A mixture of: α-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyl-ω-hydroxypoly(oxyethylene); α-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyl-ω-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyloxypoly(oxyethylene)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Sep. 23, 1987 to Dec. 12, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP. Histopathological examinations were not performed for all tissues.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1981 guideline followed, reliability scoring based on 1998 guideline

Deviations:

yes

Remarks:

histopathological examinations were not performed for all tissues

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: α-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyl-ω-hydroxypoly(oxyethylene); α-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyl-ω-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyloxypoly(oxyethylene)

EC Number:

400-830-7

EC Name:

A mixture of: α-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyl-ω-hydroxypoly(oxyethylene); α-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyl-ω-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyloxypoly(oxyethylene)

Cas Number:

104810-48-2

IUPAC Name:

14-({3-[3-(2H-1,2,3-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl]propanoyl}oxy)-3,6,9,12-tetraoxatetradecan-1-yl 3-[3-(2H-1,2,3-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl]propanoate; 14-hydroxy-3,6,9,12-tetraoxatetradecan-1-yl 3-[3-(2H-1,2,3-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl]propanoate; tris(17-({3-[3-(2H-1,2,3-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl]propanoyl}oxy)-3,6,9,12,15-pentaoxaheptadecan-1-yl 3-[3-(2H-1,2,3-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl]propanoate); tris(17-hydroxy-3,6,9,12,15-pentaoxaheptadecan-1-yl 3-[3-(2H-1,2,3-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl]propanoate); 2-(2-hydroxyethoxy)ethan-1-ol; 2-({3-[3-(2H-1,2,3-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl]propanoyl}oxy)ethyl 3-[3-(2H-1,2,3-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl]propanoate; 2-[2-(2-hydroxyethoxy)ethoxy]ethan-1-ol; 2-[2-(2-hydroxyethoxy)ethoxy]ethyl 3-[3-(2H-1,2,3-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl]propanoate; 2-hydroxyethyl 3-[3-(2H-1,2,3-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl]propanoate; 2-methoxyethan-1-ol; 2-{2-[2-({3-[3-(2H-1,2,3-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl]propanoyl}oxy)ethoxy]ethoxy}ethyl 3-[3-(2H-1,2,3-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl]propanoate; 20-({3-[3-(2H-1,2,3-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl]propanoyl}oxy)-3,6,9,12,15,18-hexaoxaicosan-1-yl 3-[3-(2H-1,2,3-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl]propanoate; 20-hydroxy-3,6,9,12,15,18-hexaoxaicosan-1-yl 3-[3-(2H-1,2,3-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl]propanoate; 3,6,9,12,15,18,21-heptaoxatricosane-1,23-diol; 3,6,9,12,15-pentaoxaheptadecane-1,17-diol; 3,6,9,12-tetraoxatetradecane-1,14-diol; bis(ethane-1,2-diol)

Details on test material:

- Lot/batch No.: EN 20043.42
- Composition of test article: confirmed and given in analytical certificate in the study report
- Storage condition of test material: undiluted at room temperature; diluted at 4 °C

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain as stated in the report: Sprague Dawley Crl CD (SD) BR
- Source: Centre d'Elevage Charles River (76140 Saint-Aubin-les-Elbeuf, France)
- Age at study initiation: 6 weeks
- Weight at study initiation: approx. 158g females and approx. 200 g males
- Housing: 2 same-sex animals from the same group per cage (wire-mesh, 43 x 21.5 x 18 cm); cages were placed in order vertically on racks and racks were moved around in a clockwise manner fortnightly.
- Diet ad libitum: pellet diet (ref. A04 C, U.A.R., 913 Villemoisson0sur-Orge, France)
- Water ad libitum: tap water filtered with 0.22 micron F.G. Millipore filters
- Acclimation period: 11 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 13
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Dilutions of test article in polyethylene glycol were prepared and homogenized with a magnetic stirrer. The preparation was performed twice a week by C.I.T. Pharmacy according to the stability results obtained before the beginning of the study.

