Registration Dossier

Administrative data

Description of key information

Repeated ingestion by both rats and dogs results in an adaptive enlargement of liver as indicated by the increase in relative liver weight with mild changes in clinical parameters. Higher dosages affect body weight, spleen and kidneys. Both for chronic feeding to rats and subchronic feeding to dogs a NOEL of 1000 ppm was determined. For dogs, this corresponds to a daily average intake of 31.7 mg/kg bw for males and of 43.6 mg/kg bw for females. For rats it is in the range of  47 —  58 mg/kg bw/day.  In a subacute study with gavage dosing, the NOEL was 30 mg/kg bw for females and less than 30 mg/kg bw for males (MHLW 2007). 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
Reason / purpose:
reference to same study
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
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.
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
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
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
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.
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.
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.
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

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
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.)

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Nov. 17, 1980 to Dec. 21, 1981
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study design appears to follow OECD Guideline 409 (1998). Contains sufficient detail to suggest GLP-like characteristics, but no statement of certification (reasonably thorough description of authors, dates, design, results and interpretation). Dated and signed Quality Assurance Inspection statements included.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity in Non-Rodents)
Deviations:
yes
Remarks:
Only one male and one female for all dose groups in recovery phase; recovery groups are optional in OECD 409
GLP compliance:
no
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 31 - 34 weeks
- Weight at study initiation: males = 8.1 kg - 12.4 kg and females = 6.4 kg - 10.7 kg
- Housing: dogs were housed in kennels equipped with underfloor heating.
- Diet (e.g. ad libitum): Pelleted standard diet; 350 g/day/animal.
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 3°C
- Photoperiod (hrs dark / hrs light): 14 hours/10 hours
Route of administration:
oral: feed
Vehicle:
other: TK 10047 was weighed and dissolved with PAG 400 before being added to feed.
Details on oral exposure:
DIET PREPARATION: TK 10047 was weighed and dissolved with PAG 400 into an Erlenmeyer flask on a Mettler balance (Type PC 4400). The pulverized dog food was then homogeneously mixed with the appropriate concentrations of the compound and about 10% of water was added before pelleting to ensure the necessary pellet quality. The pellets were subsequently air-dried.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the initiation of the study, pretest feed samples were analysed for concentration and stability of the test material. The same procedure was undertaken with the food batches applied during the test.
Duration of treatment / exposure:
3 month treatment with a 28-day recovery period for 1 dog/sex/group.
Frequency of treatment:
Once/day. 7 days/week.
Dose / conc.:
1 000 ppm
Dose / conc.:
3 000 ppm
Dose / conc.:
10 000 ppm
No. of animals per sex per dose:
6 dogs/sex/dose.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: not reported.
Positive control:
Not used.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Daily.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretest and Weeks 13 and 17.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Pretest, and Weeks 5, 9, and 13. Also Week 17 for recovery animals.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Pretest and Weeks 5, 9, and 13. Also Week 17 for recovery animals.

URINALYSIS: Yes
- Time schedule for collection of urine: Pretest and Weeks 5, 9, and 13. Also Week 17 for recovery animals.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
At the end of the 3 month test period or after an additional recovery period of 1 month all treated and control dogs were bled under T 61 (Hoechst) anaesthesia. The total weight of each animal was then determined, complete autopsy was performed and following organs were weighed: heart, liver, kidneys, adrenal glands, thyroid, pituitary, gonads and brain.

