Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

In the reproductive/developmental toxicity element of an OECD 422 screening test in rats, Oxooil LS9 elicited no evidence of impaired fertility or reproductive performance at dosages of 100, 300 or 1000 mg/kg/day. The NOAEL for fertility is 1000 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 December 2009 (animal arrival) -24 March 2010 (pathology completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study undertaken according to current OECD Test Guidelines.
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
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. This deviation was undertaken to enhance the robustness of the study.
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
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: 350-397 g males and 204-251 g females
- 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(SDS VRF 1 certified diet)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 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: 9 December 2009 (animal arrival) To: 1 February 2010 (necropsy completion)
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 Oxooil LS9 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 Oxooil LS9 in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation
Duration of treatment / exposure:
5 weeks for males; to day 4 of lactation for females (during week 7)
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
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: BBB0026). In that study, the CD rats received Oxooil LS9 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. Before the start of treatment mean bodyweights were reviewed. Control males had a low mean bodyweight in comparison to treated male groups. To average the group means to similar values, two males from the Control group were changed with two males from the middle treatment (Group 3).
- Rationale for selecting satellite groups: no satellite groups used
- Post-exposure recovery period in satellite groups: no post-exposure recovery period
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 : No

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: animals receiving 1000 mg/kg/day killed during weeks 3 or 4 of treatment. All remaining animals sacrificed after 5 weeks of treatment
- Maternal animals: One dam and litter were killed during gestation and one dam and litter during lactation for reasons of animal welfare. All remaining animals sacrificed on Day 4 of lactation.

GROSS NECROPSY
All animals were killed by carbon dioxide asphyxiation.

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
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

Male animals receiving 1000 mg/kg/day were sacrificed on humane grounds during weeks 3 or 4 (Section 7.5.1).
.
One female receiving 1000 mg/kg/day was killed for welfare reasons on Day 20 of gestation due to general poor condition. The ante-mortem clinical signs consisted of underactivity, irregular breathing, piloerection, pale eyes and whole body pallor, abnormal black staining of the perigenital and dark ventral abdomen. Macroscopic examination revealed: Abnormal black/dark viscous fluid in the vagina and right uterine horn. This female was pregnant with eight live fetuses in the left horn and five live fetuses in the right horn with two early resorptions and one late resorption. All fetuses appeared grossly normal.
A second female receiving 1000 mg/kg/day was killed for welfare reasons on Day 3 of lactation. Ante-mortem signs consisted of thin build, whole body pallor, reduced body tone and temperature and abnormal gait. Macroscopic examination revealed the female to be thin with pale and inactive mammary tissue; all pups were of thin build and had no milk in the stomach.

Forepaw paddling was observed on Day 6 of treatment and to a lesser extent on Days 10, 14, 17, 21, 24 28 and 30 of treatment and Days 0, 7, 14 and 20 of gestation in females receiving 1000 mg/kg/day. Chin rubbing and salivation was also observed periodically after dosing for females receiving 1000 mg/kg/day, and at a low incidence for animals receiving 300 mg/kg/day. Chin rubbing, salivation and forepaw paddling was observed during gestation for females receiving 300 or 1000 mg/kg/day, and chin rubbing and salivation was observed on Day one of lactation for females receiving 1000 mg/kg/day.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

Bodyweight loss and reduced food consumption were recorded for males receiving 1000 mg/kg/day and reduced bodyweight gain for rats receiving 300 mg/kg/day and dams receiving 1000 mg/kg/day. 

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)

Not applicable

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)

At 1000 mg/kg/day, 2/10 females were acyclic (compared to 0/10 in the concurrent Control group). However, all females mated within 5 days, and oestrus cycles were considered unaffected by treatment.

All females mated within 4 days (i.e. at the first oestrus opportunity after pairing) with the exception of two females receiving 1000 mg/kg/day which mated 5 days after pairing.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

Not undertaken

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

Mating performance and fertility as assessed by percentage mating, conception rate and fertility index, were considered unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)

Seminal vesicles absolute and adjusted weights were slightly low in males that received 300 mg/kg/day when compared with Controls, with adjusted weights attaining statistical significance. In the absence of any histopathological findings in this organ, the difference was considered not of toxicological importance.