VEHICLE
Concentration in vehicle: 0.4, 1, 2, 10 mg/ml PEG
Amount of vehicle: 5 ml/kg body weight/day (amount of test substance given was individually adjusted every week to the most recent body weight)
Lot/batch no.: Batches Nos. 82202 and 87163

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the dosing solutions in polyethylene glycol 300 was checked before the beginning of the study on days 0, 1, 3, and 7. The concentrations achieved were examined on week 1, 4, 8 and 12 of the study by spectrophotometry at 301 nm. The substance was stable in polyethylene glycol under study conditions and the achieved concentrations were comparable to the nominal concentrations (the difference was always lower than 9 %).

Duration of treatment / exposure:

After 91 days 12 animals of control and 20 animals from the 10 mg/kg bw/d group were saved for a 30 day recovery period. Remaining males were sacrificed after 92 days and remaining females were sacrificed after 93 days of treatment.

Frequency of treatment:

once daily, 7 days per week

Doses / concentrations

Remarks:

Doses / Concentrations:
2, 5, 10, 50 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

0 mg/kg bw/day - 16/sex
2 mg/kg bw/day - 10/sex
5 mg/kg bw/day - 10/sex
10 mg/kg bw/day - 20/sex
50 mg/kg bw/day - 10/sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not reported
- Rationale for animal assignment: random
- Details on satellite groups: after the 91-day treatment, the first surviving 6 males and 6 females from the control group and 10 males and 10 females from the 10 mg/kg bw/day group were saved for a 30-day recovery period.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CLINICAL SIGNS AND MORTALITY
The animals were observed daily for clinical signs and twice daily for mortality.
BODY WEIGHT
The weight of each animal was recorded 7 days prior, on the first day of treatment and weekly during the treatment and the recovery period.
FOOD CONSUMPTION
The food consumption by the animals of each cage was recorded weekly during treatment and recovery.
OPHTHALMOSCOPIC EXAMINATION
The animals from the control and 50 mg/kg bw/day group were subjected to ophthalmoscopic examinations before the first treatment and in week 13.
HAEMATOLOGY AND CLINICAL CHEMISTRY
In week 13 and after recovery (week 17) blood was collected under light ether anesthesia from the orbital sinus in overnight fasted animals for haematology and clinical chemistry. Parameters given in table 1 were examined.

Sacrifice and pathology:

GROSS PATHOLOGY
All surviving animals were sacrificed 18 hours after the last administration and after an 18 h fasting period after end of the recovery period, respectively. Macroscopic examinations of all animals including those that died during the study were performed. Tissues listed in table 2 were preserved in 10 % buffered formalin.
ORGAN WEIGHTS
The following organs from all animals sacrificed at termination were weighed: Adrenals, kidneys, liver, ovaries, spleen, testes, seminal vesicles and prostate. Body and organ weights were not recorded at necropsy in the animals that were found dead.
HISTOPATHOLOGY
Histopathological examinations were performed on organs given in Table 2 of all animals from the control and 50 mg/kg bw/day groups. Samples were embedded in paraplast and were stained with hemalum and eosin. All tissues not required for microscopic examinations were kept in fixative. At 2, 5 and 10 mg/kg bw/day all tissues with macroscopical abnormalities, lungs, liver, kidneys as well as seminal vesicles, prostate, testes and epididymides from the 10 mg/kg bw/d group were examined at the end of treatment. At the end of the recovery period all tissues with macroscopical abnormalities and liver, lungs, kidneys, seminal vesicles, prostate, testes and epididymides were examined in all animals.

Statistics:

A normal distribution of values in the samples was checked using KOLMOGOROV-SMIRNOV's test. In the case of an abnormal distribution, this test was performed after logarithmic transformation of the values. If a significant heterogeneity persisted after logarithmic transformation, the analysis of variances was not performed. A comparison between the treated groups and the control group in order to prove a treatment related difference was made using MANN-WHITNEY's test.
In case of normal distribution of values according to the normal law, an analysis of variances was made using BARTLETT's test (more than two samples) or FISHER's test (two samples).
A comparison between the treated and control groups in order to prove a treatment related difference was made using: DUNNETT's test, if no significant heterogeneity of the variances was established; MANN-WHITNEY's test, in the case of significant heterogeneity of the variance.
A slight difference may appear from time to time with the rounded number of the mean and standard deviation between the individual values and the summary section of the statistical tests. These differences are due to the use of different calculators in data processing, which are not processed the same way in the central memory. These differences, when they occur, are always seen in the last decimal and do not affect the scientific value of the results.