HISTOPATHOLOGY: Yes
Tissue portions of brain (cerebrum, cerebellum, brainstem), spinal cord, eye, pituitary, salivary gland, heart, thymus, thyroid, lungs (with mainstem bronchi), trachea, spleen, bone with marrow, lymph nodes (axillary and mesenteric), aorta, urinary bladder, oesophagus, stomach, small intestine (duodenum, ileum, jejunum), large intestine (colon, caecum), adrenal glands, pancreas, liver, kidneys, ovaries or testes, uterus or prostate, skeletal muscle, sciatic nerve, gall bladder, skin (mammary area) were fixed in buffered 10 % neutral formalin. The fixed tissue samples were embedded in paraplast and cut at 3-5 ym. The routine stain was haematoxylin and eosin.
Other examinations:
Hearing test was performed monthly.
Statistics:
For each time point and parameter, a uni-variate statistical analysis was conducted. Due to the routine manner of the analysis system parameter free methods were applied. Each treated group was compared to the control group in respect of dispersion and displacement. In addition a trend test was applied considering all groups.
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:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
BODY WEIGHT AND WEIGHT GAIN: The body-weight gain of group 4 (10,000 ppm) was depressed for both male and female dogs (no statistical significance)

FOOD CONSUMPTION AND COMPOUND INTAKE : The food consumption of group 4 (10,000 ppm) was reduced for both male and female dogs.

CLINICAL CHEMISTRY: The alanine-aminotransferase (GPT) increased in group 3 (3,000 ppm) and 4 (10,000 ppm) (p < 0.01). The gamma-glutamyl-transpeptidase (GGT) increased in group 4 (10,000 ppm) (p < 0.01).

PATHOLOGY: One female dog from the 10,000 ppm group was emaciated. No other gross or microscopical changes in the organs and tissues related to the treatment with TK 10047 were noted.

Haematology: Beginning at the fifth week of treatment, animals of the highest dose groups showed values for mean cell volume and mean cell haemoglobin that were slightly lower than the limit of the normal range.

ORGAN WEIGHTS:
For females and males, the relative liver weight was increased by 21% (p < 0.05 compared to control) and 28% at 10000 ppm (p < 0.01 for dose-dependent trend) at the end of the treatment period. This was not observed in animals of the recovery groups.
No experimental significance is attributed to the decreased mean thyroid to body weight ratio at the highest dose group in male dogs (p < 0.05).

OTHER FINDINGS: HEARING TEST: No impairment of the auditory perception was found.
Dose descriptor:
NOEL
Effect level:
1 000 ppm
Sex:
male/female
Basis for effect level:
other: The substances caused a reversible increase in relative liver weight with increased ALT and GGT levels, but without histopathology findings. A reduction in body weight gain occured at the highest dose.
Critical effects observed:
not specified
Conclusions:
The observations made during the study demonstrate that TK 10047 fed to dogs by daily dietary administration for a 3 months period has a "no observable effect level" of 1000 ppm.
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1972-02-24 to 1975-04-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study design appears to follow OECD Guideline 452 (1981). Dated and signed quality assurance inspection statements included (but pre-dates GLP directive). Contains sufficient detail to suggest GLP-like characteristics, but no statement of certification (reasonably thorough description of authors, dates, design, results and interpretation).
Qualifier:
according to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:
no
GLP compliance:
no
Remarks:
Study pre-dates GLP requirments
Limit test:
no
Species:
rat
Strain:
CF-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Carworth Europe, England
- Age at study initiation: 25 days old
- Weight at study initiation: individual body weights are reported.
- Fasting period before study: not reported
- Housing: the rats were housed five to a cage (unless the number was reduced by mortality), in suspended metal cages fitted with wlre-mesh floors
- Diet and water (e.g. ad libitum): free access to tap water and to quantities of powdered laboratory rat food, Spratt's Laboratory Animals Diet No. 2.
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 55
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12


Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- A premix containing 20000 ppm TK 10 047 was prepared each week, and from this the different dietary concentrations were obtained by direct dilution with further quantities of diet. Homogeneity was achieved by mixing for ten minutes in a rotary double-cone blender; all diets were stored until use in heat-sealed, opaque polythene bags.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the diet fed to the rats have been analysed for their content of TK 10 047 by spectrophotometry. The estimated relative reproducibility of this method is - 10% or better. Within the limits of error of sampling technique and of the analytical method, there was good correspondence between the concentrations found in the diets and the nominal values .

