Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Test item name: 2-(2’-hydroxy-5’-methylphenyl)benzotriazole
Purity: 99.9%
Supplier and Lot no.: Wako Pure Chemical Industries, Ltd., lot No.: DWG7396
storage: dark and cool
Verification of identity: The identity of the test substance was confirmed by comparing the infrared absorption spectrum determined using an infrared spectrophotometer (Shimadzu FTIR-8200PC) with the value in the literature.

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Japan, Inc. (Atsugi Breeding Center)
- Age at study initiation: 10 -11 weeks
- Weight at study initiation: 370 to 422 g in males, 225 to 263 g in females
- Fasting period before study: none
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): 15 to 17
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing samples were prepared once a week based on the results of the 8-day stability study conducted at the Japan Bioassay Research Center and stored in a dark place at room temperature and were used within 7 days after preparation.


VEHICLE
- Justification for use and choice of vehicle (if other than water): The substance is not soluble in water, but suspendable in olive oil
- Concentration in vehicle: adjusted to volume of 5 ml/kg bw
- Amount of vehicle (if gavage): 5 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance was checked by comparing the chromatograms determined using high-performance liquid chromatography (Hewlett Packard 1090) before and after use. The concentration and uniformity of the test substance in the dosing samples were checked by the measurement using high-performance liquid chromatography (Hewlett Packard 1090) at the time of the initial preparation.
Duration of treatment / exposure:
Males, 42 days
Females, from 14 days before mating to day 4 of lactation (42 - 53 days)
Females (satellite), 42 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Males, 12 (5 for recovery)
Females, 12; satellite females, 5
Control animals:
yes, concurrent vehicle
Details on study design:
Recovery period:
Males, 14 days
Females (satellite), 14 days
- Dose selection rationale: The doses in this study were determined based on the results of the preliminary study. The dose levels in the preliminary study were determined by setting 1000 mg/kg as the highest dose level as specified in the OECD Guideline for Testing of Chemicals, followed by 300 mg/kg, 100 mg/kg, and 30 mg/kg.

Five each of male and female Crl:CD(SD) rats aged 10 weeks per group received 2-(2’-hydroxy-5’-methylphenyl) benzotriazole orally for 14 consecutive days and were autopsied on the day after completion of administration. As a result, the test substance had no effect on the body weight or general health condition of the males and females in all treatment groups. Both the males and females given 100 mg/kg and higher showed an increase in liver weight and the females also showed an increase in total cholesterol. Increased phospholipid was also noted in the females given 300 mg/kg and higher. Based on these results, 300 mg/kg was selected as the high dose level in this study followed by 100 mg/kg as the intermediate dose level and 30 mg/kg as the low dose level.
Positive control:
not needed

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week
In the observation of females in Week 6 of treatment, however, only the satellite animals were observed to reduce the burden on the animals due to parturition and nursing.
The animals were observed for contact reaction, vocality and easiness of taking out when they were taken out from cages, easiness for handling, body temperature, fur condition, skin color, eye condition and salivation after taking out from cages, posture, activity, alert/searching behavior, gait condition, stereotypical behavior, respiration, and tremor/spasm as well as numbers of events of urination and defecation, grooming and face washing per minute on the work table.

BODY WEIGHT: Yes
- Time schedule for examinations: The males were weighed on Days 1, 8, 15, 22, 29, 36 and 42 of treatment, and the recovery animals were weighed on Days 1, 8 and 14 of recovery.
All the females were weighed on Days 1, 8 and 15 of treatment before mating. After the start of mating, they were weighed on Days 0, 7, 14 and 20 of gestation, on the parturition day and Day 4 after parturition (day 0 and 4 of nursing). The satellite females were weighed on Days 1, 8, 15, 22, 29 and 36 of treatment and Days 1, 8 and 14 of recovery.
On the autopsy day, the body weight was measured in all animals after fasting.

Food consumption: Yes
For the males, food consumption was measured between Days 1 and 8, 8 and 15, 29 and 36, and 36 and 42 in the treatment period and between Days 1 and 8, and 8 and 14 in the recovery period.
For the females, food consumption was measured for all animals before mating between Days 1 and 8, and 8 and 15. After the start of mating, food consumption was measured between Days 0 and 7, 7 and 14, 14 and 20 of gestation and between Days 0 and 4 of nursing. For the satellite females, food consumption was measured between Days 1 and 8, 8 and 15, 29 and 36, and 36 and 43 of treatment and between Days 1 and 8, and 8 and 14 of recovery.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at autopsy
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- How many animals: all
- Parameters checked: Red blood cell count, platelet count, reticulocyte ratio, white blood cell count, mean corpuscular volume (the light scattering method, hereinbefore), hemoglobin concentration (cyanmethemoglobin method), hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration (by calculation, hereinbefore) were determined by the general hematology system (ADVIA 120: Bayer), differential leukocyte classification (by Wright’s stain) using the automatic blood cell analyzer (MICROXHEG-120NA: Omron), and prothrombin time (Quick one-step method) and activated partial thromboplastin time (ellagic acid activation method) using the full automatic blood coagulation tester (Sysmex CA-5000: Sysmex).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at autopsy
- How many animals: all
- Parameters checked: for total protein (biuret method), albumin (BCG method), A/G ratio (calculation), total bilirubin (azobilirubin method), glucose (GlcK・G-6-PDH method), total cholesterol (CE・COD・POD method), triglyceride (MGLP・GK・GPO・POD method), phospholipid (PLD・ChOD・POD method), AST, ALT, γ-GTP, CK (JSCC method, hereinbefore), LDH (SFBC method), ALP (GSCC method), urea nitrogen (urease GLDH method), creatinine (Jaffé method), sodium, potassium, chlorine (ion-selective electrode method, hereinbefore), calcium (OCPC method) and inorganic phosphorus (PNP・XOD・POD method) using an automatic analyzer (Hitachi 7080: Hitachi, Ltd.).