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):

transient reduced body weight in males of the high dose group.

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

ALP, albumin, decrease in alpha-1, alpha-2, beta and/or gamma globulins

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

liver, increased at 5 mg/kg bw and above

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

dose dependently enlarged livers in males

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

slight liver hypertrophy in males of the high dose group.

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS
No treatment related clinical signs were observed. Clinical signs like piloerection, loss of body weight, chromorhinorrhea, chromodacryorrhea, round back, loud breathing or dyspnea, and decrease in activity were seen sporadically in animals of all groups. They were not considered to be of toxicological significance. Other clinical signs like cutaneous lesion or hair loss were considered to be usually observed in the laboratory rat.
MORTALITY
There were no deaths during the recovery period. Mortality during the treatment period was as follows:
0 mg/kg bw/day - 4/16 males (25%) and 1/16 females (6%)
2 mg/kg bw/day - 0/10 males (0%) and 1/10 females (10%)
5 mg/kg bw/day - 1/10 males (10%) and 0/10 females (0%)
10 mg/kg bw/day - 2/20 males (10%) and 0/20 females (0%)
50 mg/kg bw/day - 0/10 males (0%) and 1/10 females (10%)
The factors contributing to mortality were, in most cases, the same in controls and treated animals. Two deaths were due to experimental mistakes, and, in males, the mortality rate was higher in the controls than in the treated groups. Therefore, it was not considered to be of biological significance.

BODY WEIGHT AND WEIGHT GAIN
The male body weight was slightly but significantly decreased from Week 6 in males at 50 mg/kg bw/day (ranging from 94 to 84 % of control). This effect was not significant at week 13 (94%) but was considered to be treatment related. Other effects on body weight were either not dose dependent or did not reach statistical significance and were therefore not considered treatment related.

FOOD CONSUMPTION
Food consumption of treated males was similar to control males without any statistically significant difference (except in week 4 in the 50 mg/kg/day group). The food consumption of treated females was also similar to control females. The difference was occasionally statistically significant in week 7 in the 5 mg/kg/day group. These variations were not considered to be of biological significance.

OPHTHALMOSCOPIC EXAMINATION
Upon ophthalmoscopic examination no test article related abnormality was noted. However, minor changes like cornea vacuolization and persistence of hyaloid vessel were seen at 0 and 50 mg/kg bw/day, before the start of treatment and at week 13. Since these findings are not uncommon in the laboratory rat (Heywood R, Laboratory animals 7, 19-27, 1973) they were considered to be not treatment related.

HAEMATOLOGY
Findings like higher thrombocyte values in males in all treated groups, decreased mean haemoglobin content (males) and mean value of packed cell volume (male and female), lower quick time values, increased lymphocyte count or different white blood cell count (male and female) were seen. These findings were either within the historical controls, the significance was due to high individual values or changes were not dose dependent. Therefore, these findings were considered to be of no biological significance.
After recovery, no differences were observed in the 10 mg/kg bw/day group except for lower values in packed cell volume, mean cell volume, and mean cell haemoglobin, which were not considered to be of biological significance.