Duration of treatment / exposure:
104 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
100 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
300 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
3000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
4 and 6 mg/kg bw ; 14 and 17 mg/kg bw; 47 and 58 mg/kg bw; 142 and 169 mg/kg bw for males and females, respectively
Basis:
actual ingested
No. of animals per sex per dose:
50
Control animals:
yes, plain diet
Details on study design:
- Rationale for animal assignment: random
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: yes
All rats were examined daily; all signs of ill-health and toxicity, together with any behavioural changes, were recorded on the so-called Case History Sheet. Detailed descriptions were made of any skin lesions, cataracts or palpable growths, together with the progression or regression of such lesions.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: weekly for first 12 weeks, and at 4 week intervals thereafter


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


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: Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: assessed in the first instance by inspection of the water bottles


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced, and after 13, 26, 52, 75 and 102 weeks
- Dose groups that were examined: control and group 3


HAEMATOLOGY: Yes
- Time schedule for collection of blood:After 13, 26, 52, 78 and 102 weeks of treatment
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 animals/sex from control group and group 5


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 13, 26, 52, 78 and 102 weeks of treatment
- Animals fasted: No data
- How many animals: 10 animals/sex from control group and group 5


URINALYSIS: Yes
- Time schedule for collection of urine: After 13, 26, 52, 78 and 103 weeks of treatment
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All superficial tissues, including the urinogenital orifices and tail, each pinna, orbit and external auditory meatus, were examined visually and by palpation, for distortion, swelling, or other evidence of tumour formation; similar attention was given to the mammary tracts and the cervical subcutaneous structures. The nares, buccal cavity, tongue, pharynx and auditory region were then examined, and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves . After ventral mid-line incision and skin reflection, all subcutaneous tissues were examined, including regional lymph nodes, mammary and salivary glands . The abdominal viscera were examined before and after removal. The urinary bladder, from all rats dying or killed after 52 weeks of treatment, was briefly distended with fixative in situ, then removed, opened and examined
under low-power magnification. Stomach and caecum were incised and the mucosae scrutinised. The condition of the thoracic viscera was noted, with due attention to the thymus and lymph nodes; the heart was incised and each chamber examined. The oesophagus, intestines, and uterus were incised to expose the mucosae and subjected to visual appraisal in toto. The lungs were remoyed and all pleural surfaces examined under
suitable illumination, while the liver and kidneys were sectioned at intervals of a few millimetres; any lesion suggestive of neoplasia was noted, including details of location, size and multiplicity. Any abnormalities in the appearance and size of the gonads, adrenals, thyroids, intra-abdominal lymph nodes and accessory reproductive organs were recorded. Any evidence of adhesion, invasion or other interaction between presumptive neoplasia and the adjacent structures was noted.

HISTOPATHOLOGY: Yes
(all macroscopically observed lesions suggestive of neoplasia, for every animal; all macroscopically abnormal tissues from decedents in an attempt to ascertain cause of death; 10 rats of each sex from control and 3000 ppm killed at 104 weeks). The following organs were fixed and preserved: adrenals, aorta, brain (cerebrum,cerebellum and pons), caecum, colon, duodenum, eyes, femur, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (cervical and mesenteric), optic nerve, ovaries, pancreas, pituitary, prostate, rectum, sciatic nerve, skeletal muscle, skin, spinal cord, stomach (cardiac, fundus and pylorus), testes, thymus, thyroid, urinary bladder, uterus, spleen

All nodules, tissue masses and otherwise macroscopically abnormal tissues were routinely preserved and processed along with samples of adjacent tissue where appropriate.
Other examinations:
none
Statistics:
Student's 't' test was employed to assess the significance of intergroup differences where the data suggested a treatment related response . Differences in tumour incidence were compared by a 2 x 2 contingency test used as a one tailed test, computing the exact probability of the observed tumour distribution occurring or one which is more extreme.
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:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
no effects observed
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 examined
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:
BODY WEIGHT AND WEIGHT GAIN
-Slightly decreased bodyweight gain among males treated with 3000 ppm during the second year of treatment (P < 0 .05) (mean body weight decrease of 122 g during weeks 80 - 104)
FOOD CONSUMPTION AND COMPOUND INTAKE
-slightly reduced food intake among females treated with 3000 ppm during the period 53 to 80 weeks of treatment (P < 0.05 )