URINALYSIS: Yes
Fresh urine was collected before administration from all males and satellite females in Week 6 and pH, protein, glucose, ketone bodies, bilirubin, occult blood and urobilinogen were tested using a urine test paper (Multistix, Bayer).
Urinalysis was also performed for all male and female recovery animals in Week 2 of recovery as changes suspected of the effect of the test substance administration were found in the males given 100 mg/kg and higher.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: All the males were tested in Week 6 of treatment. As for the females, 5 animals that underwent parturition on the same day or closer days were selected in each group and the tests were performed on the day before autopsy in the combined repeated-dose oral toxicity and reproductive and developmental toxicity study (day 4 after parturition).
- Battery of functions tested: sensory activity, grip strength and motor activity
In the reactivity test, vision, hearing, pain sensation, pupil reflex and aerial righting reflex were examined.
Grip strength was measured using a grip strength meter (MK-380CM, Muromachi Kikai Co., Ltd.). Both the forelimbs and hindlimbs were measured twice and the means were defined as the grip strengths of the individuals.
Locomotor activity was measured for 60 minutes per animal using a locomotor measuring instrument (SCANET MV-10, Melquest).

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Autopsy was performed for the males excluding recovery animals on the day after Day 42 of treatment and for the recovery animals on the day after Day 14 of recovery. The females that underwent parturition were autopsied on Day 5 after parturition and those that did not give parturition were autopsied on the day corresponding to Day 26 of gestation to check presence or absence of conception. The satellite females were autopsied on the day after Day 14 of recovery.

HISTOPATHOLOGY: Yes
The testes, epididymides, seminal vesicles/coagulating gland, prostate (ventral lobe) and ovaries of all animals and trachea, lungs, bone marrow (femur), lymph nodes (axillary, peritoneal, etc.), thymus, spleen, heart, stomach, small intestines (including duodenum), large intestines, liver, kidneys, bladder, pituitary body, thyroid gland, parathyroid gland, adrenal glands, uterus (only in the satellite females), vagina, mammary glands, brain, spinal cord, peripheral nerve (sciatic nerve), muscle, and bone (femur) of 5 animals in each group (in the ascending order of animal numbers, excluding infertile females) and recovery animals were excised, embedded in paraffin, sectioned, and stained in hematoxylin and eosin, and observed under a light microscope.
Other examinations:
Examinations related to reproductive toxicity are described in IUCLID chapter 7.8.1
Statistics:
The chi-square test was performed for the copulation rate, conception rate, birth rate, urinalysis and histopathology results between the control group and each of the treated groups.
For the numeric data obtained in other tests, equal variation was examined preliminary by the Bartlett method using the control group as the basis. The one-way analysis of variance was performed if equal variation was shown and the Dunnett’s multiple comparison was performed for testing the means when a significant difference was detected between the groups. If the variation was not equal, the measured values were ranked across the groups and the Kruskal-Wallis rank test was performed followed by the Dunnett-type multiple comparison if a significant difference was detected between the groups. In the statistical analysis of the numeric data in the recovery animals, the F-test was performed first followed by the Student’s t-test if there was no difference in distribution and by the Aspin-Welch’s t-test if the distribution showed a difference.
The significance level was set at 5% and 1% on both sides and 5% significance level was set as the judgment criteria for the effect of the test substance.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
males only
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
males and females, partly reversible
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In the males, the protein increased in the 100 mg/kg and 300 mg/kg groups in Week 6 and the increase was significant in the 100 mg/kg group compared to the control group. Effects were reversible.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
liver and kidney
Histopathological findings: neoplastic:
no effects observed
Details on results:
No changes were observed in clinical observations, reflex/reaction, grip strength, locomotor activity, body weight and food consumption.

Urinalysis showed increased protein in the males given 100 mg/kg and above, but this change was reversible at the end of the recovery period.

No effect on hematological parameters was observed in any dose group of either sex.