Mean terminal bodyweights of lactating females that received 1000 mg/kg/day were statistically significantly lower than Controls. Unadjusted and adjusted ovary and uterus weights were unaffected by treatment.


GROSS PATHOLOGY (PARENTAL ANIMALS)

Abnormal pink coloration of the sciatic nerve, adipose tissue, seminal vesicles, prostate and urinary bladder was observed macroscopically in the treated animals given 300 or 1000 mg/kg/day(Section 7.5.1).


HISTOPATHOLOGY (PARENTAL ANIMALS)

All microscopic findings in the reproductive organs were considered to be incidental and not of toxicological importance.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted. A single case, characterized by unilateral severe atrophy of the seminiferous tubular epithelium and germ cell depletion, and was seen in the testis of one control male. Based on the single occurrence this finding was considered incidental.

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
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)

There was no effect of treatment on pregnancy outcome. All pregnant females gave birth to a live litter except for one female receiving 1000 mg/kg/day. This female was killed for welfare reasons on Day 20 of gestation due to general poor condition. This female was pregnant with eight live fetuses in the left horn and five live fetuses in the right horn with two early resorptions and one late resorption. All fetuses appeared grossly normal.
One female receiving 1000 mg/kg/day was killed for welfare reasons on Day 3 of lactation due to general poor condition. Macroscopic examination revealed she had pale and inactive mammary tissue. All pups were of thin build and had no milk in the stomach.

There were no instances of total litter loss during the post-natal period.

The following assessment of litter responses was made based on the 10, 10, 10 and 8 litters successfully reared to termination on Day 4 post partum in the 0 (Control), 100, 300 and 1000 mg/kg/day groups, respectively. Litter data, as assessed by mean numbers of implantations, total litter size, and live litter size on Day 1and Day 4 of age, were generally similar to the Control and are considered not to be affected by treatment.

Offspring survival during the pre- and post-natal period as assessed by the post implantation survival index, live birth index, viability index was unaffected by treatment.


CLINICAL SIGNS (OFFSPRING)

There were no clinical signs observed for F1 offspring that were considered to be related to treatment with Oxooil LS9.

Two litters were observed to be cold to touch and had no milk in stomach for F0 females that received 1000 mg/kg/day.

BODY WEIGHT (OFFSPRING)

Male and female offspring mean bodyweights were slightly lower for litters of F0 dams that received 1000 mg/kg/day, and overall F1 males mean bodyweight change was low for F0 dams that received 100 or 300 mg/kg/day, when compared with Controls. F1 female mean bodyweight for F0 dams that received 100 or 300 mg/kg/day were unaffected by treatment.


SEXUAL MATURATION (OFFSPRING)

Not examined

ORGAN WEIGHTS (OFFSPRING)

Not examined

GROSS PATHOLOGY (OFFSPRING)

There were no findings at macroscopic examination of F1 offspring that were considered related to treatment.

HISTOPATHOLOGY (OFFSPRING)

Not examined
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
Reproductive effects observed:
not specified
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 is 1000 mg/kg/day.
Executive summary:

An OECD 422 guideline screening study was performed to GLP standards 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 Oxooil LS9 when administered to rats by oral gavage. Three groups, each comprising five male and ten female rats, received Oxooil LS9 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 were recorded on clinical condition, detailed physical and arena observations and bodyweight. Data were 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 and 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.

Three of ten males treated at 1000 mg/kg/day were sacrificed on humane grounds during weeks 3 or 4 and the remaining males in this group killed prematurely in week 4 (Section 7.5.1). Two females treated at this dosage were killed for welfare reasons on Day 20 of gestation or Day 3 of lactation, respectively. Bodyweight loss and reduced food consumption were recorded for males receiving 1000 mg/kg/day and reduced bodyweight gain for males treated at 300 mg/kg/day and dams receiving 1000 mg/kg/day.

There was no effect of treatment on mating performance, fertility, reproductive performance, survival and development of the offspring, and there were no treatment-related pathological findings in the reproductive organs. Slightly lower male and female offspring mean bodyweights were recorded for the litters of females that received 1000 mg/kg/day which was considered to be a consequence of the low terminal bodyweight of females that received 1000 mg/kg/day and indicative of the toxicity in the dams at this dosage.