Organ Weights:
Organ weight analysis performed on rats killed after 104 weeks of treatment revealed slightly heavier thyroid/parathyroid weights among treated animals, which only attained a level of statistical significance among males treated with 1000 ppm and females treated with 1000 or 3000 ppm when related to bodyweight, and among females treated with 100, 1000 or 3000 ppm when related to brainweight.
After statistical re-evaluation of significance levels in comparison with control using Williams' test:, for the males, no significant treatment effects weredetected. For females significant increases in both absolute kidney (p < 0.05) and thyroid weights (p < 0.01) were found in both 1000 and 3000 ppm treatment groups. Although statistically significant, there is no dose-response relationship. As the values obtained for treated rats were within the normal range for CFY rats, the differences were considered to have arisen fortuitously.

Dose descriptor:
NOEL
Effect level:
1 000 ppm
Sex:
male/female
Basis for effect level:
other: Reduced body weight gain
Dose descriptor:
NOEL
Effect level:
> 47 - < 58 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Reduced body weight gain
Critical effects observed:
not specified
Executive summary:

The NOEL is 1000 ppm. This study was discussed in detail with the US FDA for the purpose of food additive petition during 1983 - 1984.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
47 mg/kg bw/day
Study duration:
chronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In the oldest study (Feron 1966) a total of 80 Wistar rats, 10 males and 10 females per dose group, were feed with 0, 0.2, 1.0 and 5.0 % for three months. It is described in detail, but not all endpoints required in the respective OECD testing guideline were assessed. Ophthalmological examinations were not performed and clinical biochemistry parameters were not evaluated.

The findings are compared to common strain characteristics, but no historical control data included in the report. No individual data for each animal and no absolute organ weights are given. Growth rate, food consumption and food efficiency of the rats fed 1 and 5 % in the rations were decreased only in the first two weeks of the study and these effects disappeared thereafter. There were no relevant changes in laboratory parameters (haematology, clinical chemistry) except from the males of the 5 % level which showed a decreased number of erythrocytes and an increased number of leukocytes. There was a dose dependent tendency of organ weight increase of the liver, kidney and spleen. The histopathological investigation revealed kidney lesions, however, nephrotic changes were noted only in the males of the 1.0 and 5 % feeding level group.

In liver, slight centrilobular hypertrophy of parenchymal cells was observed in a few animals: At 5%, each 5 males and females; 1% - 7 males and 1 female; 0.2% - 2 males; control: none. No differences in glycogen and fat content were observed. Liver findings are described as "Central zones of the lobules much less stainable than the peripheral zones. In the central zones somewhat enlarged parenchymal cells with slightly swollen nuclei". Two males of the highest dose group showed distinct pathological liver changes consisting of bile duct proliferation, an increased number of Kupffer cells, extended sinusoids and often enlarged parenchymal cells with irregular nuclei. This observation was not consistently seen. No abnormalities were seen in the following organs at microscopic examination: brain, pituitary, spinal cord, femoral nerve, bone marrow, gastrointestinal tract, testis, epididymis, ovary, adrenal, and salivary gland.

The NOEL was the 0.2 % concentration in the diet, corresponding to a daily intake of approximately 150 mg/kg bw.

 

 

 

In the subchronic feeding study with beagles (Gfeller 1981a), the substance was administered to 48 dogs (6 males and 6 females per dose group) in the diet for 3 months at dosages of 0, 1'000, 3'000 and 10'000 ppm. The study design appears to follow OECD Guideline 409 (1998) and the report contains sufficient detail to suggest GLP-like characteristics, but no statement of certification. However dated and signed Quality Assurance Inspection statements are included. For each dose group, a recovery group with each one male and one female were included.

