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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive Toxicity NOAEL (Rat): 1000 mg/kg bw/day

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-AUG-04 to TBD
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
yes
Remarks:
None of the deviations impacted the overall integrity of the study or the interpretation of the study results and conclusions.
Qualifier:
according to guideline
Guideline:
other: EPA Health Effects Test Guideline OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
yes
Remarks:
None of the deviations impacted the overall integrity of the study or the interpretation of the study results and conclusions.
GLP compliance:
yes
Limit test:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Higher Olefins & Poly Alpha Olefins vzw; Batch No. DBA0146456
- Purity, including information on contaminants, isomers, etc.: 100% (UVCB)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable in the vehicle when prepared and stored under the same conditions.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Clear colourless liquid

OTHER SPECIFICS
- Expiration date: 2023-MARCH-01
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model as it is an accepted rodent species for toxicity testing by regulatory agencies. The CRO (Charles River Den Bosch) has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males: 10-11 weeks old (at dosing); Females: 13-14 weeks old (at dosing)
- Weight at study initiation: Males: 281 to 345 grams; Females: 204 to 247 grams
- Fasting period before study: Not specified
- Housing:

On arrival; during the pretest (females only); and pre-mating period: animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm).

Mating phase: males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).

Post-mating phase: males were housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm).

Lactation phase: females were housed in Makrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams.

Locomotor activity monitoring: F0-animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.

- Use of restrainers for preventing ingestion (if dermal): no

- Diet (e.g. ad libitum): SM R/M-Z pelleted diest (SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum except during designated procedures. During motor activity measurements, animals did not have access to food for a maximum of 2 hours.

- Water (e.g. ad libitum): Municipal tap water via water bottles. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.

- Acclimation period: 8 days prior to start of the pretest period (females) or 8 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY: Analysis for nutritional components and environmental contaminants were provided by the supplier. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study. Periodic analysis of the water was performed it was considered that there were no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 22°C
- Humidity (%): 32% to 57%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2021-SEP-29 To: 2021-DEC-24
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Sigma-Aldrich (Steinheim, Germany)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared weekly and formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Dosing formulations were filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator, stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal. Dosing formulations were kept at room temperature until dosing and continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test material. No correction was made for the purity/composition of the test material.

VEHICLE

- Justification for use and choice of vehicle (if other than water): Corn oil; Corn oil selected based on trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and were not used for dosing.

- Concentration in vehicle: 0, 25, 75, or 250 mg/mL for the control, 100, 300, and 1000 mg/kg/day dose groups, respectively

- Amount of vehicle (if gavage): 4 mL/kg

- Lot/batch no. (if required): Not specified

- Purity: Not specified
Details on mating procedure:
- M/F ratio per cage: 1:1 (After 14 days of treatment)
- Length of cohabitation: maximum of 14 days was allowed for mating
- Proof of pregnancy: evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility (non-mated females (Nos. 68 and 70) were re-mated once with a male of proven fertility of the same group for a maximum of 7 days)
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol:

For one couple (Male No. 30, Female No. 70), detection of mating was not confirmed in first instance. Female No. 70 was re-mated with Male No. 29. During the re-mating period, obvious weight gain and palpation confirmed that Female No. 70 was pregnant. Consequently, the couple was separated 6 days after the start of the re-mating period. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continued di-estrous during the mating in this female. The mating date of this female was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post coitum. Based on this estimated mating date, it was determined that Male No. 30 was the male that mated with Female No. 70.

Mating was not confirmed in first instance for re-mated Female No. 68. Evidence of mating was obtained indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continued di-estrous during the extended mating period in this female. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum. It was assumed that the male used for the re-mating (Male No. 27) mated with Female No. 68. However, as the calculated Day 0 post-coitum coincided with the first day of the re-mating period and as the actual gestation time was unknown, it cannot be determined with certainty which of the two males (No. 28 or 27) mated successfully.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples (duplicate) were collected for analysis as follows:
Concentration analysis - week 1 of treatment; sample collected from approximately middle of dosing container for all dose groups; Homogeneity analysis - week 1 of treatment; sample collected from approximately top, middle, and bottom of the dosing container for dose groups 2 (100 mg/kg/day) and 4 (1000 mg/kg/day). Samples to be analyzed were transferred at room temperature under normal laboratory light conditions to the analytical laboratory and analyses were performed using a validated analytical proceure (Test Facility Study No. 20296615).


Concentration analysis:
Temperature was set to maintain 18 - 22°C and the acceptance criteria set as: mean sample concentration results within or equal to ± 15% of theoretical concentration.

Homogeneity analysis: Temperature was set to maintain 18 - 22°C and the acceptance criteria set as: relative standard deviation (RSD) of concentrations of ≤10% for each dose group.

Stability analysis: Stability analyses was performed previously in conjunction with the method development and validation study (Test Facility Study No. 20296615) and demonstrated that the test material was stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
Males: 7 days a week for a minimum of 28 days, including at least 2 weeks of treatment prior to mating and during the mating period up to and including the day before scheduled necropsy.

Females: 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy.
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 - Control (corn oil)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2 - Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3 - Intermediate dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4 - High dose
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected based on the results of a 10 Day Dose Range Finder (DRF) with oral administration of 1-Dodecene, dimers in rats (Test Facility Reference No. 20296616) and in an attempt to produce graded responses to the test material.

- Rationale for animal assignment: Animals were randomly assigned to groups at arrival. Males and females were randomized separately.

- Fasting period before blood sampling for clinical biochemistry: F0-males: Yes (overnight with a maximum of 24 hours); F0-females: No

- Justifiction for Route: The oral route of exposure was selected because this is the intended route of human exposure this is a possible route of human exposure during manufacture, handling or use of the test material.

- Justification for Dose Levels:
A Dose Range Finder (DRF) (Test Facility Reference No. 20296616) was conducted to select dose levels for the Main study (Test Facility Study No. 20286618), and to determine the peak effect of occurrence of clinical signs after dosing. For the DRF, all animal activities and necropsy were performed

The test material with vehicle was administered to the appropriate animals by once daily oral gavage for 10 consecutive days at dose levels of 600 or 1000 mg/kg/day. Based on the results of the DRF, dose levels selected for the Main study were 100, 300 and 1000 mg/kg/day.

Since no clinical signs were observed in the DRF, clinical observations were conducted and functional observations were started in the Main study after dosing at no specific time point, but within a similar time period after dosing for the respective animals.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day (except on days of receipt and necropsy where frequency was at least once daily). Arena observations were conducted once before the first administration of the test material and weekly during the Treatment Period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily, beginning during the first administration of the test material and lasting throughout the Dosing Periods up to the day prior to necropsy. During the Dosing Period, observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of all animals were recorded on Day 1 of treatment (prior to dosing) and weekly thereafter. Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: On a regular basis throughout the study by visual inspection of the water bottles. However, no quantitative investigation was introduced as no effect was suspected.

OTHER:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (F0-males: fasted overnight with a maximum of 24 hours; F0-females: No)
- How many animals: Selected F0-animals (5/sex/group)
- Parameters checked in table [No.2] were examined.

COAGULATION
Blood samples were processed for plasma, and plasma was analyzed for Prothrombin time (PT) and Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy
- Animals fasted: Yes (F0-males: fasted overnight with a maximum of 24 hours; F0-females: No)
- How many animals: Selected F0-animals (5/sex/group)
- Parameters checked in table [No.3] were examined.

SERUM HORMONES: Yes
- Time of blood sample collection: On the day of scheduled necropsy
- Animals fasted: Yes Yes (F0-males: fasted overnight with a maximum of 24 hours; F0-females: No)
- How many animals: Selected F0-animals (5/sex/group)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Males: Week 4 of treatment; Females: last week of lactation (PND 6-13)
- Dose groups that were examined: All dose groups
- Battery of functions tested: Hearing ability; Pupillary reflex; Static righting reflex; Fore- and hind-limb grip strength; Locomotor activity.
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.
Sperm parameters (parental animals):
For the testes of all selected males of Groups 1 and 4 and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

To reduce variability among the litters, on PND 4, eight pups from each litter of equal sex distribution (if possible) were selected.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals: after a minimum of 28 days of administration.
- Maternal animals: All surviving animals: PND 14-16, or after total litter loss, or failure to deliver

Unscheduled Deaths/Euthanasia
Females with total litter loss (No. 78) were weighed and deeply anesthetized using isoflurane and subsequently exsanguinated within 24 hours after the last pup was found dead or missing. They underwent necropsy, and specified tissues were retained and weighed.

Scheduled Euthanasia
Animals surviving until scheduled euthanasia (Males: after a minimum of 28 days of administration ; Females: PND 14-16, or after total litter loss, or failure to deliver) were weighed, and deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Scheduled necropsies were conducted as follows:

- Males (which sired or failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
- Females which delivered: PND 14-16.
- Females which failed to deliver: With evidence of mating (No. 56): Day 25 post-coitum.
- Without evidence of mating (No. 68): 25 days after the last day of the mating period.

All males surviving to scheduled necropsy were fasted overnight (water was available) for approximately 24 hours before necropsy. F0 females were not fasted.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera (see Table 4 and 5).

HISTOPATHOLOGY / ORGAN WEIGHTS

Organ Weights – F0 Generation
The organs identified in the tables 5 and 6 were weighed at necropsy for all scheduled euthanasia animals and females with total litter loss. Organ weights were not recorded for animals euthanized in poor condition or in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes (see Table 7 and 8)
Histopathology: F0 Generation
Representative samples of the tissues identified in table 7 and 8 below were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).

The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
- Selected animals and unscheduled deaths (sacrificed in extremis): Tissues identified in Table 7 (except animal identification, aorta, nasopharynx, esophagus, harderian gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue).
- Males that failed to sire, females that failed to deliver pups and females with total litter loss: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
- Females with total litter loss: Mammary gland
- Remaining animals: Gross lesions/masses.

For the testes of all selected males of Groups 1 and 4 and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at PND 16 days of age.
- These animals were subjected to postmortem examinations: macroscopic examination as follows:

Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital. The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Unscheduled Deaths – F1 Generation:
Recognizable fetuses of females that were euthanized in extremis (No. 43) were examined externally and sexed (both externally and internally). Live fetuses were euthanized by decapitation. Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Scheduled Euthanasia – F1 Generation:
On PND 4, the surplus pups were euthanized by decapitation. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for (complete) blood sampling were the same pups as selected for thyroid preservation.

Additionally, blood was collected from two pups per litter and the thyroid as well from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. Serum derived from the PND 14-16 pups was divided in two aliquots. One aliquot was used for measurement of thyroid hormones using the IMMULITE® 1000 analyser (TSH). The other aliquot and the pooled serum of the PND 4 pups was used for measurement of T3 and T4 using LC-MS. Measurement was performed according to the bioanalytical method validated in Test Facility Study No. 20213516.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not evaluated
Statistics:
For information on statistics please see 'Any other information on materials and methods incl. tables'.
Reproductive indices:
1. Mating index (%): (Number of females mated / Number of females paired) x 100
2. Fertility index (%): (Number of pregnant females / Number of females mated) x 100
Offspring viability indices:
1. Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100

2. Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100

3. Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100

4. Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100

5. Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100

6. Viability index 1 (%): (Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100

7. Viability index 2 (%): (Number of live offspring on Day 7 after littering / Number live offspring on Day 4 (after culling)) x 100

8. Viability index 3 (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 7 after littering) x 100

9. Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100

10. Cumulative survival index: ((Number of live offspring on Day 13 / Number of live offspring on Day 4 (after culling)) x (Number of live offspring on Day 4 (before culling) / Total number of offspring born)) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were observed either during daily detailed clinical or during the weekly arena observations.

Incidental findings observed included scabs, wounds, and alopecia. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be treatment-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No treatment-related mortality was observed in the F0 animals.

One control female (No. 43) was sacrificed in extremis on Day 23 post coitum. The female had a pale appearance and piloerection and at the point 3 pups were born of which 1 was alive and 2 were found dead. As no more pups were born and the condition of the animal deteriorated, the animal was sacrificed in extremis. At necropsy, the uterus of this female contained 3 alive and 5 dead fetuses. No other macroscopic or microscopic findings were observed. Based on this, the condition of this female was considered to be related to delivery difficulties.

One female in the 100 mg/kg/day (No. 60) dose group was sacrificed in extremis on Day 1 of lactation. The animal was found with a pale appearance and piloerection. Due to the deteriorating condition of this animal, it was sacrificed in extremis. Additionally, all 11 delivered pups were found to be dead. At necropsy, a pale liver was observed corresponding to microscopic moderate multifocal necrosis in the liver which likely contributed to the moribundity. Based on the single incidence of this event in the low dose group, the condition of this animal was not considered to be treatment-related.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain were considered to be unaffected by treatment.

Females at 1000 mg/kg/day displayed slightly lower (6% lower than control) absolute body weight on Day 4 of lactation was. As body weight gain was similar over this period and as this was only an incidental occurrence, this temporary lower body weight was not considered to be treatment-related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight was not affected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles. However, no quantitative investigation was introduced as no effect was suspected.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological parameters of rats were unaffected by treatment.

A reduction of large unstained cell (LUC) counts was observed in male rats at 300 mg/kg/day. However, in the absence of a dose-related trend, this was considered to be unrelated to treatment.

Coagulation parameters of rats were unaffected by treatment. A shorter prothrombin time (PT) observed in male rats at 100 mg/kg/day was considered to be unrelated to treatment in the absence of a dose-related trend.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical chemistry parameters of male rats were unaffected by treatment. In female rats, a reduction in total bilirubin levels (TBIL) was observed at 100, 300, and 1000 mg/kg/day (0.69x, 0.71x and 0.66x of control, respectively). In the absence of corroborative histopathological findings, this was considered not adverse. Increased sodium levels were observed in female rats at 300 mg/kg/day but this considered to be minor and in the absence of a dose-related trend, unrelated to treatment.
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
Serum levels of thyroxine (T4) and thyroid-stimulating hormone (TSH) in male and female rats were unaffected by treatment.

Serum total triiodothyronine (T3) levels in F0 males at 100 mg/kg/day were increased (Group mean values were 1.29x of control; however, an increase in the SD was also observed). Mean values were within the historical control data range and values at 300 mg/kg/day and 1000 mg/kg/day were comparable with the control group. Therefore, although treatment-related, this increase was not considered as adverse.

Serum total T3 levels were increased in F0 females at 1000 mg/kg/day (1.26x of control). No historical control data for total T3 in lactating females was available at the Testing Facility. However, since only 1/10 individual females displayed a T3 value above the highest individual control value, the mean increase of T3 at 1000 mg/kg/day was considered unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength of the fore legs (males and females), and motor activity (females only) were considered unaffected by treatment at 100 and 300 mg/kg/day.

Females at 1000 mg/kg/day displayed decreased grip strength of the hind legs (0.63x of control). In the absence of clinical signs and corroborative histopathological findings, this was not considered as adverse.

An apparent upward trend in motor activity (total movements and ambulations) observed in male rats of all treatment groups was considered to have arisen as a result of slightly low control values and therefore not considered treatment-related.

All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathology did not reveal any microscopic observations related to treatment with the test material. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no treatment-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment with all females having regular cycles of 4 to 5 days.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating Index:
Mating index was not affected by treatment with all females showing evidence of mating.

Precoital Time:
Precoital time was considered unaffected by treatment and most females showed evidence of mating within 4 days. One female (No. 61) at 300 mg/kg/day showed evidence of mating after 13 days. One female (No. 68) at 300 mg/kg/day that was probably mated during the extended mating period had an estimated precoital time of 1 day. Mating was overlooked in two females at 300 mg/kg/day (Nos. 68 and 70). As these females turned out to be pregnant and delivered a healthy litter, the mating date of these females was estimated at 21 days prior to delivery.

Number of Implantation Sites:
The number of implantation sites was considered unaffected by treatment with the mean number of implantation sites being 11.9, 11.6, 12.1, and 12.9 for the control, 100, 300, and 1000 mg/kg/day groups, respectively.

Fertility Index:
Fertility index was not affected by treatment with fertility indices being 100, 90, 100, and 100% for the control, 100, 300, and 1000 mg/kg/day groups, respectively. A lower gestation index observed at 100 mg/kg/day was the result of one female at 100 mg/kg/day (No. 56) that was not pregnant. At the low incidence and in absence of a dose related trend this finding was considered unrelated to treatment with the test material.

Parturition/Maternal Care:
One control female (No. 43) was sacrificed in extremis on Day 23 post-coitum due to presumed delivery difficulties . All other females showed no signs of difficult or prolonged parturition or deficiencies in maternal care. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were observed among pups.

Observed clinical signs like alopecia and a scab on the snout remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Viability Index:

Viability index 1 (number of live offspring on PND 4 (before culling) as percentage of the number of live offspring on PND 1) was unaffected by treatment with the test material. Viability index 1 was 100%, 100%, 98%, and 100% for the control, 100, 300, and 1000 mg/kg/day groups, respectively. Two pups at 300 mg/kg/day were found dead (on PND 3 and 4). As these were incidental findings which occurred without dose-related trend, they were not considered related to treatment.

Viability index 2 (number of live offspring on PND 7 as percentage of the number of live offspring on PND 4 (after culling)) was unaffected by treatment and viability index 2 was 100% for all groups.

Viability index 3 (number of live offspring on PND 13 as percentage of the number of live offspring on PND 7) was unaffected by treatment and viability index 3 was 100% for all groups.

Lactation Index:

The lactation index (the number of live offspring on Day 13 after littering as percentage of live offspring on Day 4 (after culling)) was not affected by treatment with the test material. No pups were found dead/missing between PNDs 5 and 13, resulting in a lactation index of 100% for all groups.

Cumulative Survival Index:

The cumulative survival index (((number of live offspring on Day 13 after litter / the number of live offspring on Day 4 (after culling)) x (number of live offspring on Day 4 (before culling) / the total number of offspring born)) x 100%) was not affected by treatment. Cumulative survival index was 100%, 88%, 98%, and 90% for the control, 100, 300, and 1000 mg/kg/day groups, respectively.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were unaffected by treatment.
Food consumption and compound intake (if feeding study):
not examined
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:
no effects observed
Description (incidence and severity):
Serum T3 and T4 levels in PND 4 pups and T3, T4 and TSH serum levels in male and female PND 14-16 pups were considered unaffected by treatment.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment with the test material.
Nipple retention in male pups:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with the test material had no effect on areola/nipple retention.