It is concluded that, the reproduction/developmental toxicity screening test elicited no evidence of impaired performance at 100, 300 or 1000 mg/kg/day Oxooil LS9. Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive/developmental toxicity within the scope of this screening test is 1000 mg/kg/day.

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

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

An OECD 422 guideline screening study was performed to GLP standards, to investigate the reproductive and developmental toxicity of the test substance Oxooil LS9 when administered to rats by oral gavage. Three groups, each comprising five male and ten female rats, received Oxooil LS9 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 were recorded on clinical condition, detailed physical and arena observations and bodyweight. Data were 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 and 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.

Three of ten males treated at 1000 mg/kg/day were sacrificed on humane grounds during weeks 3 or 4 and the remaining males in this group killed prematurely in week 4 (Section 7.5.1). Two females treated at this dosage were killed for welfare reasons on Day 20 of gestation or Day 3 of lactation, respectively. Bodyweight loss and reduced food consumption were recorded for males receiving 1000 mg/kg/day and reduced bodyweight gain for males treated at 300 mg/kg/day and dams receiving 1000 mg/kg/day.

There was no effect of treatment on mating performance, fertility, reproductive performance, survival or development of the offspring, and there were no treatment-related pathological findings in the reproductive organs. Slightly lower male and female offspring mean bodyweights were recorded for the litters of females that received 1000 mg/kg/day, which were considered to be a consequence of the low terminal bodyweight of females that received 1000 mg/kg/day and indicative of the toxicity in the dams at this dosage.

It is concluded that, the reproduction/developmental toxicity screening test elicited no evidence of impaired performance at 100, 300 or 1000 mg/kg/day Oxooil LS9. Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) in rats for reproductive toxicity within the scope of this screening test is 1000 mg/kg/day.


Effects on developmental toxicity

Description of key information

In the reproductive/developmental toxicity element of an OECD 422 screening test in rats, Oxooil LS9 elicited no evidence of developmental toxicity at dosages of 100, 300 or 1000 mg/kg/day. The NOAEL for developmental toxicity is 1000 mg/kg/day.In the complete absence of any developmental toxicity effect at the limit dosage of 1000 mg/kg/day in the OECD 422 screening study, it is considered unlikely that Oxooil LS9 will represent a developmental risk.

A pre-natal developmental toxicity study has been conducted on Oxooil LS9. The no-observed-adverse-effect level (NOAEL) was 300 mg Oxooil LS9/kg b.w./day for the dams. None of the dams died prematurely.

Noteworthy clinical observations were noted only at the high dose level (1000 mg/kg b.w./day) in form of e.g. reduced motility, prone position and piloerection. In addition, a reduced body weight and reductions in food consumption were noted at the high dose level (1000 mg/kg b.w./day). No test item-related changes were noted during the macroscopic inspection at necropsy and for the organ weights of the heart, the kidneys and the liver.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was 300 mg Oxooil LS9/kg b.w./day. At the maternal toxic dose level of 1000 mg Oxooil/kg b.w./day 2 dams were noted with a total implantation loss. Furthermore, reduced placental and fetal body weights as well as an increased incidence of delayed ossifications of the metacarpalia and the sternebra(e) were noted at the high dose level. These dose-related findings are considered to be treatment-related. No dead fetuses, no malformations and no test item-related increase in the incidence of variations were noted during the examination of the fetuses

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2 - 31, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
January 22, 2001
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 30, 2008
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 3001545196
- Expiration date of the lot/batch: At least one year upon receipt (December 03, 2015) of the test item, that is Dezember 03, 2016


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At + 10°C to +25°C, in a tightly closed container.
Blanketing with nitrogen before recapping is recommended.
- Stability under test conditions: At least one year upon receipt of the test item
- Solubility and stability of the test substance in the solvent/vehicle: An analysis of the test item-formulation indicated correctly prepared and homogenised test item vehicle mixtures,
which were stable for at least 24 hours.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item formulations were freshly prepared every day.The test item was diluted in the vehicle to the appropriate concentrations using a magnetic stirrer and was administered orally at a constant volume once daily from the 6th to the 20th day of gestation. Stirring of the formulations was continued until the last animal of the dose group had been dosed.

