2(3H)-Benzofuranone, 5,7-bis(1,1-dimethylethyl)-3-(4-hydroxy-3,5-

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Remarks:

Screening study on male sexual organ toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Principles of method if other than guideline:

The aim of this study was to obtain initial information on the effect of the test substance on male sexual organs after repeated oral administration (gavage) to male Wistar rats. The test substance was administered to the animals orally by gavage for a maximum of about 10 weeks.

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

1

Reference substance name:

2(3H)-Benzofuranone, 5,7-bis(1,1-dimethylethyl)-3-(4-hydroxy-3,5-

EC Number:

813-928-3

Cas Number:

155810-87-0

Molecular formula:

C24 H30 O3

IUPAC Name:

2(3H)-Benzofuranone, 5,7-bis(1,1-dimethylethyl)-3-(4-hydroxy-3,5-

Details on test material:

Physical state/appearance: Solid/white

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is the preferred animal species for developmental and reproductive toxicity studies according to the various test guidelines. This Wistar rat strain (Crl:Wl(Han)) is selected because extensive historical control data is available for these rats.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 7 weeks
- Weight at study initiation:
- Housing: 5/cage
- Diet: Ground Kliba maintenance diet mouse/rat "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: ad libitum
- Acclimation period: From delivery to the beginning of administration the animals were accustomed to the environmental conditions and to the
diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the test substance preparation, the specific amount of test substance was weighed, topped up with cornoil in a graduated flask and intensely mixed with a magnetic stirrer until it is completely dissolved. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 7.5 g/100 ml and 25 g/100 ml
- Amount of vehicle (if gavage): 4 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in corn oil in a refrigerator under light exclusion over a period of 7 days had been verified prior to the start of the study.
Concentration analysis:
300 mg/kg: 96.6%
1000 mg/kg: 100.1%

Duration of treatment / exposure:

10 weeks

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
A check for moribund and dead animals was made twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays. A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. During the administration period all animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal.

BODY WEIGHT: Yes
Body weights were determined once a week

FOOD CONSUMPTION
Food consumption was determined once a week.

SPERM AND SPERMATID EXAMINATIONS
After the organ weight determination, the following parameters will be determined in the right testis or right epididymis of all animals sacrificed on schedule:
• Cauda epididymis sperm motility
• Sperm morphology
• Spermatid concentration in the testis
• Sperm concentration in the cauda epididymis
The determination of the cauda epididymis sperm motility and the preparation of the specimens for sperm morphology will be carried out in a randomized sequence.

Sacrifice and pathology:

One day after the last administration all surviving animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs. Animals which die intercurrently or are sacrificed in a moribund state were necropsied as soon as possible after their death and assessed by gross pathology.

The following weights will be determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Testes
3. Epididymides
4. Prostate
5. Seminal vesicles including coagulation glands
6. Liver
7. Kidneys
8. Spleen
9. Adrenal glands
10. Pituitary gland

The following organs or tissues will be fixed in 4% formaldehyde solution or in modified Davidson's solution:
1. All gross lesions
2. Testes
3. Epididymides
4. Prostate
5. Seminal vesicles including coagulation glands
6. Liver
7. Kidneys
8. Spleen
9. Adrenal glands
10. Pituitary gland

The epididymides and testes of animals that died or were sacrificed intercurrently will be fixed in 4% buffered formaldehyde solution. The left testis and left epididymis of all animals sacrificed at scheduled dates will be fixed in modified Davidson's solution, whereas the right testis and epididymis will be used for sperm parameters. In case of macroscopic findings in the right testis or right epididymis, this testis as well as
the corresponding epididymis were fixed for histopathological examination and the left testis and epididymis will be used for sperm parameters.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation in treated animals

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

0 mg/kg: cellular debris in left epididymis (2/10), tubular degeneration in left testicle (2/10).
300 mg/kg: cellular debris in left epididymis (2/10), tubular degeneration in left testicle (2/10), tubular vacuolation in left testis (2/10)
1000 mg/kg: no findings

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No effects on sperm parameters

Effect levels

Dose descriptor:

NOAEL

Remarks:

Testes and Sperm toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no effects observed

Target system / organ toxicity

Critical effects observed:

no