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)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Aug. 4, 1992 to May. 5, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(1981)

Deviations:

no

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:

- Physical state: liquid
- Lot/batch No.: EN 20043.42
- Expiration date of the lot/batch: 12/1994
- Purity: commercial grade (see analytical certificate No. 532, 1990-02-07)
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain as stated in the report: Tif: RAI f (SPF), cross between Sprague-Dawley derived strains
- Source: Animal Production, WST-455, CIBA-GEIGY Limited, 4332 Stein, Switzerland
- Age at study initiation: Minimum 8 weeks
- Mean group weight at study initiation: 202, 202, 202, 202 g at 0, 1, 30, 150 mg/kg bw/d
- Fasting period before study: not provided
- Housing: individually in Macrolon cages Type 3
- Diet ad libitum: pelleted, certified standard feed (Nafag No. 890, Tox; Nafag, Naehr-und Futtermittel AG, Gossau, Switzerland)
- Water ad libitum: tap water
- Acclimation period: at least seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): about 16
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

- Concentration in vehicle: 0, 0.2, 6, and 30 mg of test article/ml mixture
- Amount of vehicle: 5 ml mixture/kg actual body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test solution were taken in duplicate for determination of homogeneity and stability of the test article in the vehicle and achieved concentrations of the prepared dosing mixtures. Results of analytical investigations demonstrated that the preparation of the dosing mixtures was accurate and that the test article was stable in the administration form during the dosing period.

Details on mating procedure:

Nulliparous females were mated overnight with males of proven fertility at an initial ratio of 3 females to 1 male. Successful mating was assessed by the presence of a vaginal plug or of spermatozoa in a vaginal smear. This day was designated as day 0 (of pregnancy) = day 0 post coitum (p.c). Pregnant females were removed from the mating cages and the procedure repeated for remaining females until sufficient dams were produced.

Duration of treatment / exposure:

From day 6 to 15 of gestation.

Frequency of treatment:

Daily

Duration of test:

10 days

Doses / concentrations

Remarks:

Doses / Concentrations:
1, 30, 150 mg/kg body weight
Basis:
actual ingested

No. of animals per sex per dose:

24 females per test group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a previous range-finding study (No. 924011)
- Rationale for animal assignment (if not random): Randomized by weight stratification

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No. Oral gavage 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: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: Main organs of the thoracic and abdominal cavities, in particular the genitals

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

Following removal from the uterus, the fetuses were numbered, sexed (on the basis of ano-genital distance), externally examined and weighed. They were sacrificed (subcutaneous injection of barbiturate anesthetic) and processed for visceral or skeletal examination. One fetus per litter was preserved for possible histopathological examinations in neutral buffered 4% formalin. Fetuses were assigned to either visceral or skeletal evaluation at an approximate 1:1 ratio within each litter, independent of sex (starting with skeletal). In the case of gross external anomaly or malformation, fetuses were allocated to one technique depending on the type and incidence of finding.

The viscera of approximately half of the fetuses per litter were fixed whole in Bouin's solution. Visceral examination included, in particular, morphology and position of the following organs and organ systems:
- Skin
- Central Nervous System: brain (olfactory bulbs, cerebrum, lateral and medial ventricles), spinal cord
- Eyes: lens, vitreous, retina
- Body Cavities: thorax and abdomen, including diaphragm
- Respiratory System: nasal cavity (nasal septum, turbinates, choanae), trachea, bronchi, lungs, pleura
- Digestive System: oral cavity, palate, tongue, esophagus, stomach, intestine, rectum, peritoneum
- Liver
- Endocrine System: thyroid, pancreas, adrenals, thymus, pituitary
- Circulatory System: spleen, pericardium, heart (atria, ventricles, septae), major vessels
- Excretory System: kidneys (renal papillae, renal pelvis), ureters, urinary bladder
- Genital System: testes, epididymides, vas deferens, seminal vesicles; ovaries, oviducts, uterus

Skeletal assessment in approximately half of the fetuses per litter was done according to the staining technique of Dawson (Stain Tech. 1, 123-124, 1926); after clearing with potassium hydroxide and staining with alizarin red S, the specimens were stored in glycerol. Investigation included:
- Facial bones: nasal, premaxillary, maxillary and zygomatic bones, mandibula
- Cranial bones: frontal, parietal, interparietal, occipital and exoccipital bones, fontanel
- Sternum: sternebrae 1 to 6
- Shoulder girdle: scapula and clavicle
- Forelimbs: humerus, ulna, radius, metacarpals 2-5, proximal and distal phalanges of anterior digits 1 to 5 (except proximal phalanx 1: absent)
- Pelvic girdle: ilium, ischium, pubis
- Hindlimbs: femur, tibia, fibula, calcaneus, metatarsals 1-5, proximal + distal phalanges of posterior digits 1-5 (except proximal phalanx 1: absent)
- Ribs: anteroposterior 1 to 13
- Spinal column: cervical vertebral centers and arches 1 to 7, thoracic vertebral centers and arches 1 to 13, lumbar vertebral centers and arches 1 to 6, sacral vertebral centers and arches 1 to 4

Statistics:

Continuous data: analysis of Variance Procedure (ANOVA) followed by Dunnett's t-Test in case of a significant result in the ANOVA
Categorical data: Chi-Square test followed by Fisher's Exact test in case of a significant result in the Chi-Square
Non-parametric data: Kruskal-Wallis nonparametric analysis of variance test followed by Mann-Whitney U-test

Indices:

Pregnancy; total implant loss; viable fetuses; corpora lutea; implantation site; preimplantation loss; live and dead fetuses; early and late resorption; postimplantation loss; male vs. female viable fetuses; fetal body weight

Historical control data:

Historical control data were provided for the rats used in this study (Stock: Tif:RAI f (SPF); Supplier: CIBA-GEIGY Limited) for the period Jan. 1, 1988 to Nov. 30, 1992, comprising 26 groups from 17 studies.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
No treatment related clinical signs or mortalities were observed. However, one animal of the low dose died incidentally. Some incidental clinical signs were seen such as chromodacryorrhea in 1 control and in 1 high dose dam.
One female in each of the control, low and mid, and two in the high dose group were not pregnant.
BODY WEIGHT
At 150 mg/kg bw/d mean maternal body weight and maternal body weight gain were both reduced during and after the treatment period. However, there were no statistically significant differences to control. Body weight parameters in the lower dose groups remained comparable with control values.
FOOD CONSUMPTION
A dose dependent decrease in food consumption was observed at 30 and 150 mg/kg bw/d throughout treatment (days 6 to 15). At the higher dose reduction in food consumption still was seen during the post treatment period (day 16 to 21).
The change in food consumption in the 30 mg/kg bw/d group was slight but statistically significant. In the high dose group the change was statistically significant during treatment only (details see table 1).
NECROPSY
There were no treatment related macroscopic findings in any dose group. Preimplantation losses, number of implantation sites, early and late post-implantation losses and number of live fetuses per litter were not affected by treatment. Gravid uterus weights were not affected by treatment. However, at 150 mg/kg bw/d carcass weight (264g versus 277g at control) and net body weight gain 30 g versus 41 g at control) from day 6 to 21 was significantly reduced.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

3

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

4

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL EXTERNAL EXAMINATION
The incidence and type of external findings were not affected by treatment.
FETAL VISCERAL EXAMINATION
The incidence and type of visceral findings were not affected by treatment.
FETAL SKELETAL EXAMINATION
At 150 mg/kg bw/d the incidence of litters affected with asymmetrically shaped sternebra-5 was significantly increased. This effect was attributed to a minor and reversible developmental delay in ossification and was considered to be related to maternal toxicity (details see table 2).
Other findings like increased incidence of absent ossification of the proximal phalanges of posterior digit-5 and asymmetric or bipartite sternebrae were recorded sporadically in animals of all dose groups and/or were within historical control values. Therefore, these findings were considered to be not treatment related. A summary of all fetal observations is given in Table 3.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

>= 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1: Mean maternal food consumption (g/animal/d)

Food consumption
days	Dose (mg/kg bw/d)
	0	1	30	150
0 to 6	20	20	20	19
6 to 11	24	23	22 *	21 **
11 to 16	27	27	25 *	24 **
16 to 21	29	29	28	26
*, p<0.05; **p<0.01

Table 2: Summary of skeletal anomalies – Sternebra-5

ASYMMETRICALLY SHAPED STERNEBRA-5
INCIDENCES		Dose (mg/kg bw/d)
		0	1	30	150
Fetal incidence	N	0	1	0	4
	%	0	0.6	0	2.6
Litter incidence	N	0	1	0	4*
	%	0	4.5	0	18.2
Affected fetuses per litter	Mean %	0	0.9	0	2.8
	SD	0	4.3	0	6.2
*, p<0.05

 

Table 3: Summary of all fetal observations

FETAL OBSERVATIONS
Dose (mg/kg bw/d)		0	1	30	150
TOTAL NUMBER OF LITTERS, FETUSES AND LIVE FETUSES EVALUATED
Litters		23	22	23	22
Fetuses		323	309	303	297
Live fetuses		323	309	303	297
TOTAL MALFORMATIONS
Litter incidence	N	1	1	0	0
	%	4.3	4.5	0	0
Fetal incidence	N	1	1	0	0
	%	0.3	0.3	0	0
Affected fetuses per litter	Mean %	0.31	0.45	0	0
	SD	1.5	2.1	0	0
TOTAL ANOMALIES
Litter incidence	N	4	3	4	6
	%	17.4	13.6	17.4	27.3
Fetal incidence	N	4	3	4	6
	%	1.2	1.0	1.3	2.0
Affected fetuses per litter	Mean %	1.5	1.1	1.5	2.1
	SD	3.5	2.8	3.5	3.5
TOTAL VARIATIONS
Litter incidence	N	23	22	23	22
	%	100	100	100	100
Fetal incidence	N	168	164	165	158
	%	52	53	55	53
Affected fetuses per litter	Mean %	52	53	55	53
	SD	3.1	5.3	5.2	4.9

Applicant's summary and conclusion

Conclusions:

The substance is not teratogenic in rats.