Species:
rat
Strain:
other: CD® / Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sulzfeld, Germany
- Age at study initiation:
- Weight at study initiation:
- Housing: The animals are kept singly in MAKROLON cages (type III plus).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature of 22°C ± 3 °C (maximum range)
- Humidity (%): relative humidity of 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle

IN-LIFE DATES: From: May 3, 2016 To:TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 59 days
- Weight at study initiation: 180.8 - 250.8 g
- Housing: Except during the mating period, the dams were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm.
- Diet (e.gad libitum): Commercial diet ssniff® R/Z V1324 ad libitum
- Water (e.g. ad libitum): Drinking water (in drinking bottles) was offered ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): The ventilation rate of the animal room was between fifteen to twenty air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle

IN-LIFE DATES: From:18 May 2016 To: 26 May 2016
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency):Commercial diet was offered daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test laboratory has extensive experience with the vehicle corn oil and also in combination with the test item oxooil. Furthermore the test item mixture with the vehicle was verified regarding stability, homogenicity and concentration.
- Concentration in vehicle: The measured actual concentrations of the test item in the test item vehicle mixtures were between 99.8% and 109.1% of the nominal concentrations, indicating correctly prepared and homogenized formulations which were stable at room temperature for at least 24h.
- Amount of vehicle (if gavage): 2 mL/kg b.w.
- Lot/batch no. (if required): 15296403
-Source: supplier: Caesar & Loretz GmbH, 40721 Hilden, Germany
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- Impregnation procedure: cohoused
- Sexually mature ('proved') male rats of the same breed served as partners. The female breeding partners were randomly chosen.
- M/F ratio per cage: 1 M/1 F per cage
- Length of cohabitation: Cohabitation during the dark period; If findings were negative, mating was repeated with the same partner.
- Further matings after two unsuccessful attempts: no; Rats which did not become pregnant were excluded from the analysis of the results and replaced by other animals.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Treatment period: Day 6 to 20 of gestation
Frequency of treatment:
Once daily
Duration of test:
Start of mating May 2, 2016
First mating results May 3, 2016
First administration May 9, 2016
Termination of the in-life part May 31, 2016
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control
Dose / conc.:
100 mg/kg bw/day
Remarks:
low dose
Dose / conc.:
300 mg/kg bw/day
Remarks:
intermediate dose
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
high dose
No. of animals per sex per dose:
100 females in order to have at least 20 pregnant females per group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels have been selected in agreement with the Sponsor based on the results of a preliminary dose-range-finding study in rats
- Rationale for animal assignment (if not random):
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: early in in the morning and again in the afternoon of each working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Each day the body weight will be registered always at the same time

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION : Yes
- Time schedule for examinations: Daily monitoring by visual appraisal of the drinking water bottles will be maintained throughout the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21th
- Organs examined:
Examination of the dams:
Heart, kidney and liver were weighed
Examination of the fetuses:
Macroscopic inspection (gross evaluation) and weighting of the placentae.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Weight of placentae: Yes
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [ half per litter]
- Head examinations: No
Statistics:
Toxicology (and Pathology, if applicable) data will be captured, whenever possible,
using the departmental computerized systems (Provantis®8 Integrated preclinical software, Instem LSS Ltd.). Raw data not fully compatible with the computerized systems will be maintained on paper according to appropriate SOPs.
Historical control data:
The relationship between 'Variations and Retardations' and 'Malformations' should be seen under the aspect of cumulative historical data.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At the high dose level (1000 mg Oxooil LS9/kg b.w./day) several dams were noted with signs of clinical toxicity on several test days (e.g. a reduced motility (17 of 22), salivation (21 of 22), prone position (3 of 22), piloerection (3 of 22), pale faeces (9 of 22) and an increased (4 of 22) or decreased water consumption (2 of 22)).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the high dose level (1000 mg Oxooil LS9)/kg b.w./day) a statistically significantly reduced body weight was noted from gestation day 10 until laparotomy on gestation day 21 (max.: 15.0% below the value of the control group at the day of laparotomy). Body weight gain for the whole study period was 49.7% for the dams of the high dose group in comparison to 78.4% for the dams of the control group (p ≤ 0.01).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant (p ≤ 0.01) reductions in food consumption were noted at the high dose level (1000 mg Oxooil LS9/kg b.w./day) on several gestation days after the start of treatment (max. 36.4% below the value of the control group between gestation days 6 and 7).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At the high dose level (1000 mg Oxooil/kg b.w./day) 4 dams with an increased water consumption and 2 other dams with a decreased water consumption were noted by visual appraisal.
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:
effects observed, treatment-related
Description (incidence and severity):
Uterus and carcass weights: At the high dose level (1000 mg Oxooil LS9/kg b.w./day) statistically significant reductions were noted for the gravid uterus weight (22.4% below the value of the control group) and the carcass weight (12.5% below the value of the control group).

