Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 December 2009 (animal arrival) -10 March 2010 (pathology completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study undertaken according to current OECD Test Guidelines.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
increased animal number
Principles of method if other than guideline:
The toxicity subgroup for this study comprised 5 male and 5 female rats per treatment group and the reproductive subgroup comprised 5 male and 10 female rats per treatment group. The 5 male rats in each treatment group of the toxicity subgroup were used for mating with 5 female reproductive subgroup animals of the corresponding treatment group. The ten female rats in each treatment group of the reproductive subgroup were used exclusively for the reproductive/developmental toxicity phase of the study. This deviation was undertaken to enhance the robustness of the study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tetrabutane
- Substance type: Industrial
- Physical state: clear liquid
- Impurities (identity and concentrations): no data
- Composition of test material: C16-(branched), C20-(branched) and C24-(branched)-alkanes
- Isomers composition: no data
- Purity test date: yes
- Lot/batch No.: 0649/82535
- Expiration date of the lot/batch: 8 June 2010
- Stability under test conditions: stable
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
other: Crl: CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 303-355 g
- Fasting period before study: none
- Housing: Male: 5 animals/P2000 polycarbonate cage : singly with one female in RB3 modified polyproplyene cages during mating.
Females: 5 animals/P2000 polycarbonate cage pre-mating: singly with one male in RB3 modified polypropylene cages during mating: singly in 2154 polycarbonate cages during gestation and littering
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

IN-LIFE DATES: From: 2 December 2009 (animal arrival) To: 20 January 2010 (necropsy completion)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was used as supplied. All formulations were prepared freshly weekly and were stored refrigerated (2-8°C) in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 20, 60 or 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): no data
- Purity: no data
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until detection of mating (successful for all animals up to 5 days)
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged (how): singly in 2154 polycarbonate cages
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations were analysed to assess the homogeneity and stability of the test material in the liquid matrix. The stability was assessed following storage at ambient temperature (nominally 21°C) for 0 hours, 4 hours (continual stirring) and 2 days, and refrigeration (nominally 2-8°C) for 2, 8 and 15 days. Prior to initial sampling on each day, the formulation was mixed by 20-fold inversion and magnetic stirring for a minimum of 5 minutes. At each time point, single samples (nominally 1 mL) were taken for assay from the top, middle and bottom of the magnetically stirred formulation. Stability was determined from the mean concentration of the analyte in the vehicle at each sampling point. Specimen formulations (typically 400 mL) were prepared at concentrations of 2 and 200 mg/mL and equally split between four amber glass screw-capped bottles and were confirmed for 15 days when refrigerated and for 48 hours at room temperature.
Samples of each formulation prepared for administration in the first week of the dosing procedure were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed); 2 assays from each group and 1 assay. The remainder was frozen (nominally -20°C) and retained as contingency for analysis if any result required confirmation.

The GC analytical procedure was validated with respect to linearity of detector response, precision of injection, specificity of chromatographic analysis, limit of detection, accuracy and precision.

The homogeneity and stability was confirmed for Tetrabutane in corn oil formulations at nominal concentrations of 2 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature storage for 2 days and refrigerated storage for up to 15 days. The storage times represented the maximum time from preparation to completion of administration.

The mean concentrations of Tetrabutane in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation
Duration of treatment / exposure:
5 weeks or for dams up to day 4 of lactation (daily)
Frequency of treatment:
All animals were dosed once each day at approximately the same time each day, seven days per week.
Details on study schedule:

Age at mating of the mated animals in the study:11-12 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 or 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 10 females in the reproductive toxicity subgroup, and 5 males used for mating from the toxicity subgroup
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels of 100, 300 and 1000 mg/kg/day were selected in conjunction with the Sponsor with reference to previous work with this compound performed in these laboratories (Huntingdon Life Sciences Report Number: BBB0024). In that study, the CD rats received Tetrabutane at doses of 100, 300 or 1000 mg/kg/day for seven days, there were no findings which precluded the use of these dose levels on the subsequent 4 week general toxicity and reproductive developmental toxicity screening study.

- Rationale for animal assignment (if not random):
On arrival, the animals were removed from the transit boxes and allocated to study cages. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately
- Rationale for selecting satellite groups: no satellite groups used
- Post-exposure recovery period in satellite groups: no post-exposure recovery period

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

- Time schedule:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
Daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:
Immediately before dosing
Immediately after dosing on return of the animal to its cage
On completion of dosing of each group
Between one and two hours after completion of dosing of all groups
As late as possible in the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Before treatment commenced , during each week of treatment and for females on Days 0, 7, 14 and 20 after mating and Day 4 of lacation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware (as far as practically possible) of the experimental group to which the animal belonged. For females after mating, a small number of animals were subject to these observations on each Day 4 of lactation (8, 15, 4, 8 and 4 females on each Day 4 of lactation) - the observer was aware of the experimental group and this was considered acceptable.

After removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour.

Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.


BODY WEIGHT: Yes

- Time schedule for examinations:
All toxicity and reproductive subgroup males (10 animals per treatment group) were weighed weekly throughout the study. All toxicity and reproductive subgroup females (15 animals per treatment group) were weighed weekly for the first two weeks and the toxicity subgroup females (five animals per treatment group) were weighed during Weeks 3, 4 and 5.

FOOD CONSUMPTION: Yes

The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded during weeks 1 and 2 for all male (two cages of 5 animals per treatment group) and all female (three cages of 5 animals per treatment group) toxicity and reproductive subgroups. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

WATER CONSUMPTION : Yes

Fluid intake was assessed by daily visual observation. No effect was observed and, consequently, quantitative measurements were not performed
Oestrous cyclicity (parental animals):
For two weeks before pairing, daily vaginal smears were taken from females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed
Sperm parameters (parental animals):
Not undertaken
Litter observations:
All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter. The records maintained were as follows:

Clinical signs: Daily records were maintained for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.

Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-4 of age.

Sex ratio: The sex ratio of each litter was recorded on Days 1 and 4 of age.

Bodyweight: Individual offspring bodyweights were recorded on Days 1 and 4 of age.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all animals sacrificed after 5 weeks of treatment
- Maternal animals: all surviving animals sacrificed on Day 4 of lactation. One dam and litter were killed on Day 2 of lactation for reasons of animal welfare

GROSS NECROPSY

All animals were subject to a detailed necropsy. All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. External and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative


ORGAN WEIGHTS

The following organs, taken from all animals, were dissected free of adjacent fat and other contiguous tissue and the weights recorded:

Epididymides (L&R) Seminal vesicles and coagulation gland
Ovaries with oviducts (L&R) Testes (L&R)
Prostate Uterus with cervix

L&R Bilateral organs weighed individually

Organ weights were also adjusted for terminal bodyweight using the weight recorded before necropsy

HISTOPATHOLOGY

The following tissues were examined for all animals of the Control and 1000 mg/kg/day treatment groups sacrificed on completion of the scheduled treatment period:

Epididymides (L&R) Seminal vesicles and coagulation gland
Mammary area - caudal† Testes (L&R)
Ovaries with oviducts Uterus with cervix
Prostate Vagina


For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted. For the assessment of the ovaries, one mid-line section of each ovary was examined for the presence of primordial follicles, growing follicles and corpora lutea
Postmortem examinations (offspring):
SACRIFICE
- F1 offspring were killed on Day 4 of age

- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY

For F1 offspring surviving to scheduled termination, a full macroscopic examination of the tissues was performed, after a review of the history. All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

For premature adult deaths before weaning, missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before weaning were examined as detailed above except that the cranium was not sectioned unless required to investigate a cranial abnormality. The necropsy also included an assessment for the presence of milk in the stomach, where this was possible.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not undertaken
Statistics:
See 'materials and methods' free text field below
Reproductive indices:
Mating performance and fertility
Gestation length

Offspring viability indices:
Survival indices
Sex ratio

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs, arena observations or signs related to dosing considered to be related to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female control animal was killed for welfare reasons on Day 2 of lactation after signs of piloerection, pallor, hunched posture, deep breathing and reduced body temperature. Macroscopic examination at necropsy revealed yellow amorphous adhesions on the lungs and bronchi, enlarged thymic lymph nodes, clear fluid in the thorax, and brown staining of the fur in the uro-genital region. Microscopic examination revealed evidence of oesophageal perforation, thereby indicating that the death of this Control female was due to a dosing error.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The bodyweight and bodyweight change of females receiving 1000 mg/kg/day was slightly high during Days 0-7 and 14-20 of gestation when compared with that of the Control, resulting in a slightly higher overall bodyweight gain during gestation for females receiving 1000 mg/kg/day. Bodyweight change from Day 1 to Day 4 of lactation was similar across all groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the absolute or bodyweight-adjusted organ weights
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macropathology findings that were considered to be related to treatment with Tetrabutane
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no findings that were considered to be related to treatment with Tetrabutane
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no findings that were considered to be related to treatment with Tetrabutane
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
There was a higher incidence of acyclic animals amongst females treated with Tetrabutane at 100 or 300 mg/kg/day (4/10 and 3/10 at 100 and 300 mg/kg/day, respectively, compared with 1/10 in the concurrent Control group). However, as all females mated within 5 days, and there no acyclic females in the 1000 mg/kg/day group, oestrous cycles were considered unaffected by treatment.