One male pup of the control group, and two male pups at 1000 mg/kg/day were noted with one nipple each. At the incidence observed, these observations were not considered to be treatment-related.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross necropsy did not reveal any remarkable treatment-related findings in the pups.
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Litter Size:

Litter size was not affected by treatment with the test material and live litter sizes were 10.9, 9.7, 11.5, and 10.0 living pups/litter for the control, 100, 300, and 1000 mg/kg/day groups, respectively.

The slightly lower live litter sizes at 100 and 1000 mg/kg/day could be attributed to Female Nos. 60 (at 100 mg/kg/day) and 78 (at 1000 mg/kg/day) which had total litter loss on Day 1 of lactation on which all pups were found dead at first litter check.

Sex Ratio:

Sex ratio remained unaffected post exposure to the test material.

In the 100 mg/kg/day group, the sex of one pup that was found dead at first litter check (Pup No. 11 of Litter No. 60) could not be determined (due to cannibalism). This pup was sexed as female at random. In the 1000 mg/kg/day group, the sex of four pups that were found dead at first litter check (Litter No. 78) could not be determined (due to cannibalism). These pups were sexed as 2 females and 2 males at random.

Gestation Index and Duration:

Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were unaffected by treatment with gestation indices determined to be 90%, 89%, 100%, and 90% for the control, 100, 300, and 1000 mg/kg/day groups, respectively.

The lower gestation index observed in the control group was attributed to one female that was sacrificed in extremis on Day 23 post coitum during delivery. The lower gestation index observed at 100 and 1000 mg/kg/day was related to one female in each dose group (Female Nos. 60 and 78, respectively) with a total litter loss on Day 1 of lactation. Besides (advanced) autolysis and cannibalism, no other macroscopic findings were observed in the pups of both litters. As these total litter loss events were incidental and occurred without any histopathological findings, they were considered unrelated to treatment.

Post-Implantation Survival Index:

The post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was considered unaffected in female rats treated at 100 and 300 mg/kg/day. Post-implantation survival index was 92%, 95%, and 93% for the control, 100, and 300 mg/kg/day groups, respectively. Females at 1000 mg/kg/day showed a reduced post-implantation survival index (85% compared to 92% in control). While this was considered to be treatment-related, no effects were observed on litter size and in absence of any corroborative histopathological findings, this reduction was considered not adverse.

For one female (No. 65) in the 300 mg/kg/day dose group, the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 16 days of lactation. As this was an incidental finding which occurred in absence of a dose-related trend, it was considered unrelated to treatment.

One female (No. 43) in the control group was excluded from calculation of the post-implantation survival index as this female was sacrificed in extremis during delivery on Day 23 post-coitum at which delivery status of the litter was incomplete.

Live Birth Index:

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment with the test material. The live birth indices were 100%, 88%, 100%, and 90% for the control, 100, 300, and 1000 mg/kg/day groups, respectively. The reduced live birth indices observed at 100 and 1000 mg/kg/day could mainly be attributed to Female Nos. 60 (at 100 mg/kg/day) and 78 (at 1000 mg/kg/day) which had total litter loss on Day 1 of lactation on which all pups were found dead at first litter check.

One pup at 100 mg/kg/day was found dead at first litter check. The beginning of autolysis was noted at necropsy. As the mortality incidence remained within the range considered normal for pups of this age and as no dose-related trend was observed, the death of this pups was considered unrelated to treatment with the test material.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Formulation Analysis:

 

Concentration analysis:

 

The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 85% 115% of target concentration). A small response at the retention time of the test material was observed in the chromatograms of the Group 1 formulation prepared for use. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks.

 

Homogeneity Analysis:

 

The formulations of Groups 2 and 4 were homogeneous (i.e., coefficient of variation ≤ 10%).

Table 9. Results of Formulation Analysis

Date of

analysis

Concentration (mg/mL)

Recovery (%)

Target

Nominal

Analyzed

Individual

Mean

2021-OCT-21

25.0

26.5

23.4

88

92

24.6

23.5

96

22.2

20.8

94

250

252

227

90

88

248

217

88

250

218

87

Table 10. Body Weights (grams) Result Summary

 

 

Group 1

(Control –

0 mg/kg/day)

Group 2

(100 mg/kg/day)

Group 3

(300 mg/kg/day)

Group 4

(1000 mg/kg/day)

Males

Pre-mating

 

Day 1

Week 1

Mean

311

308

317

312

SD

11.1

15.4

16.4

25.3

N

10

10

10

10

 

Day 8

Week 2

Mean

336

331

341

334

SD

13.6

19.0

17.8

29.8

N

10

10

10

10

Mating Period

 

Day 1

Week 1

Mean

352

345

357

352

SD

16.8

21.9

21.3

30.5

N

10

10

10

10

 

Day 8

Week 2

Mean

360

358

368

361

SD

19.4

22.4

22.6

31.3

N

10

10

10

10

 

Day 15

Week 3

Mean

374

372

378

376

SD

21.3

21.3

23.1

34.3

N

10

10

10

10

 

Day 22

Week 4

Mean

 

 

398

 

SD

 

 

---

 

N

 

 

1

 

Females

Pre-mating

 

Day 1

Week 1

Mean

227

227

224

220

SD

7.4

8.7

12.9

12.2

N

10

10

10

10

 

Day 8

Week 2

Mean

232

231

229

223

SD

5.9

9.2

13.6

8.4

N

10

10

10

10

Mating Period

 

Day 1

Week 1

Mean

239

238

231

232

SD

5.3

10.7

14.5

12.7

N

10

10

10

10

 

Day 8

Week 2

Mean

 

 

261

 

SD

 

 

7.1

 

N

 

 

2 x

 

 

Day 15

Week 3

Mean

 

 

262

 

SD

 

 

---

 

N

 

 

1 x

 

Post Coitum

 

Day 0

Mean

238

236

236

231

SD

7.2

9.2

17.0

11.2

N

10

9

8

10

 

Day 4

Mean

253

247

246

243

SD

7.2

11.8

16.0

12.0

N

10

9

8

10

 

Day 7

Mean

261

256

253

251

SD

6.1

10.6

16.1

12.0

N

10

9

8

10

 

Day 11

Mean

277

271

267

266

SD

7.4

13.4

14.9

13.4

N

10

9

8

10

 

Day 14

Mean

286

282

276

277

SD

7.5

11.9

14.6

14.0

N

10

9

8

10

 

Day 17

Mean

312

304

300

301

SD

8.5

9.9

14.0

17.0

N

10

9

8

10

 

Day 20

Mean

349

343

328

340

SD

14.3

12.6

28.4

20.7

N

10

9

8

10

Lactation

 

Day 1

Mean

277

272

268

263

SD

11.6

15.0

16.9

12.7

N

9

8

10

10

 

Day 4

Mean

289

286

275

271*

SD

7.3

13.6

16.5

12.5

N

9

8

10

9

 

Day 7

Mean

291

288

287

280

SD

7.4

14.1

15.8

16.6

N

9

8

10

9

 

Day 13

Mean

303

301

292

292

SD

6.9

13.2

15.3

12.9

N

9

8

10

9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

x Explanations for excluded data are listed in the tables of the individual values presented in the study report.

Table 11. Body Weight Gain (%) Result Summary

 

 

Group 1

(Control –

0 mg/kg/day)

Group 2

(100 mg/kg/day)

Group 3

(300 mg/kg/day)

Group 4

(1000 mg/kg/day)

Males

Pre-mating

 

Day 1

Week 1

Mean

0

0

0

0

SD

0.0

0.0

0.0

0.0

N

10

10

10

10

 

Day 8

Week 2

Mean

8

7

7

7

SD

1.9

1.3

1.0

1.6

N

10

10

10

10

Mating Period

 

Day 1

Week 1

Mean

13

12

13

13

SD

2.5

2.5

1.6

2.2

N

10

10

10

10

 

Day 8

Week 2

Mean

16

16

16

16

SD

3.0

2.7

2.0

1.7

N

10

10

10

10

 

Day 15

Week 3

Mean

20

21

19

21

SD

3.8

3.1

3.0

3.0

N

10

10

10

10

 

Day 22

Week 4

Mean

 

 

21

 

SD

 

 

---

 

N

 

 

1

 

Females

Pre-mating

 

Day 1

Week 1

Mean

0

0

0

0

SD

0.0

0.0

0.0

0.0

N

10

10

10

10

 

Day 8

Week 2

Mean

2

2

2

2

SD

2.0

2.1

0.8

2.2

N

10

10

10

10

Mating Period

 

Day 1

Week 1

Mean

5

5

3

6

SD

2.2

1.4

1.8

2.9

N

10

10

10

10

 

Day 8

Week 2

Mean

 

 

13

 

SD

 

 

0.8

 

N

 

 

2 x

 

 

Day 15

Week 3

Mean

 

 

16

 

SD

 

 

---

 

N

 

 

x

 

 

Day 22

Week 4

Mean

 

 

---

 

SD

 

 

---

 

N

 

 

0 x

 

 

Day 29

Week 5

Mean

 

 

---

 

SD

 

 

---

 

N

 

 

0 x

 

 

Day 36

Week 6

Mean

 

 

---

 

SD

 

 

---

 

N

 

 

0 x

 

Post Coitum

 

Day 0

Mean

0

0

0

0

SD

0.0

0.0

0.0

0.0

N

10

9

8

10

 

Day 4

Mean

6

5

4

5

SD

1.9

2.6

1.4

1.9

N

10

9

8

10

 

Day 7

Mean

10

9

7

9

SD

2.9

2.7

1.5

2.2

N

10

9

8

10

 

Day 11

Mean

16

15

13

15

SD

3.6

3.7

3.3

3.1

N

10

9

8

10

 

Day 14

Mean

20

20

17

20

SD

3.1

3.3

3.1

2.6

N

10

9

8

10

 

Day 17

Mean

31

29

27

30

SD

4.6

3.2

5.9

3.6

N

10

9

8

10

 

Day 20

Mean

47

45

39

47

SD

8.2

4.7

11.0

4.6

N

10

9

8

10

Lactation

 

Day 1

Mean

0

0

0

0

SD

0.0

0.0

0.0

0.0

N

9

8

10

10

 

Day 4

Mean

4

5

3

4

SD

2.9

3.1

3.7

1.8

N

9

8

10

9

 

Day 7

Mean

5

6

7

7

SD

2.7

2.6

9.2

3.0

N

9

8

10

9

 

Day 13

Mean

9

11

9

12

SD

4.1

4.5

6.0

3.6

N

9

8

10

9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

x Excluded data (Explanations listed in the tables of the individual values presented in the study report)

Table 12. Food Consumption (g/animal/day) Result Summary

 

 

Group 1

(Control –

0 mg/kg/day)

Group 2

(100 mg/kg/day)

Group 3

(300 mg/kg/day)

Group 4

(1000 mg/kg/day)

Males

Pre-mating

 

Days 1-8

Weeks 1-2

Mean

22

22

23

22

SD

1.7

1.4

1.7

1.3

N (Cage)

2

2

2

2

 

Days 8-15

Weeks 2-3

Mean

21

21

22

22

SD

1.8

1.2

1.1

0.8

N (Cage)

2

2

2

2

Mean of means over Pre-mating

Mean

22

21

23

22

Mating Period

 

Days 1-8

Weeks 1-2

Mean

22

21

24

23

SD

1.2

0.4

2.8

1.9

N (Cage)

2

2

2

2

 

Days 8-15

Weeks 2-3

Mean

20

19

21

21

SD

1.4

0.8

1.6

0.9

N (Cage)

2

2

2

2

Mean of means over Mating Period

Mean

21

20

23

22

Females

Pre-mating

 

Days 1-8

Weeks 1-2

Mean

15

15

15

15

SD

0.1

0.5

1.0

0.5

N (Cage)

2

2

2

2

 

Days 8-15

Weeks 2-3

Mean

15

15

15

15

SD

0.3

0.8

0.8

0.9

N (Cage)

2

2

2

2

Mean of means over Pre-mating

Mean

15

15

15

15

Post-Coitum

 

Days 0-4

Mean

19

19

18

19

SD

1.0

2.5

1.7

1.2

N

9

9

8

9

 

Days 4-7

Mean

19

19

17

18

SD

2.4

4.3

2.0

2.3

N

10

9

8

10

 

Days 7-11

Mean

19

18

18

19

SD

2.2

1.8

2.1

1.4

N

10

8

8

10

 

Days 11-14

Mean

19

19

18

20

SD

1.4

2.7

1.6

1.2

N

10

9

8

10

 

Days 14-17

Mean

21

20

20

21

SD

1.0

2.5

1.5

1.5

N

7

7

8

8

 

Days 17-20

Mean

23

22

27

22

SD

1.8

2.9

15.8

1.8

N

10

9

8

10

Mean of means

 

20

20

20

20

Lactation

 

Days 1-4

Mean

30

32

29

29

SD

7.1

4.5

6.2

3.0

N

9

8

10

9

 

Days 4-7

Mean

35

37

37

38

SD

7.3

3.2

6.0

2.9

N

9

8

10

9

 

Days 7-13

Mean

46

48

46

49

SD

10.7

3.5

7.8

2.4

N

9

8

10

9

Mean of means

 

37

39

37

39

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

x Excluded data (Explanations listed in the tables of the individual values presented in the study report)

Table 13. Relative Food Consumption (g/Kg body weight/day) Result Summary

 

 

Group 1

(Control –

0 mg/kg/day)

Group 2

(100 mg/kg/day)

Group 3

(300 mg/kg/day)

Group 4

(1000 mg/kg/day)

Males

Pre-mating

 

Days 1-8

Weeks 1-2

Mean

66

65

67

67

SD

4.4

1.7

1.5

1.4

N (Cage)

2

2

2

2

 

Days 8-15

Weeks 2-3

Mean

64

63

66

66

SD

4.6

1.2

0.1

2.9

N (Cage)

2

2

2

2

Mean of means over Pre-mating

Mean

65

64

66

67

Mating Period

 

Days 1-8

Weeks 1-2

Mean

61

59

66

65

SD

2.8

1.7

4.3

0.8

N (Cage)

2

2

2

2

 

Days 8-15

Weeks 2-3

Mean

54

52

56

56

SD

2.3

0.3

1.6

2.3

N (Cage)

2

2

2

2

Mean of means over Mating Period

Mean

57

56

61

60

Females

Pre-mating

 

Days 1-8

Weeks 1-2

Mean

64

64

64

65

SD

0.6

1.7

5.3

2.5

N (Cage)

2

2

2

2

 

Days 8-15

Weeks 2-3

Mean

65

67

66

67

SD

1.6

3.1

4.6

4.4

N (Cage)

2

2

2

2

Mean of means over Pre-mating

Mean

64

65

65

66

Post-Coitum

 

Days 0-4

Mean

75

78

75

78

SD

4.7

7.8

8.4

4.7

N

9

9

8

9

 

Days 4-7

Mean

73

74

69

71

SD

8.5

14.3

6.9

7.9

N

10

9

8

10

 

Days 7-11

Mean

69

67

69

70

SD

7.0

5.0

7.4

6.5

N

10

8

8

10

 

Days 11-14

Mean

66

67

67

71

SD

4.6

7.5

6.1

4.8

N

10

9

8

10

 

Days 14-17

Mean

67

66

67

68

SD

3.3

6.2

3.7

3.7

N

7

7

8

8

 

Days 17-20

Mean

65

65

87

64

SD

5.2

7.3

64.2

6.2

N

10

9

8

10

Mean of means

 

69

69

72

70

Lactation

 

Days 1-4

Mean

102

111

107

108

SD

24.7

15.3

23.2

12.1

N

9

8

10

9

 

Days 4-7

Mean

122

130

127

136

SD

25.7

12.5

19.1

4.8

N

9

8

10

9

 

Days 7-13

Mean

152

161

157

169

SD

35.1

13.4

24.9

6.7

N

9

8

10

9

Mean of means

 

125

134

130

138

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

x Excluded data (Explanations listed in the tables of the individual values presented in the study report)

Table 14. Functional Observations Summary

 

 

Group 1

(Control –

0 mg/kg/day)

Group 2

(100 mg/kg/day)

Group 3

(300 mg/kg/day)

Group 4

(1000 mg/kg/day)

Males

End of Treatment

 

Hearing

Score 0/1

Median

0

0

0

0

N

5

5

5

5

 

Pupil L

Score 0/1

Median

0

0

0

0

N

5

5

5

5

 

Pupil R

Score 0/1

Median

0

0

0

0

N

5

5

5

5

 

Static R

Score 0/1

Median

0

0

0

0

N

5

5

5

5

 

Grip Fore

gram

Mean

948

893

761

823

SD

140

94

200

90

N

5

5

5

5

 

Grip Hind

gram

Mean

625

493

599

556

SD

79

50

96

89

N

5

5

5

5

Females

Hearing

Score 0/1

Median

0

0

0

0

N

5

5

5

5

 

Pupil L

Score 0/1

Median

0

0

0

0

N

5

5

5

5

 

Pupil R

Score 0/1

Median

0

0

0

0

N

5

5

5

5

 

Static R

Score 0/1

Median

0

0

0

0

N

5

5

5

5

 

Grip Fore

gram

Mean

1335

1313

1200

1213

SD

257

48

126

232

N

5

5

5

5

 

Grip Hind

gram

Mean

625

513

481

396*

SD

113

138

151

85

N

5

5

5

5

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 15. Summary of Select Hematology and Coagulation Values: F0 Generation

Day: 30 Relative to Start Date (Males)

 

Reporting Hematology

Reporting Coagulation

LUC (10^9/L)

PT (sec)

Statistical Test

[G]

[G]

Group 1

(Control – 0 mg/kg/day)

Mean

0.064

17.14

SD

0.019

2.66

N

5

5

 

