tert-butyl hydroperoxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Reference 2

Reference Type:

publication

Title:

Prenatal developmental toxicity study of ethyl tertiary-butyl ether in rabbits

Author:

Asano, Y, Ishikura, T, Kudoh, K, Haneda, R and Endoh, T

Year:

2011

Bibliographic source:

Drug and Chemical Toxicology, 34, 311-317

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

ETBE was administered by oral gavage to pregnant female New Zealand White rabbits (Kbl:NZW) at dose levels of 0, 100, 300 and 1000 mg/kg bw/d on GD 6- 27, and effects on embryo-foetal development assessed.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-ethoxy-2-methylpropane

EC Number:

211-309-7

EC Name:

2-ethoxy-2-methylpropane

Cas Number:

637-92-3

Molecular formula:

C6H14O

IUPAC Name:

Tert-Butyl Ethyl Ether

Details on test material:

ETBE, lot # 2ZGTA, 99 wt% pure was supplied by Tokyo Chemical Industry Co, Ltd, Tokyo, Japan. Purity and stability characterised by GC-FID.

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Kbl:NZW
- Source: Kitayama Labs Co Ltd, Japan
- Age at study initiation: 17-18 wk
- Weight at study initiation: mean group weights approx. 3.25 kg
- Housing: single housed in aluminium cages
- Diet (ad libitum): RC4 pellets (Oriental Yeast Co. Ltd, Chiba, Japan)
- Water (ad libitum): domestic tap water
- Acclimation period: 27 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22 degrees
- Humidity (%): 50-72%
- Air changes (per hr): 10-15 per hr
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 11 June 2007 (initiation of treatment) To: 3 July 2007 (start of cesarean section)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dosing solutions prepared at 60, 180 and 600 mg/l in olive oil.
- Frequency of preparation: at least weekly

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Achieved concentration: test solutions were analyzed using GC-FID (Agilent Technologies HP6890N). Results were within 5% of target.

Details on mating procedure:

Females judged to be in oestrus (based on appearance of external genitalia) were housed together with untreated males on a 1:1 basis. Animals were considered pregnant when copulation was observed twice (designated GD 0).

Duration of treatment / exposure:

GD 6-27 (22 days)

Frequency of treatment:

daily, 7 d/wk

Duration of test:

GD 6-28

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: treatment levels were based on a preliminary study in which ETBE was administered orally to pregnant rabbits (6 / group) at 0, 30, 100, 300 and 1000 mg/kg bw/d for 22 days. Reduced food intake was noted at 1000 mg/kg bw/d but embryo-foetal development was unaffected. No maternal or foetal effects recorded at 300 mg/kg bw/d or below. Therefore the highest dose level was set at 1000 mg/kg bw/d with lower doses of 300 and 100 mg/kg bw/d.
- Rationale for animal assignment (if not random): stratified according to body weight
- Dosing: the control and test solutions were administered at 1.67 ml/kg body weight

Examinations

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS:
Dams were observed for clinical signs dosing, immediately after dosing and 1 hour post-dosing during the treatment period.

BODY WEIGHT:
Dams were weighed on GD 0 (at time of mating) and on GD 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28. Any dams sacrified early were weighed before necropsy.

FOOD CONSUMPTION:
Food intake was measured on GD 1, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28.

NECROPSY:
Animals were sacrificed by exsanguination (abdominal aorta) under pentobarbital sodium anesthesia, and the major organs in the thoracic and abdominal cavities examined macroscopically. Animals were examined to confirm pregnancy. No organ weights measurement or routine tissue sampling were performed.

Ovaries and uterine content:

The ovaries and uterus were removed from dams confirmed as pregnant, and the number of corpora lutea determined. The uterus was weighed, opened, and the number of live/dead foetuses, implantation sites, resorbed embryos, placental remnants and early/late macerated foetuses counted. The placenta was also examined. Uteri were immersed in 10 vol% ammonium sulphide solution if no signs of pregnancy were visible macroscopically.

Fetal examinations:

- Body Weight:
Recorded for all foetuses (including those with external malformations, but the data excluded from group calculations).

- Sex Determination:
Assessed from examination of the internal genital organs of all live foetuses (excluding any with external malformations).

- External examinations
Foetuses were examined for the presence or absence of external malformations, including those in the oral cavity. Any exhibiting external malformations were preserved (phosphate buffered 10 vol% formalin).

- Soft tissue examinations:
The contents of the thoracic and abdominal cavities were observed macroscopically for all live foetuses (excluding those with external malformations). The brain (Wilson technique) and heart (Nishimura microdissection technique) were excised and fixed (phosphate buffered 10% formalin).