No deaths occurred during the study. The food consumption of group 4 (10,000 ppm) was reduced for both male and female dogs. At the 3'000 and 10'000 ppm levels, the animals showed changes in blood chemistry parameters: Elevated levels of the alanin-aminotransferase (GPT) and gamma-glutamyl-transpepdidase (GGT). For females and males, the relative liver weight was increased by 21% (p < 0.05 compared to control) and 28% at 10000 ppm (p < 0.01 for dose-dependent trend) at the end of the treatment period. This was not observed in animals of the recovery groups. No experimental significance is attributed to the decreased mean thyroid to body weight ratio at the highest dose group in male dogs (p < 0.05).

No other gross or microscopical changes in the organs and tissues related to the treatment were noted. No impairment of the auditory perception was found. The NOEL was 1'000 ppm corresponding to a daily average intake of 31.7 mg/kg bw for males and of 43.6 mg/kg bw for females.

 

The key study is the 104 weeks chronic toxicity study with rats (Hunter 1975) because its design is consistent with the OECD testing guideline for chronic toxicity 452 and the reporting detail is sufficient for assessment. The study was performed prior to introduction of GLP. A total of 500 CFY rats (50 male and 50 female per dose group) were treated with the test article in the diet, at dose levels of 0, 100, 300, 1000 and 3000 ppm in the diet per day. Chemical analysis of the test item in the feed confirmed the nominal concentrations.

No clinical symptoms and no substance-related mortality were observed. At 3000 ppm a small reduction of body weight gain among males during the second year of treatment and slightly reduced food intake among females during the period 53 to 80 weeks of treatment were observed. Organ weight analysis performed on rats killed after 104 weeks of treatment revealed slightly heavier thyroid/parathyroid weights among treated animals, which only attained a level of statistical significance among males treated with 1000 ppm and females treated with 1000 or 3000 ppm when related to bodyweight, and among females treated with 100, 1000 or 3000 ppm when related to brain weight.

After statistical re-evaluation of significance levels in comparison with control using Williams' test, for the males, no significant treatment effects were detected. For females significant increases in both absolute kidney (p < 0.05) and thyroid weights (p < 0.01) were found in both 1000 and 3000 ppm treatment groups. Although statistically significant, there is no dose-response relationship. As the values obtained for treated rats were within the normal range for CFY rats, the differences were considered to have arisen fortuitously.

Based on the above findings it was concluded that 1000 ppm (47-58 mg/kg bodyweight/day) was the no-effect level. The findings at 3000 ppm are difficult to assess.

 

There are two further reports on subchronic (Carlson 1969) and subacute (Gysling 1958) exposure of rats via feed which are inadequate in quality. The results of both studies are consistent with the findings of the valid studies.

A subacute study with gavage application was performed in 2006 on behalf of MHLW. A summary report in Japanese language was prepared in 2008. This is information is compiled from the english translation. The GLP compliant study followed OECD testing guideline 422 with satellite groups for a 14 -day recovery period. Olive oil was used as vehicle. The outcome of this study confirms the effects on liver and kidney. A NOEL of 30 mg/kg bw was reported for females, with adverse findings on liver and kidney at doses of 100 mg/kg bw and 300 mg/kg bw. For males, relative liver effects were increased at the lowest dose group of 30 mg/kg bw. The puplic information does not include a table with relative organ weights. Considering that neither absolute liver weights nor body weights were affected at this dose level and that histopathology findings for liver were only observed at 300 mg/kg bw, 30 mg/kg bw are considered to be the overall NOAEL.

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 recovered by the discontinuance of dosage.

No effect on hematological parameters was observed in any dosed group of either sex. The plasma levels of phospholipid 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 dosed group of 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.

The lower effect levels in this study are attributed to bolus dosing, as the substance is efficiently taken up after ingestion. The NOEL derived from the chronic feeding studies (47 mg/kg bw) is considered to be relevant for DNEL derivation as it represents a more realistic human exposure situation.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Most relevant dosing scheme (feed more appropriate compared to gavage).

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Justification for classification or non-classification

 Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for oral repeated dose toxicity under Regulation (EC) No. 1272/2008