Group 2

(100 mg/kg/day)           

Mean

0.042

16.04*

SD

0.008

0.46

N

5

5

tCtrl

0.66

0.94

 

Group 3

(300 mg/kg/day)

Mean

0.036*

16.42

SD

0.005

0.41

N

5

5

tCtrl

0.56

0.96

 

Group 4

(1000 mg/kg/day)

Mean

0.044

16.28

SD

0.017

0.34

N

5

4

tCtrl

0.69

0.95

[G] - Anova & Dunnett: * = p ≤ 0.05

LUC: Large unstained cells

PT: Prothrombin Time

Table 16. Summary of Select Biochemistry Values: F0 Generation

Day: 51 Relative to Start Date (Females)

 

Reporting Biochemistry

TBIL (µmol/L)

NA (mmol/L)

Statistical Test

[G1]

[G1]

Group 1

(Control – 0 mg/kg/day)

Mean

3.06

143.3

SD

0.50

1.1

N

5

5

 

Group 2

(100 mg/kg/day)           

Mean

2.10*

143.0

SD

0.63

1.2

N

5

5

tCtrl

0.69

1.00

 

Group 3

(300 mg/kg/day)

Mean

2.18*

145.6*

SD

0.35

0.9

N

5

5

tCtrl

0.71

1.02

 

Group 4

(1000 mg/kg/day)

Mean

2.02*

142.4

SD

0.51

1.1

N

5

5

tCtrl

0.66

0.99

[G1] - Anova & Dunnett: * = p ≤ 0.05

TBIL: Total Bilirubin

NA: Sodium

Table 17. Summary of Macroscopic Findings (F0 Generation)

 

Group 1

(Control –

0 mg/kg/day)

Group 2

(100 mg/kg/day)

Group 3

(300 mg/kg/day)

Group 4

(1000 mg/kg/day)

Males

End of Treatment

 

Animals examined

10

10

10

10

Animals without findings

9

8

7

7

Animals affected

1

2

3

3

 

Stomach

 

            Focus/foci

0

0

1

0

Liver

 

            Right median lobe constricted

0

1

0

0

            Enlarged

0

0

0

1

Kidneys

 

            Pelvic Dilation

0

0

0

1

Testes

 

            Reduced in size

0

1

0

0

Epididymides

 

            Nodule(s)

1

0

0

0

            Reduced in size

0

1

0

0

Seminal vesicles

 

            Enlarged

0

0

0

1

Thyroid gland

 

            Enlarged

0

0

1

1

Skin

 

            Scab formation

0

0

1

0

Females

Intercurrent Death

 

Animals examined

1

1

 

1

Animals affected

1

1

 

1

 

General observations

 

      Incomplete delivery, 2 dead pups, 1 alive pup

1

0

 

0

      Total litter loss

0

1

 

1

Liver

 

       Discolouration

0

1

 

1

Uterus

 

        Contents:

1

0

 

0

 

 

End of Treatment

 

Animals examined

9

9

10

9

Animals without findings

7

7

5

9

Animals affected

2

2

5

0

 

Brain

 

           Discolouration

0

0

1

0

Lungs

 

           Focus/foci

0

1

0

0

Ovaries

 

           Cyst(s)

0

1

0

0

Thyroid gland

 

           Enlarged

1

0

0

0

           Reduced in size

0

0

1

0

Thymus

 

         Focus/foci

1

0

1

0

Mandibular lymph node

 

         Focus/foci

0

0

1

0

Skin

 

         Alopecia

0

0

1

0

Eyes

 

         Exophthalmus

0

0

1

0

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Table 18. Select Organ Weights (grams) Summary

End of Treatment

Group 1

(Control –

0 mg/kg/day)

Group 2

(100 mg/kg/day)

Group 3

(300 mg/kg/day)

Group 4

(1000 mg/kg/day)

Males

Heart

(Gram)

Mean

1.037

0.877**

1.019

0.924*

SD

1.01

0.027

0.065

0.029

N

5

5

5

5

 

Liver

(Gram)

Mean

9.14

8.60

8.42

8.04**

SD

0.39

0.49

0.24

0.69

N

5

5

5

5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 19. Select Organ/Body Weight Ratios (%) Summary

End of Treatment

Group 1

(Control –

0 mg/kg/day)

Group 2

(100 mg/kg/day)

Group 3

(300 mg/kg/day)

Group 4

(1000 mg/kg/day)

Males

Heart

(%)

Mean

0.286

0.256*

0.298

0.275

SD

0.011

0.006

0.030

0.014

N

5

5

5

5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 20. Reproduction Data Summary

 

Group 1

(Control –

0 mg/kg/day)

Group 2

(100 mg/kg/day)

Group 3

(300 mg/kg/day)

Group 4

(1000 mg/kg/day)

Females paired

10

10

10

10

Females mated

10

10

10

10

Pregnant females

10

9

10

10

Females dead on post coitum Day 23

1

0

0

0

Females with total litter loss on Day 1

0

1

0

1

Females with living pups on Day 1

9

8

10

9

 

Mating index (%):

(Females mated / Females paired) * 100

100

100

100

100

Fertility index (%):

(Pregnant females / Females mated) * 100

100

90

100

100

Gestation index (%):

(Females with living pups on Day 1 / Pregnant females) * 100

90

89

100

90

 

Table 21. Precoital Time (F0 – Generation: Post Coitum)

Day of the Pairing Period

Group 1

(Control –

0 mg/kg/day)

Group 2

(100 mg/kg/day)

Group 3

(300 mg/kg/day)

Group 4

(1000 mg/kg/day)

Number of Females Mated During the First Pairing

Day 1

1

5

-

4

Day 2

3

2

-

2

Day 3

5

2

5

1

Day 4

1

1

3

3

Day 13

-

-

1

-

 

Median Precoital Time

3

2

3

2

Mean Precoital Time

2.6

1.9

4.4

2.3

N

10

10

9

10

 

Number of Females Mated During the Second Pairing

Day 1

-

-

1

-

 

Median Precoital Time

-

-

1

-

Mean Precoital Time

-

-

1.0

-

N

0

0

1

0

+/++ Steel-test significant at 5% (+) or 1% (++) level

 

Table 22. Summary of Implantation Sites

 

Group 1

(Control –

0 mg/kg/day)

Group 2

(100 mg/kg/day)

Group 3

(300 mg/kg/day)

Group 4

(1000 mg/kg/day)

At necropsy

 

Implantations

Mean

11.9

11.6

12.4

12.9

Std Dev

3.5

2.7

3.8

2.4

N

10

9

10

10

+/++ Steel-test significant at 5% (+) or 1% (++) level

Conclusions:
Based on the lack of adverse treatment-related effects observed through the study period, the systemic and reproductive toxicity No Observed Adverse Effect Level (NOAEL) for 1-Dodecene, dimers was determined to be≥1000 mg/kg/day in the rat.
Executive summary:

A key OECD Guideline 422 combined 28-Day repeated dose toxicity study with the Reproduction / Developmental Toxicity Screening Test was conducted to evaluate the potential toxic effects of the test material (1-Dodecene, dimers; CAS# 62132-67-6) when given orally to Wistar Han rats and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.

 

The test material Wistar Han rats (10/sex/dose) were administered once daily via oral gavage in a corn oil vehicle at doses of 0, 100, 300, or 1000 mg/kg/day for a minimum of 28 days for males (including at least 2 weeks of treatment prior to mating and during the mating period up to and including the day before scheduled necropsy). Female rats were dosed 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy.

 

Parameters were evaluated in this study included mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, clinical pathology, measurement of thyroid hormones T3, T4 and TSH (F0 males and females), gross necropsy findings, organ weights and histopathologic examinations. Additionally, reproduction/developmental parameters were also determined and included mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormones T3 and T4 (PND 4 and PND 14-16 pups), and TSH (PND 14-16).

 

No treatment-related mortality was observed in the F0 animals.

 

One control female (No. 43) was sacrificed in extremis on Day 23 post coitum. The female had a pale appearance and piloerection and at the point 3 pups were born of which 1 was alive and 2 were found dead. As no more pups were born and the condition of the animal deteriorated, the animal was sacrificedin extremis. At necropsy, the uterus of this female contained 3 alive and 5 dead fetuses. No other macroscopic or microscopic findings were observed. Based on this, the condition of this female was considered to be related to delivery difficulties.

 

One female in the 100 mg/kg/day (No. 60) dose group was sacrificed in extremis on Day 1 of lactation. The animal was found with a pale appearance and piloerection. Due to the deteriorating condition of this animal, it was sacrificed in extremis. Additionally, all 11 delivered pups were found to be dead. At necropsy, a pale liver was observed corresponding to microscopic moderate multifocal necrosis in the liver which likely contributed to the moribundity. Based on the single incidence of this event in the low dose group, the condition of this animal was not considered to be treatment-related.

No treatment-related clinical signs were observed either during daily detailed clinical or during the weekly arena observations.

 

Incidental findings observed included scabs, wounds, and alopecia. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be treatment-related.

 

Body weights and body weight gain were considered to be unaffected by treatment. Females at 1000 mg/kg/day displayed slightly lower (6% lower than control) absolute body weight on Day 4 of lactation was. As body weight gain was similar over this period and as this was only an incidental occurrence, this temporary lower body weight was not considered to be treatment-related. Food consumption before or after correction for body weight was not affected by treatment.

Hearing ability, pupillary reflex, static righting reflex and grip strength of the fore legs (males and females), and motor activity (females only) were considered unaffected by treatment at 100 and 300 mg/kg/day.

 

Females at 1000 mg/kg/day displayed decreased grip strength of the hind legs (0.63x of control).In the absence of clinical signs and corroborative histopathological findings, this was not considered as adverse.

 

An apparent upward trend in motor activity (total movements and ambulations) observed in male rats of all treatment groups was considered to have arisen as a result of slightly low control values and therefore not considered treatment-related.

 

All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

 

Hematological parameters of rats were unaffected by treatment. A reduction of large unstained cell (LUC) counts was observed in male rats at 300 mg/kg/day. However, in the absence of a dose-related trend, this was considered to be unrelated to treatment. Coagulation parameters of rats were unaffected by treatment. A shorter prothrombin time (PT) observed in male rats at 100 mg/kg/day was considered to be unrelated to treatment in the absence of a dose-related trend.

 

Clinical chemistry parameters of male rats were unaffected by treatment. In female rats, a reduction in total bilirubin levels (TBIL) was observed at 100, 300, and 1000 mg/kg/day (0.69x, 0.71x and 0.66x of control, respectively). In the absence of corroborative histopathological findings, this was considered not adverse. Increased sodium levels were observed in female rats at 300 mg/kg/day but this considered to be minor and in the absence of a dose-related trend, unrelated to treatment.

 

Serum levels of thyroxine (T4) and thyroid-stimulating hormone (TSH) in male and female rats were unaffected by treatment. Serum total triiodothyronine (T3) levels in F0 males at 100 mg/kg/day were increased (Group mean values were 1.29x of control; however, an increase in the SD was also observed). Mean values were within the historical control data range and values at 300 mg/kg/day and 1000 mg/kg/day were comparable with the control group. Therefore, although treatment-related, this increase was not considered as adverse. Serum total T3 levels were increased in F0 females at 1000 mg/kg/day (1.26x of control). No historical control data for total T3 in lactating females was available at the Testing Facility. However, since only 1/10 individual females displayed a T3 value above the highest individual control value, the mean increase of T3 at 1000 mg/kg/day was considered unrelated to treatment.

 

Gross necropsy did not reveal any remarkable treatment-related findings and no treatment-related effects or alterations in organ weights were observed. Any differences, including those that reached statistical significance (males; heart at 100 mg/kg/day (absolute and relative to body weight) and 1000 mg/kg/day (absolute only); liver at 1000 mg/kg/day (absolute only)), were not considered to be treatment-related due to the direction of the change and/or a lack of dose-related trend.

 

Histopathology did not reveal any microscopic observations related to treatment with the test material. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no treatment-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

 

Except one control female (No. 43) that was sacrificed in extremis on Day 23 post-coitum due to presumed delivery difficulties, all other females showed no signs of difficult or prolonged parturition or deficiencies in maternal care. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. The length and regularity of the estrous cycle were not affected by treatment with all females having regular cycles of 4 to 5 days and mating index was also unaffected with all females showing evidence of mating.

 

Precoital time was considered unaffected by treatment and most females showed evidence of mating within 4 days. One female (No. 61) at 300 mg/kg/day showed evidence of mating after 13 days. One female (No. 68) at 300 mg/kg/day that was probably mated during the extended mating period had an estimated precoital time of 1 day. Mating was overlooked in two females at 300 mg/kg/day (Nos. 68 and 70). As these females turned out to be pregnant and delivered a healthy litter, the mating date of these females was estimated at 21 days prior to delivery. The number of implantation sites was considered unaffected by treatment with the mean number of implantation sites being 11.9, 11.6, 12.1, and 12.9 for the control, 100, 300, and 1000 mg/kg/day groups, respectively. Fertility index was not affected by treatment with fertility indices being 100, 90, 100, and 100% for the control, 100, 300, and 1000 mg/kg/day groups, respectively. A lower gestation index observed at 100 mg/kg/day was the result of one female at 100 mg/kg/day (No. 56) that was not pregnant. At the low incidence and in absence of a dose related trend this finding was considered unrelated to treatment with the test material. Overall, no reproductive toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

 

Based on the lack of adverse treatment-related effects observed through the study period, the systemic and reproductive toxicity No Observed Adverse Effect Level (NOAEL) for 1-Dodecene, dimers was determined to be≥1000 mg/kg/day in the rat.

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 May 2011 - 11 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
The body weights for males 73 and 76 performed on the 15/07/11 appear to be incorrect as they are not in line with body weight increase seen in the remaining males. In the absence of any correlates to suggest a treatment affect, it was considered that these animals were incorrectly weighed on the 08/07/11 in error. The data recorded for these animals on the 08/07/11 will therefore be suppressed.
On the 25/10/11 the male 5hr observations were recorded approximately 1.5hrs late in error.
On the 22/11/11 the residue for cage 100 was not recorded in error before the food was topped up.
These are not considered to affect the purpose or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Sprague-Dawley Crl:CD (SD) IGS BR strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
A sufficient number of male and female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. The animals were acclimatised for at least fourteen days during which time their health status was assessed. A total of two hundred and twenty-four animals (one hundred and twelve males and one hundred and twelve females) were accepted into the study. At the start of treatment, the males weighed 223 to 301g and the females weighed 157 to 217g and were approximately eight weeks old.

Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C, Teklad Global Certified Diet, Harlan Laboratories, UK, Ltd., Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd, Cheshire, UK) except for mated females during gestation and lactation.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system and print-outs of hourly mean temperatures and humidities are included in the study records. The temperature and relative humidity controls were set to achieve target values of 21±2ºC and 55 ±15% respectively. Short term deviations from these targets were considered not to affect the purpose or integrity of the study.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
The test item was prepared weekly and administered daily for 10 weeks by gavage, using a stainless steel cannula attached to a disposable plastic syringe, to three groups, each of twenty eight male and twenty-eight female F0 Generation Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twenty-eight male and twenty-eight female F0 Generation rats was dosed with vehicle alone (Arachis oil BP) 4ml/kg.

F1 generation: Selected F1 males and females (24 per dose group) were treated at the appropriate dose levels from Day 21 post partum for ten weeks during which they were evaluated for evidence of sexual maturation.
(The F2 generation were not treated, but killed after weaning.)
Details on mating procedure:
After 10 weeks of treatment, pairing of animals within each dose group was undertaken on a one male: one female basis, for a maximum of 14 days, to produce the F1 litters. At weaning of offspring from the F0 mating phase, groups of twenty-four male and twenty-four female offspring from each dose group were selected to form the F1 generation. The remaining surviving F0 females and unselected offspring were terminated at Day 21 post partum, followed by the termination of all F0 male dose groups. The offspring selected for the F1 generation were dosed for at least 10 weeks and then paired within each dose group to produce the F2 litters. At weaning of the F2 litters all surviving adults and their offspring were killed, followed by the termination of all F1 male dose groups.
Analytical verification of doses or concentrations:
yes
Remarks:
Verified within +/-10% of nominal concentration
Details on analytical verification of doses or concentrations:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results are given in Appendix 27 and show the formulations to be stable for at least twenty-one days. Formulations were therefore prepared weekly and stored at approximately +4ºC under nitrogen.
Samples were taken of test item formulations and analysed for concentration of Pentadecane, 7-methylene mixed with 1-tetradecene, dimers and trimers, hydrogenated at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Samples were diluted in tetrahydrofuran to give a final theoretical conc approx 0.1mg/ml and analysed by GC against appropriate standard concentrations. The results indicate that the prepared formulations were within ±10% of the nominal concentration.
Duration of treatment / exposure:
10 weeks
Frequency of treatment:
daily
Details on study schedule:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg of Arachis oil BP. Doses were prepared weekly, shown to be stable for at least 21 days. See below (study design) for further details of the study schedule.
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
28 (F0) 24 (F1)
Control animals:
yes, concurrent vehicle
Details on study design:
Chronological Sequence of Study
i) Groups of twenty-eight F0 male and twenty-eight F0 female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable).
ii) A vaginal smear was prepared daily for twenty-one days prior to pairing for all F0 females.
iii) During Week 11 all animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv) Following evidence of mating, the males were returned to their original cages and females were transferred to individual cages.
v) Pregnant F0 females were allowed to give birth and maintain their offspring until Day 21 post partum. Evaluation of each litter size, litter weight, mean offspring weight and clinical observations were also performed during this period.

