parenchyma. Hepatocytes within these nodules showed varying degrees of cytoplasmic change, including clear cell, eosinophilic, and basophilic alterations. Mitotic figures and varying degrees of nuclear atypia including multinucleation and hyperchromasia were noted. Brain lesions observed in rats as early as week 21 consisted of an oligodendroglioma in one low-dose male and gliomas in two low-dose males and one high-dose female. These tumors were not present in the control animals. In female rats a non-dose-related increase in the incidence of C-cell adenomas and carcinomas of the thyroid was noted. The incidence in the controls was 0/43, the low-dose group 5/43, and
the high-dose group 3/50. Most of the remaining neoplasms occurred randomly in control and dosed groups, and their incidences and types, with few exceptions, were similar to those observed historically in Fischer 344 rats. With the possible exception of the liver in male rats and the brain in male and female rats, no particular organ or system seemed to be the target of this chemical. One high-dose female had a rare and unusual neoplasm which appeared to be a primary nonchromaffin paraganglioma of the heart. The mass was visible microscopically in the wall of the left ventricle and was composed of sheets of cells arranged in small organoid-like packets. The cells had large oval and round vesicular nuclei, high nuclear cytoplasmic ratios, and a light gray-blue cytoplasm. The endocrine-like appearance of the mass suggested it might be a chemodectoma, since all efforts to
demonstrate chromaffin granules were negative. A variety of degenerative, proliferative, and inflammatory lesions were observed in the control and dosed rats, but none were believed to be related to administration of 1H-benzotriazole.

Mice:
In female mice, a non-dose-related increase in the incidence of alveolar/bronchiolar adenomas and carcinomas was noted. The incidence in the controls was 0/49, the low-dose group 10/49, and the high-dose group 4/49. The remaining neoplasms occurred randomly in control and dosed groups, and their incidence and type, with few exceptions, were similar to those observed historically in B6C3F1 mice. No particular organ or system seemed to be the target of this chemical. A variety of degenerative, proliferative, and inflammatory lesions were seen in the control and dosed mice.

Effect levels

1

Dose descriptor:

LOAEL

Effect level:

ca. 6 700 ppm

Based on:

test mat.

Remarks:

nominal in diet, time weighted average in rats

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Mean body weights of the dosed male and female (both species) were lower than those of the corresponding controls throughout most of the bioassay

2

Dose descriptor:

LOAEL

Effect level:

ca. 11 700 ppm

Based on:

test mat.

Remarks:

nominal in diet, time weighted average in mice

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Mean body weights of the dosed male and female (both species) were lower than those of the corresponding controls throughout most of the bioassay

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Survival of rats

Group	absolute numbers	Survival
high-dose males	36/50	72 %
low-dose males	34/50	68 %
control males	32/50	64 %
high-dose females	43/50	86 %
low-dose females	40/50	80 %
control females	36/50	72 %

Table 2: Survival of mice

Group	absolute numbers	Survival
high-dose males	42/50	84 %
low-dose males	36/50	72 %
control males	33/50	66 %
high-dose females	47/50	94 %
low-dose females	39/50	78 %
control females	30/50	60 %

The recalculation of the dosage from ppm in food to mg/kg bw/day is performed according to Lehman, A.J. (1954). Association of Food and Drug Officials Quarterly Bulletin 18: 66.

rat: 6700 ppm = 335 mg/kg bw/day

12100 ppm = 605 mg/kg bw/day

Applicant's summary and conclusion

Conclusions:

Concerning the body weight gain, a LOAEL of 6700 ppm in rats and 11700 ppm in mice was observed. Organo-toxicological effects related to the substance were not observed. The LOAEL of 6700 ppm is equivalent to 335 mg/kg bw/day, 11700 ppm to 1755 mg/kg bw/day.
Although only two dose groups were administerd, the study is rated as reliable with restrictions. This deviation is considered as acceptable as long as a reasonable Assessment Factor is applied in order to account for Dose-response relationship.
Although administration of this chemical to rats may be associated with neoplastic nodules of the liver in male Fischer 344 rats, there was no convincing evidence, based on the histopathologic examination, that administration of 1H-benzotriazole was carcinogenic under the conditions of this bioassay.
In the B6C3F1 mouse, based on the histopathologic examination, there was no evidence for the carcinogenicity of IH-benzotriazole under the conditions of this bioassay.

Executive summary:

For Benzotriazole a well-conducted in vivo study is available showing a LOAEL of 325 mg/kg bw / day for the chronic repeated dose toxicity. The study was designed as carcinogenicity study.

This means that a similar result for Sodium Benzotriazolate can be anticipated.

 Sodium Benzotriazolate has a LOAEL of 325 mg/kg bw/day for chronic repeated dose toxicity based on a read-across approach.

 

A DNEL for oral, dermal and/or inhalation route can be based on this information.

Classification and labelling are / are not needed for this endpoint.

A risk characterisation will be performed because the substance is classified for oral toxicity.