The plasma levels of phospholipids in the group of both sexes given 300 mg/kg, and of total cholesterol in the females given 300 mg/kg were increased. These changes had disappeared by the end of the recovery period.

Relative liver weight was increased in the males given 30 mg/kg and above. Absolute liver weight was increased in the males given 300 mg/kg alone. In the females, both absolute and relative organ weights were increased in the liver of the 100 mg/kg and 300 mg/kg-dosed
groups, and in the kidneys of the 300 mg/kg-dosed group. These changes had disappeared by the end of the recovery period.

In the histological examination, liver and kidney lesions occurred in the both sexes. The hepatic lesions were characterized by increased incidences of hypertrophy of the centrilobular hepatocytes in the males given 300 mg/kg and in the females given 100 mg/kg and above. Theses changes were not observed at the end of the recovery period. The kidney lesions exhibited a gender difference, as characterized by increased incidences of an eosinophilic body of proximal tubules in the males given 300 mg/kg, and hydropic change and regeneration of proximal tubules in the females given 100 mg/kg and above. The eosinophilic body of proximal tubules remained affected in the recovery group of males given 300 mg/kg, whereas female kidney lesions were not observed in the satellite females given 300 mg/kg.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: reversible increase in relative liver weight without histopathology or clinical chemistry correlate.
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: reversible liver and kidney findings at 100 and 300 mg/kg bw.

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
kidney
liver
Treatment related:
yes
Dose response relationship:
yes

Any other information on results incl. tables

Table 1: Relevant organ weights of males

  control 30 mg/kg 100 mg/kg 300 mg/kg recovery control recovery high dose
Liver (g) 13 13.8 13.7

15.1*

13.2 13.5
Liver (%) 2.57 2.81 2.8

3**

2.5 2.7
kidney (g) 3.16 3.12 3.01 3.33 3.31 3.2
kidney (%) 0.63 0.64 0.62 0.67 0.62 0.64
Testes (g) 3.61 3.28** 3.44 3.51 3.32 3.87
Testes (%) 0.71 0.67 0.71 0.7 0.63 0.68
Epididymides (g) 1.263 1.132** 1.147* 1.17 1.21 1.287*
Epididymides (%) 0.25 0.231 0.236 0.235 0.228 0.256*

Table 2: Relevant organ weight of pregnant females

  control 30 mg/kg 100 mg/kg 300 mg/kg recovery control recovery high dose
Liver (g) 10 10.7 11* 11.4** 7.4 8.1
Liver (%) 3.25 3.45 3.66** 3.77** 2.5 2.74
kidney (g) 1.9 1.94 1.96 2.08* 1.95 1.86
kidney (%) 0.61 0.63 0.65 0.69** 0.66 0.64

Table 3: Histopathology of relevant organs in males

  control 30 mg/kg 100 mg/kg 300 mg/kg recovery control recovery high dose
number of animals examined 5 5 5 5 5 5
hepatocellular hyerptrophy, central grade + 0 0 0

4*

0 0
grade 2+ 0 0 0 0 0 0
liver, fatty change, central grade + 1 0 0 0 0 0
Kidney
eosinophilic body grade + 0 0 0

1*

0

2
grade 2+ 0 0 1

4*

0 0
regeneration, proximal tubule grade + 0 0 0 0 0

1*

number of animals examined 7 12 12 7 5 5
Testes
germ cell necrosis grade + 0 1 0 0 0 0
spermatogenic granuloma grade + 0 0 1 1 0 0

Table 4: Histopathology of relevant organs in females

  control 30 mg/kg 100 mg/kg 300 mg/kg recovery control recovery high dose
number of animals examined 5 5 5 5 5 5
liver - hepatocellular hyerptrophy, central grade + 0 0 2

2*

0 0
grade 2+ 0 0 1

2*

0 0
liver - fatty change, central grade + 0 0 0 0 0 0
Kidney
eosinophilic body grade + 0 0 0 0 0 0
grade 2+ 0 0 0 0 0 0
regeneration, proximal tubule grade + 0 0 1 1 0 0
hydrophobic change, proximal tubule grade + 0 0 1 2 0 0

Applicant's summary and conclusion

Executive summary:

For the repeated dose toxicity, the endpoints for deciding NOEL was relative liver weight in the males given 30 mg/kg and above, hydropic change and regeneration of renal proximal tubules in the females given 100 mg/kg and above. For the reproductive/developmental toxicity, there were no effects in both sexes and offspring of 300 mg/kg-dosed group. It was concluded that the NOEL for the repeated dose toxicity of 2-(2’-hydroxy-5’-methylphenyl)benzotriazole was below 30 mg/kg/day for males and 30 mg/kg/day for females.

Findings of this study are in principle consistent with those observed in the chronic feeding studies. In this study, they are observed at similar or slightly lower doses despite the shorter duration. This is attributed to the different application modes (bolus dose in this study versus continous uptake in the chronic feeding studies.)