CLINICAL CHEMISTRY
Increased alkaline phosphatase activity was seen in males and females at 50 mg/kg bw/day (factor of 1.4 and 2.9 for females and males, respectively) and in males at 10 mg/kg bw/day (factor of 1.5). An increase in albumin levels was noted in males and females from the 10 and 50 mg/kg bw/day groups, with a decrease in alpha-1, alpha-2, beta and/or gamma globulins. These decreases were also noted in females from the 2 and/or 5 mg/kg bw/day groups. Further details are given in table 3. Although these changes were not accompanied by any organic lesions in the organ that could be the source, they were considered related to the administration of the test article. At the end of the recovery period, these abnormalities were totally reversible.
Other significant changes were:
Decreased sodium levels (142 and 142 mmol/l compared to 144 mmol/l at control) and chloride levels (105 and 106 compared to 109 mmol/l) in males at 10 and 50 mg/kg bw/d and increased levels of inorganic phosphorus and glucose in males at 50 mg/kg bw/d (2.8 versus 2.3 and 8.7 vs. 7.3 mmol/l at control, respectively). Males also had a lower bilirubin level (2 and 2 vs. 3 µmol/l at control) as well as increased albumin/globulin ratios (1.7 and 2.1 vs. 1.3 at control) and alkaline phosphatase activity (224 and 428 vs. 148 UI/l) at 10 and 50 mg/kg bw/d.
Further changes in females consisted of decreased calcium levels at 2, 5 10, 50 mg/kg bw/d (2.8, 2.7, 2.8, 2.7 vs. 2.9mmol/l at control), decreased total bilirubin level (2, 2, 2 vs. 3µmol/l at control) at 5, 10 and 50 mg/kg bw/day, increased albumin/globulin ratios at 2, 5, 10 and 50 mg/kg bw/d (2.1, 2.1, 2.2, 2.6 vs. 1.7 at control), increased alkaline phosphatase (ALP) activity at 50 mg/kg bw/d (108 vs. 78 UI/l at control), as well as decreased aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) activity at 2, 5, 10, 50 mg/kg bw/d (54, 59, 62, 59 vs. 75 UI/l at control and 28, 29, 24 and 25 vs. 53 UI/l at control, respectively).
The decreases in ALAT and ASAT mean group values were due to individual high values in the control group. The individual values in the treated groups were comparable to those of the controls and to the normal historical values, therefore they were considered to be of no biological significance. Other changes seen in clinical biochemistry like decreased cholesterol in females at 10 mg/kg bw/d were not dose dependent.
All effects at 10 mg/kg bw/day were partially (beta-globulin, alpha-1-globulin) or fully (ALP activity, albumin, alpha-2-globulin) reversible.

GROSS PATHOLOGY
Macroscopic examination at the end of treatment revealed dose dependently enlarged livers in males (1/10, 7/10 and 10/10 at 5, 10, and 50 mg/kg bw/day). These findings were correlated with hepatic cell hypertrophy in 3/10 males from the 50 mg/kg bw/day group at the end of treatment. No abnormality was observed in females and in both sexes after the recovery period. The hepatotropic effect observed at all dose levels in males was considered to indicate an adaptive change of a functional nature.

ORGAN WEIGHTS
A dose dependent increase in the net and relative liver weight in males and females from all treated groups was noted at the end of the treatment period. These variations were correlated with enlarged livers and hepatic cell hypertrophy at 50 mg/kg bw/day. They were not present at the end of the recovery period. A decrease in the net adrenal weight and an increase in the relative kidney and testes weights were noted in males from the 50 mg/kg bw/day group. In the absence of any microscopic findings, these changes could be related to the decrease in body weight in these animals. Other findings like a significantly increased relative and absolute spleen weight at 5 mg/kg bw/day in females was not associated with histopathological findings and was considered to be of no biological significance.

HISTOPATHOLOGY
A minimal to slight hepatic cell hypertrophy was found in 3/10 males at 50 mg/kg bw/day. This change could be related to the administration of the test substance. The incidence, severity, and morphological characters of other microscopic changes like congestions of adrenal glands in one female each of the 0, 2 and 10 mg/kg bw/day group did not suggest a treatment relationship.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The substance affects the liver. As shown in separate mechanistic studies, it is a potent peroxisome proliferator.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 3: Selected biochemical parameters