vi) At Day 21 post partum, wherever possible, one male and one female offspring from the F0 mating phase were selected to form the next generation (F1). Surviving adult F0 females and unselected offspring were killed and examined macroscopically. Selected organs were weighed and/or preserved for histopathological examination for F0 females and selected organs were weighed for one unselected male offspring and one unselected female offspring.
vii) Following completion of the F0 female gestation and lactation phases, the F0 male dose groups were killed and examined macroscopically. Selected organs were weighed and/or preserved for histopathological examination for F0 males. A sample of epididymal semen was analysed for sperm motility and a sample of semen was prepared for morphological assessment. Testicular and epididymal samples were frozen for subsequent homogenisation resistant spermatid enumeration.
viii) Selected F1 males and F1 females were treated at the appropriate dose levels from Day 21 post partum for ten weeks during which they were evaluated for evidence of sexual maturation.
ix) A vaginal smear was prepared daily for twenty-one days prior to pairing for all F1 females.
x) Following ten weeks of treatment all F1 animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
xi) Following evidence of mating, the males were returned to their original cages and females were transferred to individual cages.
xii) Pregnant F1 females were allowed to give birth and maintain their offspring until Day 21 post partum. Evaluation of each litter size, litter weight, mean offspring weight and clinical observations were also performed during this period.
xiii) At Day 21 post partum, all surviving F1 females and offspring were killed and examined macroscopically. Selected organs were weighed and/or preserved for histopathological examination for F1 females and selected organs were weighed for one male and one female offspring.
xiv) Following completion of the F1 female gestation and lactation phases, the F1 male dose groups were killed and examined macroscopically. Selected organs were weighed and/or preserved for histopathological examination for F1 males. A sample of epididymal semen was analysed for sperm motility and a sample of semen was prepared for morphological assessment. Testicular and epididymal samples were frozen for subsequent homogenisation resistant spermatid enumeration.
Parental animals: Observations and examinations:
Morbidity and mortality, clinical signs, body weight development, food and water consumption were monitored during the study.
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.
Oestrous cyclicity (parental animals):
Prior to pairing of females for the F0 and F1 mating phases, a vaginal smear was taken daily for twenty-one days and a sample was placed on a glass slide. The smears were allowed to dry and then stained using a diluted giemsa stain. The smears were examined microscopically and the stage of oestrous was recorded.
Sperm parameters (parental animals):
At necropsy of adult F0 and F1 males the following evaluations were performed:
i) The left testis and epididymis were removed, dissected from connective tissue and weighed separately.
ii) For the testis, the tunica albuginea was removed and the testicular tissue stored frozen at approximately -20ºC. At an appropriate later date the tissues were thawed, re-weighed and homogenised in a suitable saline/detergent mixture.
Samples of the homogenate were stained with a DNA specific fluorescent stain and a sub-sample was analysed for numbers of homogenisation resistant spermatids.
iii) For the epididymis the distal region was incised and a sample of the luminal fluid collected and transferred to a buffer solution for analysis of sperm motility and sperm morphology. Approximately 200 individual sperm were assessed using an automated semen analyser, to determine the number of motile, progressively motile and non-motile sperm. The characteristics of motile sperm were also identified using the computer assisted sperm analyser (Hamilton-Thorne TOX IVOS system).
iv) A sample of semen was preserved in formalin and then stained with eosin. A subsample was placed on a glass slide with a coverslip and a morphometric analysis of 200 sperm* was performed manually.

* = preserved in Bouins fluid then transferred in 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later
v) The cauda epididymis was separated from the body of the epididymis, and then weighed. The cauda epididymis was then frozen at approximately -20ºC. At an appropriate later date the tissues were thawed, re-weighed and homogenised in an appropriate saline/detergent mixture. Samples of the homogenate were stained with eosin and a sub-sample was analysed for homogenisation resistant spermatids.
vi) For ii), iii), iv) and v) above samples from Groups 1 (Control) and 4 (high dose group) were evaluated.
Litter observations:
Litter Size, Sex Ratio and Viability.
Offspring Growth and Development: litter weights, offspring body Weight on Day 1, subsequent gain to weaning (Day 21 of age).
Sexual Maturation (F1 only).
Litter Observations. Clinical signs
Postmortem examinations (parental animals):
3.4.2 Pathology
Surviving adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 21 post partum. Surviving offspring (with the exception of offspring selected to form the F1 generation) were terminated via carbon dioxide asphyxiation. Following the termination of all adult females and offspring, all surviving males were killed by intravenous overdose of sodium pentobarbiturate agent followed by exsanguination.
In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted. In the case of non-pregnant females, the procedure was enhanced by staining the uteri with a 0.5% ammonium polysulphide solution where applicable. All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

3.4.2.1 Organ Weights
The following organs, removed from all F0 males and F0 females from each treatment group that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals Pituitary (post fixation)
Brain Seminal vesicles (with coagulating gland and fluids)
Epididymides (total and caudal) Spleen
Kidneys
Testes
Liver
Thyroid/Parathyroid gland (post fixation)
Ovaries Uterus (with cervix and oviducts)
Prostate

The following organs were weighed from one male and one female offspring from the F0 and F1 pairings (where available):
Brain Spleen Thymus Uterus

3.4.2.2 Histopathology
The following tissues were preserved from all F0 males and females from each dose group, in buffered 10% formalin except where stated:
Adrenals
Prostate
Right Epididymis *
Seminal vesicles (with coagulating gland)
Ovaries Uterus (with Oviducts and Cervix)
Right Testis* Vagina
Pituitary gland
Gross lesions
All tissues were despatched to the histology processing Test Site (Harlan Laboratories
Ltd, Zelgliweg 1, CH-4452 Itingen, Switzerland) (Principal Investigator: S Gäehler). The
tissues from all F0 and F1 control and high dose group animals, any animals dying during
the study and any animals which did not achieve a pregnancy were prepared as paraffin
blocks, sectioned at nominal thickness of 5m and stained with haematoxylin and eosin
for subsequent microscopic examination. In addition, sections of testes and
epididymides from all control and 1000 mg/kg bw/day males were also stained with
Periodic Acid-Schiff (PAS) stain and examined.

Microscopic examination was conducted by the Study Pathologist (Principal Investigator:Dr K Heider at AnaPath GmbH). A complete histopathology phase report, including methods is presented in Appendix 25.

Postmortem examinations (offspring):
The following organs were weighed from one male and one female offspring from the F0 and F1 pairings (where available):
Brain Spleen Thymus Uterus
Macroscopic abnormalities
Statistics:
Quantitative data was analysed to detect the significance of intergroup differences from control; statistical significance was achieved at p<0.05. The following parameters were analysed:
Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Absolute Organ Weights, Body Weight-Relative Organ Weights, Sperm Concentration and Sperm Homogeneity Resistance.

Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module:

The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Transformed data were analysed to find the lowest treatment level with a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows non-homogeneity of means, the data was analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric).

Probability values (p) were calculated as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p>=0.05 (not significant)
Reproductive indices:
Live birth index (No of offspring alive on day 1 / No of offspring born * 100
Offspring viability indices:
Viability Index 1 (%) Number of offspring alive on Day 4/ Number of offspring alive on day 1 * 100
Viability Index 2 (%) Number of offspring alive on Day 7/ Number of offspring alive on Day 4 * 100
Viability Index 3 (%) Number of offspring alive on Day 14/ Number of offspring alive on Day 7 * 100
Viability Index 4 (%) Number of offspring alive on Day 21/ Number of offspring alive on Day 14 * 100
Viability Index 5 (%) Number of offspring alive on Day 21 / Number of offspring alive on day 1 * 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinically observable signs of toxicity were detected in test or control animals throughout the study period. The following clinical signs were considered unrelated to test item toxicity:

The F0 male treated with 300 mg/kg bw/day, the F1 male treated with 1000 mg/kg bw/day, one of the F1 control females and the F1 female treated with 100 mg/kg bw/day that were found dead showed no clinical signs prior to death. The remaining F1 control female that was found dead on Day 32 had an increased respiratory rate, ptosis, lethargy and chromodacryorrhea on Day 31. The male treated with 300 mg/kg bw/day that was killed in extremis on Day 101 of the F0 generation had a mass, tiptoe gait, a red/brown stained snout, hunched posture and chromodacryorrhea prior to termination on Day 101. As previously discussed, microscopic examination revealed the deterioration of this animal was most likely to be due to an abscess formation in the right forelimb. The male treated with 1000 mg/kg bw/day that was killed in extremis on Day 106 of the F0 generation showed a red/brown stained snout, noisy and laboured respiration, a decreased respiratory rate, lethargy, hunched posture and increased salivation prior to termination.

The control female that was killed in extremis on Day 97 of the F0 generation showed a red/brown stained snout, pilo-erection, lethargy and hunched posture on the day of termination. As previously discussed, microscopic examination revealed the deterioration of this animal was considered to be due to the development of thrombosis in the uterus after pregnancy. The male treated with 1000 mg/kg bw/day that was killed in extremis on Day 106 of the F1 generation had a mass, laboured respiration, a decreased respiratory rate and lethargy on the day of termination. As previously discussed, microscopic examination revealed the cause of this death was most likely due to the abscess formation in the right forelimb and the pleuritis which was possibly caused by a mal-dose. Incidents of red/brown staining of the external body surface, generalised fur loss, wound/scab formation, exophthalmos, chromodacryorrhea and noisy respiration were detected throughout the treatment period in treated and/or control animals from the F0 generation. Episodes of generalised fur loss and scab formation were also evident throughout the treatment period in treated animals from the F1 generation. These observations are commonly observed in laboratory maintained rats on long term studies and are considered of no toxicological significance. Isolated incidences of mass formation were noted in a few treated and control animals in both generations during the treatment period. In the F0 generation these masses correlated either with lipoma in the thoracic region or mammary gland, decidual proliferation in uterus, abscess formation in the thoracic region or on the forelimb or adenocarcinoma of mammary gland. In the F1 generation these masses correlated either with adenoma in mammary gland, decidual proliferation in uterus, pleuritis or abscess formation on the forelimb. All these findings were considered to be incidental or possibly caused by a mal-dose and therefore are considered not to represent a toxic effect of treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no deaths considered to be related to test item throughout the study.

The following unscheduled deaths were seen during the treatment period:
One male treated with 1000 mg/kg bw/day was killed in extremis on Day 106 of the F0 generation; however the aetiology of this death was not established. One male treated with 300 mg/kg bw/day was killed in extremis on Day 101 of the F0 generation. The deterioration of this animal was most likely to be due to an abscess formation in the right forelimb. A further male from this treatment group was found dead on Day 53 of the F0 generation; however the aetiology of this death was not established. One control female was killed in extremis on Day 97 of the F0 generation. The deterioration of this animal was considered to be due to the development of thrombosis in the uterus after pregnancy.

One male treated with 1000 mg/kg bw/day was killed in extremis on Day 106 of the F1 generation. The cause of this death was most likely due to the abscess formation in the right forelimb and the pleuritis which was possibly caused by a mal-dose. A further male from this treatment group was found dead on Day 118 of the F1 generation; however the aetiology of this death was not established. One control female was found dead on Day 32 of the F1 generation. The cause of this death was considered to be pleuritis that was possibly caused by a mal-dose. A further control female was found dead on Day 99 of the F1 generation. The cause of this death was possibly due to the marked decidual proliferation with thrombosis in the uterus.

One female treated with 100 mg/kg bw/day was found dead on day 104 of the F1 generation; however the aetiology of this death was not established.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males from the F0 generation treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in body weight gain during Week 7 of treatment. F0
generation males treated with 300 and 100 mg/kg bw/day also showed a statistically significant increase in body weight gain during Week 15 of treatment. An increase in body weight gain is considered not to represent an adverse effect of treatment and in the absence of true dose related responses during Week 15 the intergroup differences were considered not to be toxicologically significant. F0 generation males treated with 1000 mg/kg bw/day showed a statistically significant reduction in body weight gain during Week 15 of treatment. In isolation and in the absence of an effect on overall body weight gain the intergroup difference was considered not to be of toxicological importance.

F0 generation females treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in body weight gain during Week 3 of treatment. F0 generation females treated with 100 mg/kg bw/day also showed a statistically significant increase in body weight gain during Week 10 of treatment. An increase in body weight gain is considered not to represent an adverse effect of treatment and in the absence of a true dose related response during Week 10 the intergroup differences were considered not to be toxicologically significant. F0 generation females treated with 100 mg/kg bw/day showed a statistically significant reduction in body weight gain during Week 5 of treatment. In the absence of a true dose related response the intergroup difference was considered not to be toxicologically significant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Males:
No adverse effect on food consumption or food efficiency was detected in treated males in either generation when compared to control animals.

Females:
Pre-pairing Food Consumptions: No adverse effect on food consumption or food efficiency was detected in treated females in either generation during the maturation phases. Gestation Food Consumptions: No adverse effect on food consumption was detected in treated females in either generation during the gestation phases.
Lactation Food Consumptions: No adverse effect on food consumption was detected in treated females in either generation during the lactation phases.
Food efficiency:
no effects observed
Description (incidence and severity):
No adverse effect on food consumption or food efficiency was detected in treated animals in either generation.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No adverse effect on water consumption was detected.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on female oestrous cycles or on the type or proportion of females with anomalous oestrous cycles.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
No adverse effect of treatment on sperm concentration, motility or morphology was apparent for males in either generation.
F0 males treated with 1000 mg/kg bw/day showed a statistically significant increase in the percent of sperm with normal morphology. An increase in this parameter is not considered to represent an adverse effect of treatment.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
From the F0 generation, one female treated with 100 mg/kg bw/day and one female treated with 300 mg/kg bw/day was non-pregnant following positive evidence of mating, a further female from each of these treatment groups was also non-pregnant however both females did not show any positive evidence of mating and one female treated with 1000 mg/kg bw/day was non-pregnant following positive evidence of mating. No histopathological correlates were evident in the male or female reproductive organs to suggest the cause of the non-pregnancies.

Gestation: There were no differences in gestation lengths in either generation. The distribution for treated females was comparable to controls. The gestation lengths were between 22 and 24½ days.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinically observable signs of toxicity were detected in test or control animals throughout the study period. The following clinical signs were considered unrelated to test item toxicity:

The F1 male treated with 1000 mg/kg bw/day, one of the F1 control females and the F1 female treated with 100 mg/kg bw/day that were found dead showed no clinical signs prior to death. The remaining F1 control female that was found dead on Day 32 had an increased respiratory rate, ptosis, lethargy and chromodacryorrhea on Day 31.

The male treated with 1000 mg/kg bw/day that was killed in extremis on Day 106 of the F1 generation had a mass, laboured respiration, a decreased respiratory rate and lethargy on the day of termination. As previously discussed, microscopic examination revealed the cause of this death was most likely due to the abscess formation in the right forelimb and the pleuritis which was possibly caused by a mal-dose. Episodes of generalised fur loss and scab formation were evident throughout the treatment period in treated animals from the F1 generation. These observations are commonly observed in laboratory maintained rats on long term studies and are considered of no toxicological significance. Isolated incidences of mass formation were noted in a few treated and control animals in both generations during the treatment period. In the F1generation these masses correlated either with adenoma in mammary gland, decidual proliferation in uterus, pleuritis or abscess formation on the forelimb. All these findings were considered to be incidental or possibly caused by a mal-dose and therefore are considered not to represent a toxic effect of treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no deaths considered to be related to test item throughout the study. The following unscheduled deaths were seen during the treatment period:

One male treated with 1000 mg/kg bw/day was killed in extremis on Day 106 of the F1 generation. The cause of this death was most likely due to the abscess formation in the right forelimb and the pleuritis which was possibly caused by a mal-dose. A further male from this treatment group was found dead on Day 118 of the F1 generation; however the aetiology of this death was not established. One control female was found dead on Day 32 of the F1 generation. The cause of this death was considered to be pleuritis that was possibly caused by a mal-dose. A further control female was found dead on Day 99 of the F1 generation. The cause of this death was possibly due to the marked decidual proliferation with thrombosis in the uterus. One female treated with 100 mg/kg bw/day was found dead on day 104 of the F1 generation; however the aetiology of this death was not established.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
F1 generation females treated with 1000 and 100 mg/kg bw/day showed a statistically significant increase in body weight gain during Days 4 and 7 of lactation. The pattern of body weight gain for the parent female during lactation is influenced by the increasing demands of milk production from the growing litter. It is normally anticipated that females in lactation will show body weight gain during the first two weeks of lactation, although gain will lessen during the second week, and by the third week of lactation body weight will remain static or loss is expected. The increased intergroup difference is therefore considered not to be of toxicological importance.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant effects were detected in the organ weights measured in either generation.

For F1 males treated with 1000 mg/kg bw/day a statistically significant increase in kidney weight both absolute and relative to terminal body weight was evident. In the absence of a similar effect detected in F0 animals the intergroup difference was considered to be incidental and unrelated to treatment.

