Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline No. 408, 21 September 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Vehicle:
corn oil
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 100 and 300 mg/kg bw/day
Basis:
actual ingested
the dose-levels are expressed in terms of the active material (3 hydroxy 1,1 dimethylbutyl peroxyneodecanoate) and correspond to the doses of 11.12, 111.2 and 333.6 mg Luperox 610/kg/day, respectively, taking into account the correction factor of 1.112
No. of animals per sex per dose:
16 (control and 1000 mg/kg bw/d)
10 (10 and 100 mg/kg bw/d)
Control animals:
yes, concurrent vehicle
Details on study design:
Monitoring of estrous cycle
The estrous cycle stage was determined for each female planned to be sacrificed at the end of the treatment period, from a fresh vaginal lavage (stained with methylene blue), daily for 21 consecutive days before the end of the treatment period.
As no treatment-related changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.

SEMINOLOGY
At the end of the treatment period, just before sacrifice, each male was deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and the left epididymis was removed. Animals were then sacrificed.
As no treatment-related changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.
- Epididymal sperm
Sperm from the cauda epididymis was sampled for motility and morphology investigations (see § Epididymal sperm motility, and § Epididymal sperm morphology).
The cauda of the left epididymis was separated from the corpus using a scalpel and subsequently kept at 20°C pending further investigation (see § Epididymal sperm count).
- Epididymal sperm motility
The sperm was evaluated on a slide, after appropriate dilution. The number of motile and immotile spermatozoa from a sample of 200 spermatozoa was evaluated under a microscope using a 40-fold magnification. Results were expressed as the proportion of motile and non-motile spermatozoa.
- Epididymal sperm morphology
The morphology was determined from a sperm smear, after eosin staining and counting of 100 spermatozoa per slide. Results were expressed as the proportion of spermatozoa in each of the following categories:
normal,
normally shaped head separated from flagellum,
abnormal head separated from flagellum,
abnormal head with normal flagellum,
abnormal head with abnormal flagellum,
normally shaped head with abnormal flagellum.
- Epididymal sperm count
After thawing, the left cauda epididymis was weighed, minced and homogenized in a saline-triton solution using a Polytron.
An aliquot of the suspension was sampled and the number of spermatozoa was counted in a microscope slide counting chamber.
Results were expressed as the number of spermatozoa per cauda and per gram of cauda.
- Testicular sperm
At necropsy, the left testis was sampled and frozen at -20°C for further sperm count investigation. After thawing, the left testis was weighed (without the albuginea) and ground. The resulting preparation was diluted and sperm heads resistant to homogeneization (i.e. elongated spermatids and mature spermatozoa) were counted in a microscope slide counting chamber.
Results were expressed as a number of sperm heads per gram of testis and the daily sperm production rate was calculated (using a time divisor of 6.10; Blazak et al., 1993).

- Organ weights
Right and left epididymides, ovaries, right and left testes and uterus were weighed wet as soon as possible after dissection.

- Microscopic examination
A microscopic examination was performed on: right epididymides, mammary glands/area, ovaries (including oviducts), prostate (dorso-lateral and ventral), seminal vesicles (including coagulation gland), right testes, uterus (horns and cervix) and vagina for the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period and for animal found dead,

In addition testicular staging was performed for control and high-dose males (groups 1 and 4). A detailed examination of the testes was performed, using a thorough understanding of tubule development through the different stages of the spermatogenic cycle. Transverse sections of the testes were stained with hematoxylin: PAS in order to detect retained spermatids, missing germ cell layers, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.

Results and discussion

Effect levels

Dose descriptor:
NOAEL
Remarks:
Reproductive organs
Effect level:
> 300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No effects were observed

Observed effects

- Estrous cycle
No effects were observed on the estrous cycle.

- Seminology
No test item-related effects were noted at seminology investigations.

- Organ weights
No test item-related effects were noted.

- Microscopic examination
No test item-related effects were noted.

Any other information on results incl. tables

Mean sperm analysis data 

Sex

Male

Dose-level (mg/kg/day)

0

10

100

300

% of motile sperm

98.1

98.2

97.6

94.4

% of morphologically normal sperm

96.0

96.7

95.6

93.9

Mean number of spermatozoa
(106/cauda of epididymis)

165.9

183.7

150.5

171.9

Mean number of sperm heads
(106/g of testis)

123.7

117.3

109.0

110.5

 

Applicant's summary and conclusion

Executive summary:

In the frame of the OECD 408 study (Haag, 2015), the estrous cycle was determined on all females daily for 21 consecutive days at the end of the treatment period. Seminology investigations (count, motility and morphology) were performed on all males at time of sacrifice at the end of the treatment period. Histopathological examinations of the male and female reproductive organs were performed on control and 300 mg/kg bw/day groups at time of sacrifice. No effects were observed on these reproductive parameters.