Further organ weights: The weighing of the heart, the kidneys and the liver revealed no test item-related differences between the dams of the control group and the treatment groups.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
A total resorption of all implants (a postimplantation loss of 100%) was noted for 2 of 22 dams of the high dose group (1000 mg Oxooil LS9/kg b.w./day), leading to a statistically significantly (p ≤ 0.01) increased post-implantation loss (10.9% in comparison to 4.7% in the control group).
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
pre and post implantation loss
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significantly (p ≤ 0.01) decreased fetal weight was noted at the high dose level (1000 mg Oxooil LS9/kg b.w./day). The fetal weight 13.4% below the value of the control group (male and female fetuses together).
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): A statistically significantly (p ≤ 0.01) decreased fetal weight was noted at the high dose level (1000 mg Oxooil LS9/kg b.w./day).
The fetal weight was 13.4% below the value of the control group (male and female fetuses together).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No malformation was noted during the macroscopic examination at laparotomy (external inspection and inspection of the inner organs and tissues for gross lesions).
Skeletal malformations:
no effects observed
Description (incidence and severity):
No malformation was noted during the skeletal examination according to DAWSON.
Visceral malformations:
no effects observed
Description (incidence and severity):
No malformation was noted during the soft tissue examination according to WILSON.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
A statistically significantly (p ≤ 0.01) decreased placental weight was noted at the high dose level (1000 mg Oxooil LS9/kg b.w./day). The placental weight at the high dose level was 9.9% below the value of the control group (male and female placentae combined).

Retardations: At the high dose level (1000 mg Oxooil LS9/kg b.w./day) the skeletal examination according to DAWSON revealed an increased incidence of delays in ossification for the metacarpalia and the sternebra(e) that are considered to be test item related.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
other: retardations: Increased incidence of delayed ossifications of the metacarpalia and the sternebra(e) at the high dose level
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg Oxooil LS9/kg b.w./day for the dams.
The no-observed-adverse-effect level (NOAEL) for the fetal organism was 300 mg Oxooil LS9/kg b.w./day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a prenatal developmental toxicity study, the test item Oxooil LS9 was administered orally to female rats at dose levels of 100, 300 or 1000 mg/kg b.w./day from the 6th to 20th day of pregnancy.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg Oxooil LS9/kg b.w./day for the dams.

None of the dams died prematurely.

Noteworthy clinical observations were noted only at the high dose level (1000 mg/kg b.w./day) in form of e.g. reduced motility, prone position and piloerection.

In addition, a reduced body weight and reductions in food consumption were noted at the high dose level (1000 mg/kg b.w./day).

No test item-related changes were noted during the macroscopic inspection at necropsy and for the organ weights of the heart, the kidneys and the liver.

 

The no-observed-adverse-effect level (NOAEL) for the fetal organism was 300 mg Oxooil LS9/kg b.w./day.

At the maternal toxic dose level of 1000 mg Oxooil/kg b.w./day 2 dams were noted with a total implantation loss. Furthermore, reduced placental and fetal body weights as well as an increased incidence of delayed ossifications of the metacarpalia and the sternebra(e) were noted at the high dose level. These dose-related findings are considered to be treatment-related.

No dead fetuses, no malformations and no test item-related increase in the incidence of variations were noted during the examination of the fetuses.

Justification for classification or non-classification

Within the scope of an OECD 422 reproductive/developmental screening study and a prenatal developmental toxicity study according to OECD 414, no classification for reproductive or developmental toxicity is indicated for Oxooil LS9 according to the general classification and labelling requirements for dangerous substances and preparations (Directive 67-548-EEC) or the classification, labelling and packaging (CLP) regulation (EC) No 1272/2008.