F1 males treated with 100 mg/kg bw/day showed a statistically significant reduction in pituitary weight both absolute and relative to terminal body weight. F1 females treated with 1000 and 100 mg/kg bw/day also showed a statistically significant reduction in pituitary weight both absolute and relative to terminal body weight whilst females treated with 300 mg/kg bw/day showed a statistically significant increase in absolute and relative pituitary weight. In the absence of a true dose related response or in the absence of any histopathological correlates the intergroup differences were considered not to be of toxicological importance. F1 males treated with 300 mg/kg bw/day showed a statistically significant increase in thyroid weight both absolute and relative to terminal body weight. In the absence of a true dose related response the intergroup difference was considered not to be of toxicological importance. F1 females treated with 1000 mg/kg bw/day showed a statistically significant reduction in thyroid weight both absolute and relative to terminal body weight.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Interim Deaths: The male treated with 1000 mg/kg bw/day that was killed in extremis on Day 106 had a mass in the thoracic region, of which the heart, lungs and thymus were all adhered to. The male from this treatment group that was found dead on Day 118 had a mass under the right forelimb. The female treated with 100 mg/kg bw/day that was found dead on Day 104 did not show any macroscopic abnormalities. One control female that was found dead on Day 32 had enlarged adrenals, a fluid filled thoracic cavity and white fibrous adhesions on the lungs. The other control female that was found dead on Day 99 had a mass in the left horn of the uterus. In the absence of any similar findings in terminal kill animals the intergroup differences were considered to be incidental and unrelated to treatment.
Terminal Kill: One male treated 100 mg/kg bw/day had an enlarged left testis. One female treated with 300 mg/kg bw/day had a pale right kidney, an enlarged spleen and a malformed uterus. In the absence of true dose related reponses the intergroup differences were considered not to be of toxicological importance. One female treated with 100 mg/kg bw/day had a mass in the thoracic cavity and in the mammary tissue. Microscopic examination revealed adenoma of the mammary gland and was considered to be an incidental finding and of no toxicological importance.
Neuropathological findings:
not examined
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects in either generation on female oestrous cycles or on the type or proportion of females with anomalous oestrous cycles.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Sperm Analysis. There were no toxicologically significant effects on the concentration, motility or morphology of samples of epididymal sperm. There were no treatment-related effects on the concentration of homogenisation resistant epididymal or testicular spermatid counts.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
From the F1 generation, one female treated with 300 mg/kg bw/day and one female treated with 1000 mg/kg bw/day was non-pregnant following positive evidence of mating. No histopathological correlates were evident in the male or female reproductive organs to suggest the cause of the non-pregnancies. One female treated with 300 mg/kg bw/day showed positive evidence of mating but failed to give birth to any live offspring. Microscopic examination of this female showed a moderate unilateral pyelonephritis and a marked pyometra in one uterine horn with surrounding peritonitis and adhesions to duodenum and arteritis/periarteritis. These inflammatory processes were accompanied by a marked reactive extramedullary hematopoiesis in the spleen. The male partner to this female did not reveal any relevant findings, therefore the different inflammatory processes in the female were considered to be the reason for the non-pregnancy. A pseudopregnancy cannot however be totally excluded because in both the cervix and vagina a mucification was noted.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed
Clinical signs:
no effects observed
Description (incidence and severity):
The type, incidence and distribution of clinical observations observed for offspring in both generations were consistent with that normally expected in offspring of the age examined and did not indicate any adverse effect of treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F1 Generation
Interim Deaths: The male treated with 1000 mg/kg bw/day that was killed in extremis on Day 106 had a mass in the thoracic region, of which the heart, lungs and thymus were all adhered to. The male from this treatment group that was found dead on Day 118 had a mass under the right forelimb. The female treated with 100 mg/kg bw/day that was found dead on Day 104 did not show any macroscopic abnormalities. One control female that was found dead on Day 32 had enlarged adrenals, a fluid filled thoracic cavity and white fibrous adhesions on the lungs. The other control female that was found dead on Day 99 had a mass in the left horn of the uterus. In the absence of any similar findings in terminal kill animals the intergroup differences were considered to be incidental and unrelated to treatment.
Terminal Kill: One male treated 100 mg/kg bw/day had an enlarged left testis. One female treated with 300 mg/kg bw/day had a pale right kidney, an enlarged spleen and a malformed uterus. In the absence of true dose related reponses the intergroup differences were considered not to be of toxicological importance. One female treated
with 100 mg/kg bw/day had a mass in the thoracic cavity and in the mammary tissue.
Microscopic examination revealed adenoma of the mammary gland and was considered to be an incidental finding and of no toxicological importance.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males:
There were no toxicologically significant effects detected in body weight development in either generation.

Females
Pre-pairing Body weights: There were no toxicologically significant effects detected in body weight development in either generation during the maturation phases.

Gestation Body weights: There were no treatment related effects detected in body weight development in either generation during the gestation phases.
Lactation Body weights: There were no toxicologically significant effects detected in body weight development in either generation during the lactation phases.
F1 generation females treated with 1000 and 100 mg/kg bw/day showed a statistically significant increase in body weight gain during Days 4 and 7 of lactation. The pattern of body weight gain for the parent female during lactation is influenced by the increasing demands of milk production from the growing litter. It is normally anticipated that females in lactation will show body weight gain during the first two weeks of lactation, although gain will lessen during the second week, and by the third week of lactation body weight will remain static or loss is expected. The increased intergroup difference is therefore considered not to be of toxicological importance.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the attainment of sexual maturation.
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
For F1 males treated with 1000 mg/kg bw/day a statistically significant increase in kidney weight both absolute and relative to terminal body weight was evident. In the absence of a similar effect detected in F0 animals the intergroup difference was considered to be incidental and unrelated to treatment.

F1 males treated with 100 mg/kg bw/day showed a statistically significant reduction in pituitary weight both absolute and relative to terminal body weight. F1 females treated with 1000 and 100 mg/kg bw/day also showed a statistically significant reduction in pituitary weight both absolute and relative to terminal body weight whilst females treated with 300 mg/kg bw/day showed a statistically significant increase in absolute and relative pituitary weight. In the absence of a true dose related response or in the absence of any histopathological correlates the intergroup differences were considered not to be of toxicological importance.

F1 males treated with 300 mg/kg bw/day showed a statistically significant increase in thyroid weight both absolute and relative to terminal body weight. In the absence of a true dose related response the intergroup difference was considered not to be of toxicological importance.

F1 females treated with 1000 mg/kg bw/day showed a statistically significant reduction in thyroid weight both absolute and relative to terminal body weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant macroscopic abnormalities detected in both the F0 and F1 animals. The following macroscopic abnormalities were considered to be unrelated to treatment with the test item:


Interim Deaths: The male treated with 1000 mg/kg bw/day that was killed in extremis on Day 106 had a mass in the thoracic region, of which the heart, lungs and thymus were all adhered to. The male from this treatment group that was found dead on Day 118 had a mass under the right forelimb. The female treated with 100 mg/kg bw/day that was found dead on Day 104 did not show any macroscopic abnormalities. One control female that was found dead on Day 32 had enlarged adrenals, a fluid filled thoracic cavity and white fibrous adhesions on the lungs. The other control female that was found dead on Day 99 had a mass in the left horn of the uterus. In the absence of any similar findings in terminal kill animals the intergroup differences were considered to be incidental and unrelated to treatment.
Terminal Kill: One male treated 100 mg/kg bw/day had an enlarged left testis. One female treated with 300 mg/kg bw/day had a pale right kidney, an enlarged spleen and a malformed uterus. In the absence of true dose related reponses the intergroup differences were considered not to be of toxicological importance. One female treated
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic findings were within normal background range and not treatment-related.
Behaviour (functional findings):
no effects observed
Developmental immunotoxicity:
not examined
In total, for the F0 generation there were 27, 25, 25 and 27 females at 0 (control), 100, 300 and 1000 mg/kg bw/day respectively who gave birth to a live litter and successfully reared young to weaning (Day 21 of age). From the F1 generation, there were 21, 23, 22 and 23 females at 0 (Control), 100, 300 and 1000 mg/kg bw/day respectively who gave birth to a live litter and successfully reared young to weaning (Day 21 of age) and have been included in the assessment of litter responses.
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The type, incidence and distribution of clinical observations observed for offspring in both generations were consistent with that normally expected in offspring of the age examined and did not indicate any adverse effect of treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
From the F1 generation, one control female showed total litter loss post partum. As there was no consistency between the two generations and also no increase in mortality for offspring for those litters surviving to weaning in either generation, these isolated occurrences were considered to be incidental.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no adverse effect of treatment on litter weights, offspring body weight on Day 1 for either sex or subsequent body weight gain of each sex to weaning (Day 21 of age) in either generation.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Reproductive effects observed:
no

F2 generation were sacrificed after weaning at day 21 therefore sexual maturation was not examined and F2 animals were not treated with the test item.

Conclusions:
The oral administration of Pentadecane, 7-methylene mixed with 1-tetradecene, dimers and trimers, hydrogenated to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The ‘No Observed Effect Level’ (NOEL) for adult toxicity was therefore considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity for both F0 and F1 generations and offspring was considered to be 1000 mg/kg bw/day.
Executive summary:

The oral administration of Pentadecane, 7-methylene mixed with 1-tetradecene, dimers and trimers, hydrogenated to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, did not result in any toxicologically significant effects. The ‘No Observed Effect Level’ (NOEL) for adult toxicity was therefore considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity for both F0 and F1 generations and offspring was considered to be 1000 mg/kg bw/day.

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-05-27 to 2006-03-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable without restriction because it was conducted according to GLP, is scientifically acceptable and appears to have adhered to the OECD guideline recommendations.
Qualifier:
according to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (U.K.) Limited
- Age at study initiation: (P): 6 to 8 weeks; (F1): 3 weeks
- Weight at study initiation: (P) Males: 205 to 280 grams; Females: 148 to 205 grams
- Fasting period before study: No
- Housing: Groups of four in polypropylene cages prior to mating; one male to one female during mating; Post-mating, males returned to original cages and females housed individually during gestation and lactation
- Diet (e.g. ad libitum): Rodent PMI 5002 (Certified) diet (BCM IPS Limited, London, UK) ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2ºC
- Humidity (%): 55 ±15%
- Air changes (per hr): At least 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

IN-LIFE DATES: From: 2005-06-22 To: 2005-10-20
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test material was prepared on a weekly basis at the appropriate concentrations as a solution in Arachis oil BP and stored at 4ºC in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not reported
- Concentration in vehicle: 0, 12.5, 62.5, or 250 mg/mL depending on the dosage
- Amount of vehicle (if gavage): 4 mL/Kg
- Purity: Not reported
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 21 days
- Proof of pregnancy: Vaginal plug in situ and/or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: No. There is no data on repeated mating attempts in the study
- After successful mating each pregnant female was caged (how): Females were transferred to individual cages and dosing continued during the subsequent gestation and lactation phases of the study.
- Any other deviations from standard protocol: None reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of dosing formulations at 250 mg/ml were also analysed for achieved concentration on a weekly basis during the study. The results indicatethat prepared high dose formulations were within ± 12% of the nominal concentration.
Duration of treatment / exposure:
Approximately 139 days (~20 weeks)
Frequency of treatment:
Daily (except for females during parturition)
Details on study schedule:
- F0 parental animals not mated until 10-11 weeks post-exposure.
- Age at mating of the mated animals in the study: 13 weeks for females; 16 to 18 weeks for males
Remarks:
Doses / Concentrations:
0, 50, 250, 1000 mg/kg bw/day
Basis:
other:
No. of animals per sex per dose:
24 per sex per dose
Control animals:
yes
Details on study design:
- Dose selection rationale: The dose levels were chosen by the Sponsor based on available toxicological data.
- Rationale for animal assignment (if not random): Animals were allocated to dose groups using a randomisation procedure based on stratified
bodyweights (Table No. 1).
Positive control:
A positive control was not employed in the study design.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Prior to dosing and at weekly intervals before pairing, ten animals/sex from each dose group were observed cage-side for functional and behavioural changes.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately prior to dosing and then 1 to 5 hours post-dosing from Monday through Friday. On the weekend, animals were observed prior to dosing, immediately after dosing and at 1 hour post dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Males: Day 0 and once a week thereafter; Females: Weekly during maturation and daily during mating; Post-mating bodyweights were recorded on days 1, 4, 7, 14, and 21 post-coitum and on Days 1, 4, 7, 14 and 21 post partum for females that littered.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, During the maturation period, weekly food consumption was recorded for each cage of adults. Food consumption was not recorded during the period of co-habitation during the mating phase but was re-instigated on a weekly basis for males once this was completed. For females showing evidence of mating, food consumption was recorded for the periods covering days 1 to 7, 7 to 14, and 14 to 21 post coitum and following littering for the periods covering days 1 to 7, 7 to 14 and 14 to 21 post partum.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, weekly food conversation efficiency (bodyweight gain/food intake) was calculated for both sexes for the pre-pairing maturation phase of the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt change.

OTHER: Ophthalmoscopic Examination - Eyes of 10 animals/sex from the control and high-dose group were examined pre-treatment and prior to mating (during Week 10).
Oestrous cyclicity (parental animals):
A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm
within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating.
Sperm parameters (parental animals):
Parameters examined in 10 [F0] male parental generations from each treatment group: Testis weight, epididymis weight, sperm count in epididymides; atrophy of testes
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 1 postpartum: Yes
- All live pups were examined.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number and sex of pups, still births, live births, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving adult males were killed shortly before termination of the females and litters commenced (week 17).
- Maternal animals: Non-pregnant adult females were killed on or after day 25 post coitum. Surviving adult females were killed at weaning (day 21 post partum).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Additionally, any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table No. 2 and Table No. 3 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 21 days post partum.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: A full external and internal examination was conducted and any macroscopic abnormalities were recorded.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table No. 2 and Table No. 3 were prepared for microscopic examination and weighed, respectively.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain, litter weights, offspring bodyweights and quantitative functional performance data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dennett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test. Non-parametric methods were used to analyse implantation loss, offspring sex ratio and developmental landmarks and reflexological responses. Chi-squared analysis was used for differences in the incidence of lesions occurring with an overall frequency of 1 or greater while the Kruskal-Wallis one-way non-parametric analysis of variance was utilized for the comparison of severity grades for the more frequently observed graded conditions.
Reproductive indices:
1] Pre-coital interval - Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

2] Fertility Indices – For each dose group, 2 indices as indicated below were calculated:

Mating Index (%): (Number of animals mated/Number of animals paired) x 100

Pregnancy Index (%): (Number of pregnant females/ Number of animals mated) x 100

3] Gestation length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition. Where the start of parturition occurred overnight, the total was adjusted by subtracting half a day.

4] Parturition Index (%): (Number of females delivering live offspring/Number of pregnant females) x 100

Offspring viability indices:
1] Live birth and viability indices: The following indices were calculated for each group from group mean data:

Live Birth Index (%) = (Number of offspring alive on Day 1/ Number of offspring born) x 100

Viability Index 1 (%) = (Number of offspring alive on Day 4/ Number of offspring alive on Day 1) x 100

Viability Index 2 (%) = (Number of offspring alive on Day 7/ Number of offspring alive on Day 4) x 100

Viability Index 3 (%) = (Number of offspring alive on Day 14/ Number of offspring alive on Day 7) x 100

Viability Index 4 (%) = (Number of offspring alive on Day 21/ Number of offspring alive on Day 14) x 100

Viability Index 5 (%) = (Number of offspring alive on Day 21/ Number of offspring alive on Day 1) x 100

2] Sex ration (% males): Group mean values calculated from each litter value on Day 1 and 21 using the following formula:

Sex ration (% males) = (Number of male offspring/ Total number of offspring) x 100

3] Implantation losses: Group mean percentile pre-implantation and post implantation loss were calculated as follows:

% pre - implantation loss = (Number of Corpora Lutea - Number of implantation sites/ Number of corpora lutea) x 100

% post - implantation loss = (Group number of implantation sites - Number of offspring/ Number of implantation sites) x 100
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): There were two unscheduled deaths during the study, neither of which were considered treatment-related. No clinically observable signs of toxicity were detected in test or control animals throughout the study period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Bodyweight and bodyweight gain of males and females (including the gestation and lactation phases) at dosages of up to 1000 mg/kg/day were unaffected by treatment throughout the study. Food consumption of males during the pre-pairing and post pairing phases of the study was unaffected by treatment at dosages of up to 1000 mg/kg/day. Food consumption of females during the pre-mating, gestation and lactation periods of the study was also not adversely affected by treatment at dosages up to 1000 mg/kg/day.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): No data

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): The distribution of pre-coital intervals for treated animals was essentially similar to control with the majority of animals showing evidence of mating within four days of pairing. The pattern of mating observed for all groups was consistent with females showing a normal oestrous cycle and mating at the first oestrus opportunity, and this indicated that oestrous cycling had been unaffected by treatment at dosages up to 1000 mg/kg/day.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): There were no effects on the reproductive function of male rats observed during the study

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): There were no treatment-related effects on mating performance or fertility.

ORGAN WEIGHTS (PARENTAL ANIMALS): Intergroup differences in absolute and bodyweight-relative organ weights for either sex were not indicative of any adverse effect of treatment.

GROSS PATHOLOGY (PARENTAL ANIMALS): Neither the type, incidence nor distribution of macroscopic finding observed at necropsy for decedent animals or for animals killed at scheduled termination indicated any adverse effect of treatment for either sex.

HISTOPATHOLOGY (PARENTAL ANIMALS): No treatment-related changes were observed.

Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Based on the lack of treatment-related effects observed throughout maturation, mating, gestation and lactation phases of the study.
Remarks on result:
other: Generation: P and F1
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
VIABILITY (OFFSPRING): Litter size and offspring survival were considered to have been unaffected by maternal treatment at dosages of up to 1000 mg/kg/day. Mean numbers of corpora lutea, implantation, offspring at birth and live offspring at Day 1 post partum in treated groups were essentially similar to control. Subsequent offspring survival to weaning in treated groups was also similar to control. Sex ratio for offspring at birth and live
offspring at Day 1 of age and weaning (day 21) was similar in all groups and did not indicate any selective effect of survival for either sex.

CLINICAL SIGNS (OFFSPRING): No toxicologically significant clinical findings were observed.

BODY WEIGHT (OFFSPRING): No adverse treatment-related effects on offspring bodyweight were observed through the study period.

SEXUAL MATURATION (OFFSPRING): There were no adverse effects on sexual maturation observed in the offspring.

GROSS PATHOLOGY (OFFSPRING): The macroscopic findings observed for interim and terminal kill offspring throughout the dose groups were consistent with normally expected low incidence findings in pups of the age examined and were of no toxicological importance.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Based on the lack of treatment-related effects observed throughout maturation, mating, gestation and lactation phases of the study.
Reproductive effects observed:
not specified
Conclusions:
The oral administration of1-dodecene, trimer to rats by gavage at a maximum dose level of 1000 mg/kg/day, throughout maturation, mating, gestation and lactation resulted in no treatment-related effects. The ‘No Observed Effect Level’ for adult toxicity and reproductive and developmental toxicity
was therefore considered to be 1000 mg/kg/day.
Executive summary:

In a one-generation reproduction study,1 -dodecene, trimer was administered orally, once daily, by gavage to three groups each of twenty-four male and twenty-four female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, at dose levels of 1000, 250 and 50 mg/kg/day. A further group of twenty-four male and twenty-four female rats received the vehicle alone to serve as a control.

 

There were two unscheduled deaths on the study, occurring in the control and 250 mg/kg/day dosage groups, neither of which was associated with treatment. There were no signs of clinical toxicity observed in either sex at any of the doses tested. Behavioural and functional performance remained unaffected in male and female rats treated with 1 -dodecene, trimer. Sensory reactivity, body weight, food and water consumption were unaffected as were fertility and mating performance. Haematological and clinical chemistry assessments revealed no significant treatment-related effects on male and female rats.