MALES after 13 week treatment
Dose mg/kg bw/day	Albumin		A1-Globulin		A2-Globulin		Beta-Globulin		G-Globulin
	%	g/l	%	g/l	%	g/l	%	g/l	%	g/l
0	56.3	42.5	9.7	7.3	5.8	4.3	18.4	13.9	9.9	7.4
2	58.8	44.4	7.8	6.0	4.7	3.6	18.2	13.8	10.4	7.8
5	59.9 *	45.7	8.2	6.3	4.8	3.6	16.8 *	12.9	10.3	7.9
10	62.7 **	47.5 **	7.4 *	5.6 *	5.0	3.9	16.0 **	12.1 **	9.0	6.8
50	67.7 **	52.5 **	6.5 **	5.0 **	3.8 **	2.9 **	14.0 **	10.8 **	8.2	6.3
MALES after recovery (17 weeks)
0	62.1	50.4	3.8	3.1	4.6	3.8	18.3	14.9	11.2	9.2
10	63.3	50.7	5.6 **	4.5 *	4.3	3.4	16.6 *	13.3 *	10.2	8.2
FEMALES after 13 week treatment
Dose mg/kg bw/day	Albumin		A1-Glubulin		A2-Globulin		Beta-Globulin		G-Globulin
	%	g/l	%	g/l	%	g/l	%	g/l	%	g/l
0	62.6	48.3	5.8	4.5	4.1	3.2	17.7	13.6	9.9	7.6
2	67.7 *	51.0	4.3	3.2 *	3.3	2.5	15.8	11.9 *	9.0	6.8
5	66.8 *	51.1	3.9 *	3.0 *	3.2	2.4 *	15.8	12.0 *	10.3	7.8
10	68.5 **	53.5 **	4.2 *	3.3 *	3.0 **	2.3 **	15.4 **	11.9 **	9.0	6.9
50	71.6 **	57.9 **	3.5 **	2.8 *	3.6	2.9	13.8 **	11.2 **	7.4 *	6.0 *
FEMALES after recovery (17 weeks)
0	67.9	55.8	3.5	2.9	3.4	2.8	15.5	12.7	9.7	7.9
10	69.4	56.9	3.9	3.2	3.1	2.5	13.6	11.1 *	10.1	8.2
*, p<0.05; **, p<0.01

Table 4: Mean body weights, absolute and relative mean organ weights (g and g/100g bw, respectively) after 13 weeks.

MALES
		Dose (mg/kg bw/d)
		0	2	5	10	50
Body weight		458	430	443	446	411 *
Sem. Ves.	Absolute	1.706	1.590	1.633	1.669	1.559
	relative	0.373	0.372	0.370	0.374	0.378
Testes	Absolute	3.539	3.488	3.511	3.539	3.540
	relative	0.774	0.816	0.796	0.795	0.865 *
Prostate	Absolute	1.456	1.457	1.411	1.385	1.328
	relative	0.319	0.338	0.319	0.310	0.322
Adrenal glands	Absolute	0.066	0.058	0.062	0.061	0.055 *
	relative	0.014	0.014	0.014	0.014	0.013
Liver	Absolute	12.355	12.744	14.659 *	16.390 **	22.882 **
	relative	2.691	2.960 *	3.320 **	3.674 **	5.549 **
Spleen	Absolute	0.959	0.817	0.981	0.856	0.830
	relative	0.209	0.189	0.223	0.192	0.201
Kidneys	Absolute	3.693	3.657	3.649	3.979	3.740
	relative	0.809	0.853	0.826	0.891	0.910 *
FEMALES
		Dose (mg/kg bw/d)
		0	2	5	10	50
Body weight		257	251	258	259	249
Ovaries	Absolute	0.117	0.117	0.119	0.126	0.133
	relative	0.046	0.046	0.046	0.049	0.054
Adrenal glands	Absolute	0.067	0.068	0.064	0.067	0.062
	relative	0.026	0.027	0.025	0.026	0.025
Liver	Absolute	7.315	7.476	8.407 *	8.374 *	10.694 **
	relative	2.838	2.974	3.268 **	3.243 **	4.292 **
Spleen	Absolute	0.557	0.622	0.697 *	0.610	0.573
	relative	0.217	0.248	0.272 *	0.237	0.231
Kidneys	Absolute	2.150	2.161	2.119	2.188	2.277
	relative	0.839	0.859	0.825	0.846	0.914
*, p<0.05; **,p<0.01