- Skeletal examinations:
All live foetuses (excluding those with external malformations) were fixed (95% alcohol), stained with Alizarin red S (Dawson method), and examined for skeletal malformations or variations. The degree of ossification was determined by counting the number of ossified metacarpals, metatarsals and digital phalanges and sacral/caudal vertebrae together with the degree of ossification of the sternebrae.

Statistics:

Body weight, body weight gain, food consumption, uterine weight, number of corpora lutea, number of implantations, number of live foetuses, sex ratio, body weight and ossification data (phalanges and vertebrae) were analyzed by Bartletts test followed by Dunnetts test if the data were homogeneous, with a Dunnett-type mean rank test used if the results were heterogeneous. Implantation indices, embryo-fetal deaths, external malformations, visceral malformations, visceral variations, skeletal malformations and skeletal variations were calculated and the data were analyzed by Dunnett-type mean rank test.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
GENERAL:
There were 1 or 2 were non-pregnant animals in the control, 100 and 300 mg/kg bw/d groups (all animals pregnant at 1000 mg/kg bw/d). No deaths occurred in any group. Abortions occurred in the control (1 animal on GD 28), 300 mg/kg bw/d (2 animals on GD 19 and GD 26) and 1000 mg/kg bw/d groups (1 animal on GD 25), but no dose-dependence apparent. One control animal and one animal that aborted in the 300 mg/kg bw/d group exhibited reductions in body weight and food intake for at least 4 days before abortion whereas clinical signs, body weight and food consumption were unremarkable in the other animals.

CLINICAL SIGNS:
Apart from reduced production of faeces (recorded in 14, 10, 7 and 13 animals from the 0, 100, 300 and 1000 mg/kg bw/d groups) there were no findings of note.

BODY WEIGHT:
Body weight was statistically significantly decreased by 4-5% in the 1000 mg/kg bw/d group relative to controls on GD 12, 14 and 16 but was unremarkable thereafter; body weight gain in this group on GD 6-28 was approx. half that of the control, however the effect was not statistically significant. Body weight and weight gain in the 100 and 300 mg/kg bw/d groups was unremarkable.

FOOD CONSUMPTION:
Food consumption was decreased significantly by 25-30% at 1000 mg/kg bw/d on GD 8, 10, 12 and 14, but was comparable to the control group from GD 20 onwards. Food intake for the 100 and 300 mg/kg bw/d groups was similar to that of the controls.

NECROPSY:
Dark red discoloration of the lung was observed in 1 animal from the 300 and 1000 mg/kg bw/d groups with mild to moderate haemorrhage and inflammatory cell infiltration noted microscopically; cause not known but judged of limited limited relevance. There were no other findings of note.

CESAREAN SECTION DATA:
The number of corpora lutea, implantations and implantation index was similar in the control and treated groups. Uterus weight was comparable between the groups.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

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:
GENERAL:
There were no significant differences in the index of embryo-foetal deaths, number of live foetuses, sex ratio, body weight of live foetuses or the type or incidence of external malformations.

VISCERAL EXAMINATION:
No significant differences in the incidence of visceral malformations or variations, with any findings present occurring in control and treated groups with no obvious dose relationship present.

SKELETAL EXAMINATION:
No significant differences in the incidence of skeletal malformations or variations and extent of ossification with any findings present occurring in control and treated groups with no obvious dose relationship present.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

  Selected results:

Treatment (mg/kg bw/d)	0	100	300	1000
Mean no. corpora lutea	10.3	10.4	10.1	9.8
Mean no. implantations	8.4	8.7	9.0	7.7
Mean implantation index (%)	80.4	84.7	89.3	77.1
Mean no. resorbed foetuses	11.0	11.3	7.0	8.7
Total no. early resorptions	8	7	10	11
Total no. late resorptions	5	10	2	8
Mean no. live foetuses	7.8	7.9	8.4	6.9
Sex ratio	0.50	0.54	0.54	0.42
Male foetal bw (g)	33.5	33.4	33.9	32.3
Female foetal bw (g)	31.2	31.4	32.0	30.1
No. foetuses examined	171	173	167	158
No. visceral malformations	1	1	1	3
No. visceral variations	3	5	5	3
No. skeletal malformations	5	4	3	8
No. skeletal variations	9	11	6	15

 

Applicant's summary and conclusion

Conclusions:

Dams in the 1000 mg/kg bw/d group showed small but statistically significant reductions in body weight on GD 8-14 (the period of organogenesis) with significant reductions in food intake over the same period; overall body weight gain on GD 6-28 was decreased by one half. The maternal NOAEL was therefore 300 mg/kg bw/d. There were no effects of ETBE-treatment on pregnancy or foetal development (NOAEL = 1000 mg/kg bw/d).