 

No treatment-related effects on offspring growth or development were detected. Litter sizes from birth to weaning were essentially similar across all dose groups. Gross necroscopy did not reveal any remarkable findings and neither did histopathology.

 

The oral administration of 1 -dodecene, trimer to rats by gavage at a maximum dose level of 1000 mg/kg/day, throughout maturation, mating, gestation and lactation resulted in no treatment related effects. Thus, the NOAEL for adult toxicity and reproductive and developmental toxicity was considered to be 1000 mg/kg/day.

 

This study received a Klimisch score of 1 and is classified as reliable without restriction because it was conducted according to GLP, is scientifically acceptable and appears to have adhered to the OECD guideline recommendations.

 

This study will influence the DNEL.

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993-06-01 to 1993-11-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because it was compliant with GLPs and appeared to follow OECD 415 guidelines.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Principles of method if other than guideline:
This study was the preliminary part of a 91-day subchronic study with in utero exposure. Males and females were mated for 4 weeks prior to mating and through mating until sacrifice, which for females included through gestation and lactation day 20.
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina
- Age at study initiation: (P) 10 weeks; The F1 generation was used for a separate subchronic study and not reproduction
- Weight at study initiation: (P) Males: 319 to 422 grams; Females: 202 to 274 grams; F1 animals were not used as a part of the reproduction study
- Housing: Individually except during cohabitation
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 17 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 40 to 70%
- Air changes (per hr): 10 to 12 per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light


IN-LIFE DATES: From: 1995-06-01 To: 1993-08-11
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Specific amount of test compound was weighed into a beaker with the appropriate amount of vehicle. The mixture was stirred by hand then sufficient vehicle was added to achieve the desired concentration. Solutions were then stirred for 30 minutes using a stir bar.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Not reported
- Concentration in vehicle: 0, 20, 100, or 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg of vehicle or solution was administered
- Lot/batch no. (if required): 920897 and 933384
- Purity: Not reported
Details on mating procedure:
- M/F ratio per cage: 1 to 1 ratio
- Length of cohabitation: 15 days
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male from the same group.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged in nesting boxes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to study initiation, homogeneity and stability tests were performed. All test mixtures were analysed for verification of the test article concentration. Samples were found to be homogeneous and stable. All concentrations used were found to be within 20% of the nominal concentration.
Duration of treatment / exposure:
Males and females were dosed for 4 weeks prior to mating and through mating until sacrifice, which for females included through gestation and lactation day 20.
Frequency of treatment:
Daily
Details on study schedule:
- F1 animals not mated.
Remarks:
Doses / Concentrations:
0, 100, 500, or 1000 mg/kg/day
Basis:
other: nominal in polyethylene glycol 400
No. of animals per sex per dose:
Thirty animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Not reported
- Rationale for animal assignment (if not random): Random
Positive control:
No positive control was used.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once a day
- Cage side observations included mortality, morbidity, and overt signs of toxicity.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly


BODY WEIGHT: Yes
- Time schedule for examinations: Males: weekly; Females: Gestational days 0, 7, 14, and 20 and lactation days 1, 7, 14, and 21


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


WATER CONSUMPTION: No


Oestrous cyclicity (parental animals):
Estrous cyclicity was not examined.
Sperm parameters (parental animals):
Sperm parameters were not examined.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of eight pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, and clinical signs


GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at the end of breeding.
- Maternal animals: All surviving animals on lactation day 21.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY / ORGAN WEIGHTS: Only abnormal tissues were preserved for possible microscopic examination. No organs were specified as weighed.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected for subchronic study were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations macroscopic examination as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY / ORGAN WEIGHTS: No histopathology or organ weights were conducted.
Statistics:
Continuous data were analysed using a one way analysis of variance followed by a Dunnett's test (sometimes a modified Dunnett's test). Count data were analysed using a Chi-square or a Mann-Whitney U test.
Reproductive indices:
Gestation and lactation body weight, copulation index, fertility index, precoital interval, gestation length, gravid and non-gravid females that did not deliver, and presence of corpora lutea
Offspring viability indices:
Alive pups on day 1, 4, 8, 14, and 21; dead and live pups on lactation day 0; sex ratio; mean litter size; and body weight
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
not specified
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): Soft stools and faecal staining occurred in all groups including the control and were considered related to the vehicle used. There were no clinical signs related to treatment.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): There were no treatment-related effects.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): There were no treatment-related effects.


GROSS PATHOLOGY (PARENTAL ANIMALS): There were no treatment-related effects.


Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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 were no treatment-related effects on the viability parameters measured.


CLINICAL SIGNS (OFFSPRING): There were no clinical signs related to treatment.


BODY WEIGHT (OFFSPRING): There were no treatment-related effects on pup body weight.


GROSS PATHOLOGY (OFFSPRING): There were no gross pathology findings related to treatment.


Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Overall effects
Reproductive effects observed:
not specified
Conclusions:
Dec-1-ene, homopolymer, hydrogenated did not appear to have any effects on reproduction.
Executive summary:

In a one-generation reproduction study, dec-1 -ene, homopolymer, hydrogenated was administered to 30 Sprague-Dawley Crl:CD®BR VAF/Plus® rats/sex/dose by gavage at dose levels of 0, 100, 500, or 1000 mg/kg bw/day. Both males and females were treated for 4 weeks prior to mating and through mating. At the end of mating, males were sacrificed. Females were treated through gestation and until lactation day 21. On lactation day 4, litters were culled to 8 pups (4 per sex).

 

There were no treatment-related effects on clinical signs, mortality, body weight, or gross pathology in the parental generation or in the pups through lactation day 21. There were no treatment related effects on reproduction or pup viability. Some pups were used further in a subchronic study with the remainder sacrificed on lactation day 21. There is no parental or offspring systemic or reproduction LOAEL, based n the lack of effects. The parental systemic and reproduction NOAEL is 1000mg/kg bw/day in males and females. The offspring NOAEL is 1000 mg/kg bw/day even after the additional 91 day subchronic exposure.

 

This study received a Klimisch score of 1 and is classified as reliable with restrictions because it was compliant with GLPs and appeared to follow OECD 415 guidelines. It should be noted that this study provides information on the reproduction in rats, but was part of a subchronic study with exposure in utero and therefore did not examine all aspects of reproduction. Consequently, it does not meet REACh's requirement for a two-generation study. This study will not influence the DNEL.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Species:
rat
Quality of whole database:
Four key read across studies available for assessment.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A key OECD Guideline 422 combined 28-Day repeated dose toxicity study with the Reproduction / Developmental Toxicity Screening Test was conducted to evaluate the potential toxic effects of the test material (1-Dodecene, dimers; CAS# 62132-67-6) when given orally to Wistar Han rats and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development (Charles River Laboratories Den Bosch BV, 2022).

 

The test material Wistar Han rats (10/sex/dose) were administered once daily via oral gavage in a corn oil vehicle at doses of 0, 100, 300, or 1000 mg/kg/day for a minimum of 28 days for males (including at least 2 weeks of treatment prior to mating and during the mating period up to and including the day before scheduled necropsy). Female rats were dosed 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy.

 

Parameters were evaluated in this study included mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, clinical pathology, measurement of thyroid hormones T3, T4 and TSH (F0 males and females), gross necropsy findings, organ weights and histopathologic examinations. Additionally, reproduction/developmental parameters were also determined and included mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormones T3 and T4 (PND 4 and PND 14-16 pups), and TSH (PND 14-16).

 

No treatment-related mortality was observed in the F0 animals.

 

One control female (No. 43) was sacrificed in extremis on Day 23 post coitum. The female had a pale appearance and piloerection and at the point 3 pups were born of which 1 was alive and 2 were found dead. As no more pups were born and the condition of the animal deteriorated, the animal was sacrificedin extremis. At necropsy, the uterus of this female contained 3 alive and 5 dead fetuses. No other macroscopic or microscopic findings were observed. Based on this, the condition of this female was considered to be related to delivery difficulties.

 

One female in the 100 mg/kg/day (No. 60) dose group was sacrificed in extremis on Day 1 of lactation. The animal was found with a pale appearance and piloerection. Due to the deteriorating condition of this animal, it was sacrificed in extremis. Additionally, all 11 delivered pups were found to be dead. At necropsy, a pale liver was observed corresponding to microscopic moderate multifocal necrosis in the liver which likely contributed to the moribundity. Based on the single incidence of this event in the low dose group, the condition of this animal was not considered to be treatment-related.

 

No treatment-related clinical signs were observed either during daily detailed clinical or during the weekly arena observations.

 

Incidental findings observed included scabs, wounds, and alopecia. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be treatment-related.

 

Body weights and body weight gain were considered to be unaffected by treatment. Females at 1000 mg/kg/day displayed slightly lower (6% lower than control) absolute body weight on Day 4 of lactation was. As body weight gain was similar over this period and as this was only an incidental occurrence, this temporary lower body weight was not considered to be treatment-related. Food consumption before or after correction for body weight was not affected by treatment.

 

Hearing ability, pupillary reflex, static righting reflex and grip strength of the fore legs (males and females), and motor activity (females only) were considered unaffected by treatment at 100 and 300 mg/kg/day.

 

Females at 1000 mg/kg/day displayed decreased grip strength of the hind legs (0.63x of control).In the absence of clinical signs and corroborative histopathological findings, this was not considered as adverse.

 

An apparent upward trend in motor activity (total movements and ambulations) observed in male rats of all treatment groups was considered to have arisen as a result of slightly low control values and therefore not considered treatment-related.

 

All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

 

Hematological parameters of rats were unaffected by treatment. A reduction of large unstained cell (LUC) counts was observed in male rats at 300 mg/kg/day. However, in the absence of a dose-related trend, this was considered to be unrelated to treatment. Coagulation parameters of rats were unaffected by treatment. A shorter prothrombin time (PT) observed in male rats at 100 mg/kg/day was considered to be unrelated to treatment in the absence of a dose-related trend.

 

Clinical chemistry parameters of male rats were unaffected by treatment. In female rats, a reduction in total bilirubin levels (TBIL) was observed at 100, 300, and 1000 mg/kg/day (0.69x, 0.71x and 0.66x of control, respectively). In the absence of corroborative histopathological findings, this was considered not adverse. Increased sodium levels were observed in female rats at 300 mg/kg/day but this considered to be minor and in the absence of a dose-related trend, unrelated to treatment.

 

Serum levels of thyroxine (T4) and thyroid-stimulating hormone (TSH) in male and female rats were unaffected by treatment. Serum total triiodothyronine (T3) levels in F0 males at 100 mg/kg/day were increased (Group mean values were 1.29x of control; however, an increase in the SD was also observed). Mean values were within the historical control data range and values at 300 mg/kg/day and 1000 mg/kg/day were comparable with the control group. Therefore, although treatment-related, this increase was not considered as adverse. Serum total T3 levels were increased in F0 females at 1000 mg/kg/day (1.26x of control). No historical control data for total T3 in lactating females was available at the Testing Facility. However, since only 1/10 individual females displayed a T3 value above the highest individual control value, the mean increase of T3 at 1000 mg/kg/day was considered unrelated to treatment.

 

Gross necropsy did not reveal any remarkable treatment-related findings and no treatment-related effects or alterations in organ weights were observed. Any differences, including those that reached statistical significance (males; heart at 100 mg/kg/day (absolute and relative to body weight) and 1000 mg/kg/day (absolute only); liver at 1000 mg/kg/day (absolute only)), were not considered to be treatment-related due to the direction of the change and/or a lack of dose-related trend.

 

Histopathology did not reveal any microscopic observations related to treatment with the test material. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no treatment-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

 

Except one control female (No. 43) that was sacrificed in extremis on Day 23 post-coitum due to presumed delivery difficulties, all other females showed no signs of difficult or prolonged parturition or deficiencies in maternal care. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. The length and regularity of the estrous cycle were not affected by treatment with all females having regular cycles of 4 to 5 days and mating index was also unaffected with all females showing evidence of mating.

 

Precoital time was considered unaffected by treatment and most females showed evidence of mating within 4 days. One female (No. 61) at 300 mg/kg/day showed evidence of mating after 13 days. One female (No. 68) at 300 mg/kg/day that was probably mated during the extended mating period had an estimated precoital time of 1 day. Mating was overlooked in two females at 300 mg/kg/day (Nos. 68 and 70). As these females turned out to be pregnant and delivered a healthy litter, the mating date of these females was estimated at 21 days prior to delivery. The number of implantation sites was considered unaffected by treatment with the mean number of implantation sites being 11.9, 11.6, 12.1, and 12.9 for the control, 100, 300, and 1000 mg/kg/day groups, respectively. Fertility index was not affected by treatment with fertility indices being 100, 90, 100, and 100% for the control, 100, 300, and 1000 mg/kg/day groups, respectively. A lower gestation index observed at 100 mg/kg/day was the result of one female at 100 mg/kg/day (No. 56) that was not pregnant. At the low incidence and in absence of a dose related trend this finding was considered unrelated to treatment with the test material. Overall, no reproductive toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

 

Based on the lack of adverse treatment-related effects observed through the study period, the systemic and reproductive toxicity No Observed Adverse Effect Level (NOAEL) for 1-Dodecene, dimers was determined to be ≥1000 mg/kg/day in the rat.

Additionally, two read-across studies were identified for poly alpha olefins and their structural analogue, 1-dodecene, trimer: a 91-day study which assessed the systemic toxicological effects of treatment with 1-decene, homopolymer, hydrogenated on rats previously treated in utero with the same chemical and a 90-day study 1-dodecene, trimer which assessed fertility and developmental effects in a one-generation study (OECD 415). Details of the studies are presented below.

 

In a one-generation reproduction study, 1-decene, homopolymer, hydrogenated was administered to 30 Sprague-Dawley Crl: CD®BR VAF/Plus® rats/sex/dose by gavage at dose levels of 0, 100, 500, or 1000 mg/kg bw/day (Daniel, 1994). Both males and females were treated for 4 weeks prior to mating and through mating. At the end of mating, males were sacrificed. Females were treated through gestation and until lactation day 21.

 

There were no treatment-related effects on clinical signs, mortality, body weight, or gross pathology in the parental generation or in the pups through lactation day 21. There were no treatment related effects on reproduction or pup viability. Some pups were used further in a subchronic study with the remainder sacrificed on lactation day 21. There is no parental or offspring systemic or reproduction LOAEL, based n the lack of effects. The parental systemic and reproduction NOAEL is 1000mg/kg bw/day in males and females. The offspring NOAEL is 1000 mg/kg bw/day even after the additional 91 day subchronic exposure.

 

In a one-generation reproduction study, 1-dodecene, trimer was administered orally, once daily, by gavage to three groups each of twenty-four male and twenty-four female Sprague-Dawley Crl: CD® (SD) IGS BR strain rats, at dose levels of 1000, 250 and 50 mg/kg/day (Knox et al., 2007). A further group of twenty-four male and twenty-four female rats received the vehicle alone to serve as a control.

 

There were two unscheduled deaths on the study, occurring in the control and 250 mg/kg/day dosage groups, neither of which was associated with treatment. There were no signs of clinical toxicity observed in either sex at any of the doses tested. Behavioural and functional performance remained unaffected in male and female rats treated with 1 -dodecene, trimer. Sensory reactivity, body weight, food and water consumption were unaffected as were fertility and mating performance. Haematological and clinical chemistry assessments revealed no significant treatment-related effects on male and female rats.

 

No treatment-related effects on offspring growth or development were detected. Litter sizes from birth to weaning were essentially similar across all dose groups. Gross necroscopy did not reveal any remarkable findings and neither did histopathology.

 

The oral administration of 1-dodecene, trimer to rats by gavage at a maximum dose level of 1000 mg/kg/day, throughout maturation, mating, gestation and lactation resulted in no treatment related effects. Thus, the NOAEL for adult toxicity and reproductive and developmental toxicity was considered to be 1000 mg/kg/day.

 

No adverse fertility effects were reported in a one-generation study with 1-dodecene, trimer; a structural analogue of dec-1-ene, dimers, hydrogenated, at the limit dose of 1000 mg/kg/day (Knox et al., 2007).  No treatment-related effects were reported in rats treated in utero and then subsequently treated an additional 91 days after birth with 1-decene homopolymer, hydrogenated (Daniel, 1994). The NOAEL was 1000 mg/kg/day for both studies (highest dose tested).

The weight of evidence presented by these studies suggests that poly alpha olefins, as a group, are unlikely to present a significant hazard potential to fertility; therefore a DNEL for this endpoint is not necessary.

 

Justification for Read Across

 

Several criteria justify the use of the read across approach to fill data gaps for poly alpha olefins using 1-dodecene, trimer as an analogue. 1 -dodecene, trimer, like other compounds in this category, is a poly alpha olefin, i. e., highly branched isoparaffinic chemicals produced by oligomerization of oct-1-ene, dec-1-ene, and/or dodec-1-ene. Therefore its physiochemical and toxicological properties are expected to be similar to those of other poly alpha olefins.

Effects on developmental toxicity

Description of key information

Developmental Toxicity NOAEL (Rat): 1000 mg/kg bw/day (OECD 414)

Not expected to cause developmental toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-JUL-11 to 2021-OCT-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau
Version / remarks:
November 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Chevron Phillips Chemical Company LP; Batch No. DBA0146456
- Purity, including information on contaminants, isomers, etc.: 100 % (UVCB)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature (15 to 25°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable for one day at ambient temperature (15 to 25°C) and 17 days when stored refrigerated (2 to 8°C)

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Clear and bright liquid

OTHER SPECIFICS
- Expiration date: 2023-MAY-01
Species:
rat
Strain:
Wistar
Remarks:
RccHan®:WIST
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation: 169 to 238 grams
- Fasting period before study: Not specified
- Housing: Individually in polycarbonate cages with a stainless steel mesh lid
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet ad libitum
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes ad libitum
- Acclimation period: at least 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2021-JUL-15 To: 2021-AUG-06
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared weekly and required amount of the test material was weighed out. Starting with the lowest concentration, approximately 50% of the final volume of vehicle was added to the test material and magnetically stirred until it was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous. The formulation was then transferred to the final containers, via syringe, whilst being magnetically stirred. A series of formulations at the required concentrations were prepared by dilution of individual weighing’s of the test material and stored refrigerated at 2 to 8°C.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified
- Concentration in vehicle: 0, 25, 75, or 250 mg/mL for the control, 100, 300, and 1000 mg/kg/day dose groups, respectively
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no. (if required): Not specified
- Purity: Not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity:
Homogeneity and stability of formulations during storage were confirmed as part of another study (Labcorp Study Number 8457609). In that study, formulations in the range 2.5 mg/mL to 250 mg/mL were determined to be stable for one day at ambient temperature (15 to 25°C); 17 days when stored refrigerated (2 to 8°C).