The majority of animals mated within 4 days (i.e. at the first oestrus opportunity after pairing). Two females mated within 5 days.
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
Not undertaken
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance and fertility were unaffected by treatment.
The duration of gestation was unaffected by treatment, with all females having a gestation length within the expected range of 22-23 days. The gestation index was 100% in all groups

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive/developmental toxicity up to a limit dosage of 1000 mg/kg/day

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs amongst the offspring which were considered related to maternal treatment with Tetrabutane
Mortality / viability:
no mortality observed
Description (incidence and severity):
The number of uterine implantation sites, total litter size at Day 1 of age and live litter size at Days 1 and 4 were unaffected by parental treatment.
Neither peri- nor post-natal offspring survival was affected by parental treatment with Tetrabutane
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Assessment of offspring bodyweight data showed that bodyweight at Day 1 of age and subsequent growth to Day 4 appeared slightly low for the offspring of all treated groups. This is likely to be a consequence of the slightly higher litter size in these groups, and not selectively related to the parental administration of Tetrabutane.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of the small number of offspring dying before scheduled termination on Day 4 of age showed absence of milk in the stomach as the predominant finding. This is a common observation for offspring dying at such a young age and was considered not to indicate any adverse effect of maternal treatment with Tetrabutane.
There were no treatment related findings among offspring examined at scheduled termination on Day 4 of age.
Histopathological findings:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive/developmental toxicity up to day 4 of lactation

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

TABLE 1

 

Bodyweight change – group mean values (g) for reproductive subgroup females during gestation and lactation

 

Group

:

 1

 2

 3

 4

Compound

:

Control

Tetrabutane

Tetrabutane

Tetrabutane

Dose (mg/kg/day)

:

0

100

300

1000

 

 

 

Gestation

 

Lactation

Group

 

Days

Days

Days

Days

 

Days

/Sex

 

0-7

7-14

14-20

0-20

 

1-4

Statistical test:

 

 Wi

 Wi

 Wi

 Wi

 

 Wi

1F

Mean

35

34

77

145

 

9

 

SD

3.7

6.3

14.5

15.6

 

4.3

 

N

10

10

10

10

 

9

 

 

 

 

 

 

 

 

2F

Mean

32

33

80

145

 

6

 

SD

6.8

6.4

10.2

14.8

 

6.3

 

N

10

10

10

10

 

10

 

 

 

 

 

 

 

 

3F

Mean

31

32

84

147

 

11

 

SD

5.6

6.1

6.9

12.0

 

10.2

 

N

10

10

10

10

 

10

 

 

 

 

 

 

 

 

4F

Mean

41

35

87

162*

 

9

 

SD

9.7

7.8

13.3

24.1

 

6.6

 

N

10

10

10

10

 

10

 

 

 

 

 

 

 

 

 

Wi    Treated groups compared to Control using Williams’ test.

 

 

 

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive/developmental toxicity within the scope of this screening test was 1000 mg/kg/day.
Executive summary:

An OECD 422 guideline screening study was performed at the Laboratories of Huntingdon Life Sciences, Eye, on behalf of Evonik Oxeno GmbH., to investigate the reproductive and developmental toxicity of the test substance Tetrabutane when administered to rats by oral gavage. Three groups, each comprising five male and ten female rats, received Tetrabutane once daily at dosages of 100, 300 or 1000 mg/kg/day. An additional five males from each treatment group of the toxicity phase of this combined study were used for mating with corresponding females of the reproductive phase. The males were treated daily for a period of five complete weeks before termination. The females were treated daily for two weeks before pairing, throughout pairing and gestation and for three days of lactation, until termination on Day 4 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose, for the same duration.

Throughout the study, data was recorded on clinical condition, detailed physical and arena observations and bodyweight. Data was recorded on food consumption for all animals prior to mating, and during gestation and lactation for mated females. For females, data were recorded on oestrous cycles, mating performance and fertility and gestation length.  Organ weight, macroscopic and microscopic pathology investigations were undertaken. The clinical condition of F1 offspring, litter size and survival, sex ratio and bodyweight were assessed and macroscopic pathology investigations were undertaken. The study was conducted to GLP.

There was no effect of treatment on mating performance, fertility or gestation and there were no pathology findings or changes considered to be related to treatment. There was a slightly increased overall bodyweight gain during gestation for females receiving 1000 mg/kg/day, when compared with controls. Bodyweight change from Day 1 to Day 4 of lactation was similar across all groups.

The reproduction/developmental toxicity screening test found no evidence of impaired performance at 100, 300 or 1000 mg/kg/day Tetrabutane. Based on the results of this study, it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive/developmental toxicity within the scope of this screening test was 1000 mg/kg/day.

The study is considered acceptable for classification and satisfies the guideline requirements for a rat reproductive/developmental toxicity screening test.