Concentration analysis:
Samples of each formulation prepared for administration in the first and last weeks of treatment were analyzed for achieved concentration of the test material using gas chromatography with flame ionization detection.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Natural mating with Han Wistar of established fertility at the supplier’s facility. Males and females were not related. Positive evidence of mating was considered to be Gestation Day 0. Animals arrived 2 days after mating.
Duration of treatment / exposure:
Day 6 to Day 20 (inclusive) after mating
Frequency of treatment:
Once daily
Duration of test:
Day 6 to Day 20 (inclusive) after mating
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (Control - Corn oil)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2 (Low dose)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3 (Intermediate dose)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4 (High dose)
No. of animals per sex per dose:
22/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels selected were based on the results of a preliminary study (Labcorp Study Number 8457611) which investigated doses of 0, 100, 300, and 1000 mg/kg/day. There was no evidence of maternal toxicity, on the outcome of pregnancy, and all fetuses were observed to be macroscopically normal. Adjusted maternal body weight change (GD 6 21) at 300 or 1000 mg/kg/day was low (48% or 63% of Control, respectively) but attributed at 1000 mg/kg/day to the marginally high litter size, which was due to chance, when compared with Control.

The high-dose level should produce some maternal and/or developmental toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity. Therefore, doses of 0, 100, 300, and 1000 mg/kg/day were investigated in the current OECD 414 study.

- Rationale for animal assignment (if not random): Randomly to each group on the day of arrival

- Fasting period before blood sampling for (rat) dam thyroid hormones: No overnight deprivation of food

- Time of day for (rat) dam blood sampling: time not specified but to minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparisons.

- Other:
- Animal model: The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The RccHan®:WIST. (Han Wistar) strain was used because of the historical control data available at the CRO.
- Route of administration: The oral gavage route of administration was chosen to simulate the conditions of possible human exposure
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment and cages were inspected daily for evidence of animal ill-health.

Signs associated with dosing: Detailed observations were recorded daily during the treatment period (Pre-dose ; One to two hours post-dosing; As late as possible in the working day)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 3, 6, 12, 18, and 21 after mating.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was determined on Days 3 and 6-21 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
The weight of food supplied to each adult, that remaining, and an estimate of any spilled was recorded for the following periods: Days 3-5, 6-8, 9-11, 12-14, 15-17, and 18-20 after mating inclusive.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: Gravid uterine weight (including cervix and ovaries) and Thyroid weighed.

All adult animals were sacrificed via carbon dioxide asphyxiation and subjected to a detailed necropsy. A full macroscopic examination of the tissues was performed and all external features and orifices examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded. For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

OTHER:
Histology:
Tissues were routinely preserved in 10% Neutral Buffered Formalin, dehydrated, embedded in paraffin wax, and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required. Sections were stained with hematoxylin and eosin.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: Yes (including cervix and ovaries)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Fetuses (live and dead)
Blood sampling:
- Serum: Yes
- Volume collected : 1.0 mL

Thyroid Hormone Analysis:
Blood samples were collected from all adult animals at termination on GD 21 (necropsy). 1.0 mL of blood was collected from the sublingual vein under isoflurane anaesthesia and stored in tubes with clotting activator. Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes and then centrifuged at 2000 g for ten minutes at 4°C. Serum was then transferred to appropriately labelled polypropylene “cryo” tubes using plastic disposable pipettes and mixed by gentle ten-fold inversion. Following mixing, each serum sample was divided in two aliquots (aliquot 1: 0.2 mL serum for T3/T4; aliquot 2: residual serum for TSH). The samples were then analyzed for Triiodothyronine (T3); Thyroxine (T4); and Thyroid stimulating hormone (TSH) concentrations.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: yes
Statistics:
Please see 'Any other information on materials and methods incl. tables' for information on statistics.
Indices:
1) Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations) / (Number of corpora lutea) x 100

2) Post-implantation loss (%) = (Number of implantations - Number of live fetuses) / (Number of implantations) x 100
Historical control data:
Historical control data is presented in Tables 13 to 15 in the section 'Any other information on results incl. tables'.
Clinical signs:
no effects observed
Description (incidence and severity):
No adverse treatment-related clinical signs of toxicity were observed through the study period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Overall body weight gain (GD 6-21) and adjusted body weight change (GD 6-21) was unaffected by treatment at 100, 300 or 1000 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Overall food consumption was unaffected by treatment at 100, 300 or 1000 mg/kg/day.

While overall food consumption was statistically significantly high at 300 or 1000 mg/kg/day, the differences were marginal (106% or 112% of Control, respectively) and therefore considered to be incidental rather than treatment-related.
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
Endocrine findings:
no effects observed
Description (incidence and severity):
No effect of treatment was observed on serum concentrations of T3, T4, and TSH.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed in the thyroids (with parathyroids).
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross necropsy did not reveal any remarkable treatment-related findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no microscopic findings in the thyroids.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The number of implantations and the extent of pre- and post-implantation losses (%) were unaffected by treatment at 100, 300 or 1000 mg/kg/day.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The number of resorptions were unaffected by treatment at 100, 300 or 1000 mg/kg/day.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The number of resorptions were unaffected by treatment at 100, 300 or 1000 mg/kg/day.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
One Control animal (No. 11), two animals treated at 100 mg/kg/day (No’s 33 and 39), one animal treated at 300 mg/kg/day (No. 65), and one animal treated at 1000 mg/kg/day (No. 84) were not pregnant.
Other effects:
no effects observed
Description (incidence and severity):
Placental weights were unaffected by treatment at 100, 300, or 1000 mg/kg/day.
Key result
Dose descriptor:
NOEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: Systemic Toxicity
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Fetal weights were unaffected by treatment at 100, 300, or 1000 mg/kg/day.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of live young was unaffected by treatment at 100, 300, or 1000 mg/kg/day.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The ratio of male to female fetuses was unaffected by treatment at 100, 300, or 1000 mg/kg/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Total litter weights were unaffected by treatment at 100, 300, or 1000 mg/kg/day.

One Control animal (No. 11), two animals treated at 100 mg/kg/day (No’s 33 and 39), one animal treated at 300 mg/kg/day (No. 65) and one animal treated at 1000 mg/kg/day (No. 84) were not pregnant and one animal at 300 mg/kg/day had no live fetuses. Therefore, assessment of litter response was performed on 21 litters at 0 or 1000 mg/kg/day and on 20 litters at 100 or 300 mg/kg/day.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
Ano-genital distance was unaffected subsequent to parental exposure to the test material at 100, 300, or 1000 mg/kg/day.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
No major or minor treatment-related fetal abnormalities were observed.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Two female fetuses in one litter (Dam/Litter No. 67) at 1000 mg/kg/day exhibited abnormalities of the head (one fetus had Microphthalmia (left eye) and one fetus had Brachygnathia and other related cranial abnormalities (jaw, palate, ear and nose)). However, given that only a single litter was affected and in the absence of related aetiology to other malformations at 1000 mg/kg/day or lower, these observations were not considered treatment-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
An increase in the incidence of the minor visceral abnormality subdural brain haemorrhage compared to control animals was observed at 1000 mg/kg/day (six fetuses in five litters). This exceeded the Historical Control Data (HCD) range (fetuses; 0-3, litters; 0-3) but was considered to have occurred during the necropsy procedure and therefore, was unrelated to treatment with the test material.
Other effects:
not examined
Key result
Dose descriptor:
NOEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental Toxicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Formulation Analysis

Analyzed mean concentrations of 1-Dodecene, dimers were within 7% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 2%, confirming precise analysis.

Table 2. Results of Formulation Analysis

Occasion

Group

Nominal concentration (mg/mL)

Analyzed concentration

(mg/mL)

RME (%)

Difference from mean (%)

Procedural recoveries

Analysis 1

Analysis 2

Mean

Analyzed (%)

Mean

First Week

1

0

ND

ND

-

-

-

-

100.4

2

25

27.1

26.1

26.6

+6.4

±1.90

108.01

3

75

78.1

77.3

77.7

+3.6

±0.47

100.4

4

250

265

262

264

+5.6

±0.47

100.3

 

 

 

 

 

 

 

Last Week

1

0

ND

ND

-

-

-

-

100.4

2

25

25.6

26.0

25.7

+2.8

±0.28

108.01

3

75

69.3

71.1

77.1

+2.8

±1.28

100.4

4

250

242

239

241

-3.6

±0.51

100.3

RME    Relative mean error, representing the deviation from nominal

ND       Not detected

1           Result above the range established during the validation therefore excluded as per SOP.

Table 3. Body weight and Body weight change - Group mean values (g) during Gestation

Dose

Group

 

Day

Change

3

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

3-6

6-21

Statistics

Test

 

Av

Av

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Av

Wi

Group 1

Control

0

mg/kg/day

Mean

208

222

222

224

228

233

237

241

243

247

254

262

271

282

290

300

306

14

85

SD

15.7

15.3

16.0

16.6

15.5

16.3

15.8

15.2

16.3

16.9

17.9

19.0

18.9

21.0

21.7

23.1

23.7

4.4

14.4

N

21

21

21

21

21

21

21

21

21

21

21

21

21

21

21

21

21

21

21

 

Group 2

100

mg/kg/day

Mean

202

214

216

219

222

227

232

236

239

243

249

257

265

276

285

294

302

12

87

SD

10.3

10.1

9.8

9.6

10.2

10.2

11.3

11.0

11.3

12.1

12.5

12.6

13.2

15.9

17.1

17.8

18.3

4.5

16.5

N

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

 

Group 3

300

mg/kg/day

Mean

204

217

216

219

223

227

233

238

241

245

252

259

269

280

288

299

304

13

88

SD

17.1

16.2

16.0

17.9

18.0

18.7

18.3

18.3

18.2

18.5

19.7

19.9

20.5

21.3

21.7

23.1

23.8

3.3

11.9

N

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

 

Group 4

1000

mg/kg/day

Mean

204

214

215

219

223

226

231

235

238

242

249

255

265

277

286

296

304

10

90

SD

14.1

14.1

14.2

14.5

14.3

14.7

14.4

14.3

13.7

14.3

14.6

15.1

15.9

16.6

17.8

18.2

18.7

5.1

7.8

N

21

21

21

21

21

21

21

21

21

21

21

21

21

21

21

21

21

21

21

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests.

Wi: Treated groups compared with Control using Williams’ test

Table 4. Gravid uterine weight, adjusted body weight and adjusted body weight change - group mean values (kg) on Day 21 of gestation

Dose Group

(mg/kg/day)

 

Body Weight

Day 6

Terminal Body Weight

Day 21

Body Weight Change

Day 6-21

Gravid Uterine Weight

Adjusted Body Weight

Day 21

Adjusted Body Weight Change

Day 6-21

Statistics test

 

Av

Wi

Wi

Wi

Wi

Wi

Group 1

(Control –

0 mg/kg/day)

Mean

222

306

84

68.7

237

16

SD

15.3

23.5

14.0

15.63

18.1

11.0

N

21

21

21

21

21

21

 

Group 2

(Control –

100 mg/kg/day)

Mean

214

302

87

63.6

238

24

SD

10.1

18.4

16.5

20.18

13.6

8.7

N

20

20

20

20

20

20

 

Group 3

(Control –

300 mg/kg/day)

Mean

217

304

87

67.1

237

20

SD

16.2

24.0

12.0

15.57

22.4

11.0

N

20

20

20

20

20

20

 

Group 4

(Control –

1000 mg/kg/day)

Mean

214

303

89

71.7

231

17

SD

14.1

18.9

7.8

10.13

14.2

7.9

N

21

21

21

21

21

21

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests.

Wi: Treated groups compared with Control using Williams’ test

Table 5. Food consumption - Group Mean Values (g/animal/day) during Gestation (Females)

Dose

Group

 

Day

Mean

3-6

6-9

9-12

12-15

15-18

18-21

6-21

Statistics

Test

 

Av

Wi

Wi

Wi

Wi

Sh

Wi

Group 1

(Control –

0 mg/kg/day)

Mean

17

16

17

18

20

16

17

SD

1.8

2.1

1.1

1.5

2.0

1.9

1.2

N

21

21

21

21

21

21

21

 

Group 2

(Control –

100 mg/kg/day)

Mean

16

15

17

18

19

17

17

SD

2.5

1.4

1.5

1.6

2.1

1.1

1.2

N

20

20

20

20

20

20

20

 

Group 3

(Control –

300 mg/kg/day)

Mean

17

16

19*

19

20

18

18*

SD

1.7

1.8

1.8

1.9

1.8

2.6

1.7

N

20

20

20

20

20

20

20

 

Group 4

(Control –

1000 mg/kg/day)

Mean

17

17*

18*

19

21

18

19**

SD

1.8

1.8

1.8

1.6

2.0

3.9

1.4

N

21

21

21

21

21

21

21

* p<0.05

** p<0.01

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests.

Wi: Treated groups compared with Control using Williams’ test

Sh: Treated groups compared with Control using Shirley’s test

Table 6. Group Mean Serum T3, T4, and TSH Concentration Data in Pregnant Rats

Dose Group

(mg/kg/day)

Parameters

Gestational Day 21 Termination

T3 (pg/mL)

T4 (pg/mL)

TSH (pg/mL)

Group 1

(Control –

0 mg/kg/day)

Mean

470

21700

690

SD

127

5280

308

CV%

27.0

24.3

44.6

N

21

21

21

 

Group 2

(Control –

100 mg/kg/day)

Mean

473

21900

639

SD

162

6310

247

CV%

34.2

28.8

38.7

N

20

20

20

 

Group 3

(Control –

300 mg/kg/day)

Mean

522

21000

635

SD

130

5100

261

CV%

24.9

24.3

41.0

N

20

20

21

 

Group 4

(Control –

1000 mg/kg/day)

Mean

509

22100

696

SD

96.3

4570

215

CV%

18.9

20.7

31.0

N

21

21

21

Table 7. Litter data - Group Mean Values on Day 21 of Gestation

Group

 

Corpora Lutea

Implantations

Resorptions

Implantation Loss (%)

Live Young

Sex Ratio

(% M)

Early

Late

Total

Pre-

Post-

Male

Female

Total

Statistics Test

 

Wi

Wi

 

 

 

Wi

Wi

 

 

Wi

Wi

Group 1

(Control –

0 mg/kg/day)

Mean

11.7

10.1

0.2

0.1

0.4

14.4

3.2

4.0

5.7

9.7

41.9

SD

1.85

2.74

0.54

0.36

0.59

16.89

5.02

2.01

2.13

2.51

17.77

N

21

21

21

21

21

21

21

21

21

21

21

 

Group 2

(Control –

100 mg/kg/day)

Mean

12.2

9.6

0.3

0.2

0.4

21.2

4.1

4.4

4.8

9.2

51.0

SD

1.51

3.12

0.91

0.37

0.94

24.75

10.28

1.96

2.40

3.20

18.98

N

20

20

20

20

20

20

20

20

20

20

20

 

Group 3

(Control –

300 mg/kg/day)

Mean

11.8

10.5

0.8

0.2

1.0

12.4

8.2

5.2

4.4

9.6

53.4

SD

1.54

2.69

0.91

0.41

1.00

19.30

8.48

2.11

1.76

2.42

14.21

N

20

20

20

20

20

20

20

20

20

20

20

 

Group 3

(Control –

1000 mg/kg/day)

Mean

12.3

11.0

0.6

0.0

0.6

11.6

4.9

5.0

5.4

10.4

49.8

SD

1.77

1.90

0.87

0.00

0.87

13.21

7.42

1.63

2.29

1.86

17.39

N

21

21

21

21

21

21

21

21

21

21

21

Wi: Treated groups compared with Control using Williams’ test

Table 8. Placental, Litter and Fetal weights - group mean values (g) on Day 21 of gestation

Dose Group

(mg/kg/day)

 

Placental Weight

Total Litter Weight

Male Fetal Weight

Female Fetal Weight

Overall Fetal Weight

Statistics test

 

Wi

Wi

Sh

Sh

Sh

Group 1

(Control –

0 mg/kg/day)

Mean

0.47

48.68

5.22

4.99

5.09

SD

0.066

12.359

0.689

0.686

0.678

N

21

21

21

21

21

 

Group 2

(Control –

100 mg/kg/day)

Mean

0.46

47.58

5.38

5.09

5.26

SD

0.071

16.028

0.382

0.398

0.381

N

20

20

20

20

20

 

Group 3

(Control –

300 mg/kg/day)

Mean

0.47

49.79

5.37

5.06

5.22

SD

0.053

12.391

0.288

0.307

0.302

N

20

20

20

20

20

 

Group 4

(Control –

1000 mg/kg/day)

Mean

0.46

53.47

5.31

5.00

5.16

SD

0.035

8.396

0.255

0.336

0.289

N

21

21

21

21

21

Wi: Treated groups compared with Control using Williams’ test

Sh: Treated groups compared with Control using Shirley’s test

Table 9. Ano-genital Distance - Group Mean Absolute and Adjusted Values for Fetuses on Day 21 of Gestation

Dose Group

(mg/kg/day)

 

Fetal Weight (g)

Ano-genital / distance (mm)

Statistics test

Sh

Sh

Males

Group 1

(Control –

0 mg/kg/day)

Mean

5.22

3.47

SD

0.69

0.21

N

21

21

 

Group 2

(Control –

100 mg/kg/day)

Mean

5.38

3.48

SD

0.38

0.16

N

20

20

 

Group 3

(Control –

300 mg/kg/day)

Mean

5.37

3.61

SD

0.29

0.40

N

20

20

 

Group 4

(Control –

1000 mg/kg/day)

Mean

5.31

3.46

SD

0.26

0.19

N

21

21

Females

Group 1

(Control –

0 mg/kg/day)

Mean

4.99

2.21

SD

0.69

0.11

N

21

21

 

Group 2

(Control –

100 mg/kg/day)

Mean

5.09

2.25

SD

0.40

0.14

N

19

19

 

Group 3

(Control –

300 mg/kg/day)

Mean

5.06

2.23

SD

0.31

0.15

N

20

20

 

Group 4

(Control –

1000 mg/kg/day)

Mean

5.00

2.24

SD

0.34

0.13

N

21

21

Sh: Treated groups compared with Control using Shirley’s test

Table 10. Summary of Fetal examinations - Major Abnormalities – Group Incidences

 

 

Fetuses

Litters

Group

Group 1

0

mg/kg/day

Group 2

100

mg/kg/day

Group 3

300

mg/kg/day

Group 4

1000 mg/kg/day

Group 1

0

mg/kg/day

Group 2

100

mg/kg/day

Group 3

300

mg/kg/day

Group 4

1000 mg/kg/day

Number Examined

204

183

191

219

21

20

20

21

Total Number Affected

1

0

1

2

1

0

1

1

Head

 

Skeletal

Fused premaxillae

0

0

0

1

0

0

0

1

Single medial lower incisor socket

0

0

0

1

0

0

0

1

Absent upper incisor socket(s)

0

0

0

1

0

0

0

1

Fused mandibles

0

0

0

1

0

0

0

1

Displaced tympanic annula

0

0

0

1

0

0

0

1

Brachygnathia

0

0

0

1

0

0

0

1

Basisphenoid partially fused to tympanic annula

0

0

0

1

0

0

0

1

Misshapen basisphenoid

0

0

0

1

0

0

0

1

Partially fused nasals

0

0

0

1

0

0

0

1

Misshapen palatine bones

0

0

0

1

0

0

0

1

Visceral

Microphthalmia

0

0

0

1

0

0

0

1

Cervical/Thoracic

 

Visceral

Complete situs inversus

0

0

1

0

0

0

1

0

Appendicular

 

External

Malrotated hindlimb(s)

1

0

0

0

1

0

0

0

Table 11. Summary of Fetal examinations - Minor Skeletal Abnormality and Variants Findings – Group Incidences

Group

 

Fetuses

Litters

Group 1

0

mg/kg/day

Group 2

100

mg/kg/day

Group 3

300

mg/kg/day

Group 4

1000 mg/kg/day

Group 1

0

mg/kg/day

Group 2

100

mg/kg/day

Group 3

300

mg/kg/day

Group 4

1000 mg/kg/day

Number Examined

98

87

91

105

21

20

20

21

Minor skeletal abnormalities

 

Cranial

sutural bone(s)

1

0

0

0

1

0

0

0

Ribs

medially thickened/kinked

1

2

2

0

1

2

2

0

Sternebrae

bipartite ossified

0

0

1

0

0

0

1

0

misaligned ossification sites

1

2

1

1

1

2

1

1

Costal cartilage

2nd not connected to sternum

0

0

0

1

0

0

0

1

interrupted

3

3

0

0

3

3

0

0

Total affected by one or more of the above

 

6

7

4

2

5

6

4

2

Rib and vertebral configuration

 

Cervical rib

short supernumerary

3

5

2

1

2

3

2

1

13thRib

short without costal cartilage

0

0

1

2

0

0

1

2

Number of 14thRibs

short supernumerary

22

8

17

22

11

5

9

10

full supernumerary

0

0

0

1

0

0

0

1

total

22

8

17

22

11

5

9

10

Thoracolumbar vertebrae

20

1

0

0

0

1

0

0

0

Pelvic girdle

unilateral caudal shift

0

0

1

2

0

0

1

1

Delayed/Incomplete ossification/unossified

 

Cranial

Cranial centres

3

0

0

1

2

0

0

1

Sternebrae

5th and/or 6th

11

7

4

4

6

5

2

3

1st to 4th

0

1

2

1

0

1

2

1

total

11

7

6

4

6

5

4

3

Vertebrae

Cervical

7

0

0

2

2

0

0

1

Thoracic

1

0

1

2

1

0

1

2

Caudal

1

0

0

0

1

0

0

0

Appendicular

metacarpals

2

0

0

0

1

0

0

0

Increased ossification

 

Cranial

fused/partially fused jugal to maxilla

5

8

3

5

5

5

2

4

Table 12. Summary of Fetal examinations - Minor Visceral Abnormality and Necropsy Findings – Group Incidences

Group

 

Fetuses

Litters

Group 1

0

mg/kg/day

Group 2

100

mg/kg/day

Group 3

300

mg/kg/day

Group 4

1000 mg/kg/day

Group 1

0

mg/kg/day

Group 2

100

mg/kg/day

Group 3

300

mg/kg/day

Group 4

1000 mg/kg/day

Number Examined

106

96

99

114

21

20

20

21

Number of Heads Examined at Detailed Visceral Examination

 

106

96

100

114

21

20

20

21

Head abnormalities (fixed visceral)

 

Eyes

folded retina

1

0

0

0

1

0

0

0

dilated orbital sinus

0

1

1

0

0

1

1

0

Brain

subdural haemorrhage

0

1

1

6

0

1

1

5

Total affected by one or more of the above

 

1

2

2

6

1

2

2

5

Necropsy observations

(fresh visceral)

 

Thymus

partially undescended lobe

1

0

0

0

1

0

0

0

Umbilical artery

left

2

7

6

8

2

5

5

5

Total affected by one or more of the above

 

3

7

6

8

3

5

5

5

Table 13. Fetal examinations - Major Historical Control Data (July 2015 – present)

 

 

Study # 8457610

HCD Range

 

 

Fetuses

Litters

Fetuses

Litters

Group

Group 1

0

mg/kg/day

Group 2

100

mg/kg/day

Group 3

300

mg/kg/day

Group 4

1000 mg/kg/day

Group 1

0

mg/kg/day

Group 2

100

mg/kg/day

Group 3

300

mg/kg/day

Group 4

1000 mg/kg/day

Number Examined

204

183

191

219

21

20

20

21

Head

 

 

 

Skeletal

Fused premaxillae

0

0

0

1

0

0

0

1

0-0

0-0

Single medial lower incisor socket

0

0

0

1

0

0

0

1

0-1

0-1

Absent upper incisor socket(s)

0

0

0

1

0

0

0

1

0-0

0-0

Fused mandibles

0

0

0

1

0

0

0

1

0-0

0-0

Displaced tympanic annula

0

0

0

1

0

0

0

1

0-0

0-0

Brachygnathia

0

0

0

1

0

0

0

1

0-1

0-1

Basisphenoid partially fused to tympanic annula

0

0

0

1

0

0

0

1

0-0

0-0

Misshapen basisphenoid

0

0

0

1

0

0

0

1

0-0

0-0

Partially fused nasals

0

0

0

1

0

0

0

1

0-0

0-0

Misshapen palatine bones

0

0

0

1

0

0

0

1

0-1

0-1

Visceral

Microphthalmia

0

0

0

1

0

0

0

1

0-0

0-0

Cervical/Thoracic

 

 

 

Visceral

Complete situs inversus

0

0

1

0

0

0

1

0

0-0

0-0

Table 14. Fetal examinations - Minor Skeletal Historical Control Data (July 2015 – present)

Group

 

Study # 8457610

HCD Range

Fetuses

Litters

Fetuses

Litters

Group 1

0

mg/kg/day

Group 2

100

mg/kg/day

Group 3

300

mg/kg/day

Group 4

1000 mg/kg/day

Group 1

0

mg/kg/day

Group 2

100

mg/kg/day

Group 3

300

mg/kg/day

Group 4

1000 mg/kg/day

Number Examined

98

87

91

105

21

20

20

21

441

77

Minor skeletal abnormalities

 

 

 

Sternebrae

bipartite ossified

0

0

1

0

0

0

1

0

0-0

0-0

Costal cartilage

2nd not connected to sternum

0

0

0

1

0

0

0

1

0-0

0-0

Rib and vertebral configuration

 

13thRib

short without costal cartilage

0

0

1

2

0

0

1

2

0-0

0-0

Number of 14thRibs

full supernumerary

0

0

0

1

0

0

0

1

0-3

0-2

Pelvic girdle

unilateral caudal shift

0

0

1

2

0

0

1

1

0-4

0-3

Delayed/Incomplete ossification/unossified

 

Sterrnebrae

1stto 4th

0

1

2

1

0

1

2

1

0-13

0-4

Table 15. Fetal examinations - Minor Visceral Historical Control Data (July 2015 – present)

Group

 

Study # 8457610

HCD Range

Fetuses

Litters

Fetuses

Litters

Group 1

0

mg/kg/day

Group 2

100

mg/kg/day

Group 3

300

mg/kg/day

Group 4

1000 mg/kg/day

Group 1

0

mg/kg/day

Group 2

100

mg/kg/day

Group 3

300

mg/kg/day

Group 4

1000 mg/kg/day

Number Examined

106

96

99

114

21

20

20

21

774

77

 

106

96

100

114

21

20

20

21

360

68

Head abnormalities (fixed visceral)

 

 

 

Eyes

dilated orbital sinus

0

1

1

0

0

1

1

0

0-0

0-0

Brain

subdural haemorrhage

0

1

1

6

0

1

1

5

0-3

0-3

Necropsy observations (fresh visceral)

 

Umbilical artery

Left

2

7

6

8

2

5

5

5

3-17

2-12

Conclusions:
Based on the lack of adverse treatment-related effects observed, the No Observed Effect Level (NOEL) of 1-Dodecene, dimers (CAS No. 62132-67-6) for maternal toxicity, embryo-fetal survival, growth and development in rats was determined to be 1000 mg/kg/day.
Executive summary:

A key OECD Guideline 414 pre-natal developmental toxicity study was conducted to evaluate the potential effects of the test material (1-Dodecene, dimers (CAS No. 62132-67-6)) on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the rat.

 

The test material was administered to time-mated female Hans Wistar rats (22/dose) once daily via oral gavage in a corn oil vehicle at doses of 0, 100, 300, or 1000 mg/kg bw/day from Day 6 to 20 after mating. The control group received corn oil only. Clinical observations, body weight, and food consumption were recorded and animals were sacrificed on Day 21 after mating. Adult females were examined macroscopically at necropsy on Day 21 after mating, blood samples were taken for thyroid hormone analysis and the gravid uterus and thyroid weights were recorded. All fetuses were examined macroscopically at necropsy and the ano-genital distance measured. Fetal pathology investigations were undertaken on all fetuses by detailed internal visceral examination or skeletal examination. 

 

 

No mortality or adverse treatment-related clinical signs of toxicity were observed through the study period. Overall body weight gain (GD 6-21), adjusted body weight change (GD 6-21), gravid uterine weight, and overall food consumption was unaffected by treatment at 100, 300 or 1000 mg/kg/day. While overall food consumption was statistically significantly high at 300 or 1000 mg/kg/day, the differences were marginal (106% or 112% of Control, respectively) and therefore considered to be incidental rather than treatment-related. There was no effect of treatment observed on serum concentrations of T3, T4, and TSH and no treatment-related changes were observed in the thyroids (with parathyroids). Gross necropsy did not reveal any remarkable treatment-related findings and no microscopic effects were observed in the thyroids.

 

Implantation losses, the numbers of resorptions and live young and the ratio of male to female offspring was unaffected by treatment.

 

Placental, fetal and total litter weights were also unaffected by treatment and no effect of treatment was observed on fetal ano‑genital distance. No major or minor treatment-related fetal abnormalities were observed. Two female fetuses in one litter (Dam/Litter No. 67) at 1000 mg/kg/day exhibited abnormalities of the head (one fetus had Microphthalmia (left eye) and one fetus had Brachygnathia and other related cranial abnormalities (jaw, palate, ear and nose)). However, given that only a single litter was affected and in the absence of related aetiology to other malformations at 1000 mg/kg/day or lower, these observations were not considered treatment-related. An increase in the incidence of the minor visceral abnormality subdural brain haemorrhage compared to control animals was observed at 1000 mg/kg/day (six fetuses in five litters). This exceeded the Historical Control Data (HCD) range (fetuses; 0-3, litters; 0-3) but was considered to have occurred during the necropsy procedure and therefore, was unrelated to treatment with the test material.

 

Based on the lack of adverse treatment-related effects observed, the No Observed Effect Level (NOEL) of 1-Dodecene, dimers (CAS No. 62132-67-6) for maternal toxicity, embryo-fetal survival, growth and development in rats was determined to be 1000 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17/01/2012 - 14/09/2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Clinical Observations

On Day 18 of the study the five hour observations for animal numbers 85 to 90 were not recorded in error.

Post Mortem Studies

The macroscopic observations for the litter from female number 36 were not recorded due to a technician error.

These deviations do not affect the purpose or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Details on test animals or test system and environmental conditions:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD(SD) IGS BR strain rats obtained from Charles River (UK) Limited, Margate, Kent
At the start of the study the females weighted 186 to 291 g and were approximately ten to twelve weeks old

The animals were housed individually in solid-floor polypropylene cages with stainless steel lids furnished with softwood flakes in a single air-conditioned room within the Harlan Laboratories
The rate of air exchange was at least 15 air changes per hour and low intensity fluorescent lighting was controlled to give 12 hours continuous light and 12 hours darkness
The temperature and relative humidity were set to achieve target values of 21 ± 2ºC
and 55 ± 15% respectively.
A pelleted diet was used
Main drinking water supplied from polycarbonated bottles attached to the cage
The diet and drinking water did not contain any contaminant at a level that might have affected the purpose or integrity of the study
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
The animals were randomly allocated to treatment groups using a randomisation procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of Pentadecane, 7-methylene mixed with 1-tetradecene, dimers and trimers, hydrogenated in the test item formulations was determined by gas chromatography (GC) using an external standard technique.

The standard and sample solutions were analysed by GC using the following conditions:
GC system: Agilent Technologies 5890, incorporating autosampler and workstation
Column: DB-5 (30 m x 0.25 mm id x 0.25 µm film)
Oven temperature program: initial 200 ºC for 1 mins (rate 15ºC/min; final 325 ºC for 10 mins)
Injection temperature: 300 ºC
Flame ionisation detector temperature: 300 ºC
Injection volume: 1 µl
Retention time: Profile of peaks from ~ 7 to 8.5 mins
Duration of treatment / exposure:
Treatment was administered for 14 days, from Day 5 to Day 19.
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
The foetuses were killed by subcutaneous injection of a suitable barbiturate agent. Foetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate foetuses were identified using an indelible marker and placed in Bouin’s fixative. Foetuses were transferred to 90% industrial methylated spirits (IMS) in distilled water and examined for visceral anomalies under a low power binocular microscope. The remaining foetuses were identified using colour coded wires and placed in 70% IMS in distilled water. The foetuses were eviscerated, processed and the skeletons stained with alizarin red. The foetuses were examined for skeletal development and anomalies. Following examination foetuses that were examined for skeletal development were placed in 100% glycerol.
Ovaries and uterine content:
The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Foetal sex
iv) External foetal appearance
v) Foetal weight
vi) Placental weight
vii) Gravid uterus weight
Fetal examinations:
Implantation types were divided into:

Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/foetal and placental tissue visible
Dead Foetus: A foetus that had died shortly before necropsy. These were included as late deaths for reporting purposes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant clinical observations detected in any treated females.
One female treated with 1000 mg/kg bw/day had generalised fur loss between Days 11 and 20. In isolation this observation is considered not to be of toxicological significance.
Dermal irritation (if dermal study):
not examined
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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
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:
effects observed, non-treatment-related
Description (incidence and severity):
One female treated with 1000 mg/kg bw/day had generalised fur loss at necropsy. This was an isolated incident and is considered not to be of toxicological significance. One female treated with 300 mg/kg bw/day had a mass in the left mammary gland. As similar observations were not apparent in animals treated with 1000 mg/kg bw/day, this was considered to be an isolated finding and is considered not to be of toxicological significance.

Animals treated with 300 mg/kg bw/day showed a statistically significant (p<0.01) increase in total corpora lutea when compared to control animals. In the absence of a true dose related response or any associated effects in the uterine parameters examined the intergroup difference is considered not to be of toxicological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
not examined
Pre- and post-implantation loss:
not examined
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
not examined
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No effects observed.
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
not examined
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
not examined
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Key to tables

M – Male

F – Female

sd – standard deviation

n – number of animals/litters

NF – number of foetuses

NL – number of litters

† - group mean per litter

%† - group mean percent

N/A – not applicable

● - no data available

Table 1 summary of female performance

Category

Number of female at dose level (mg/kg bw/day)

0

100

300

1000

Initial Group Size

24

24

24

24

Pregnant

24

24

24

24


Table 2 clinical observations – group incidences

 

Dose level

Number of animals

Clinical observations

Number showing effect(days post coitum affected)

0 (control)

24

No abnormalities detected

-

100

24

No abnormalities detected

-

300

24

No abnormalities detected

-

1000

24

No abnormalities detected

1 (days 11-20)

 

Table 3 body weight during gestation – group mean values

Dose level (mg/kg bw/day)

Number of animals

Body weight (g) at daypost coitum

 

3

5

6

7

8

11

14

17

20

0 (control)

24

Mean

241

250

251

256

261

281

300

330

370

sd

26

26

25

26

26

28

28

32

37

100

24

Mean

243

252

255

259

264

285

304

332

371

sd

20

19

19

19

20

22

24

27

31

300

24

Mean

241

250

252

256

261

280

300

329

369

sd

18

19

18

18

18

20

21

25

30

1000

24

Mean

240

250

251

256

260

279

297

324

363

sd

20

20

19

19

19

19

21

24

28

 

Table 4 body weight change during gestation – group mean values

Dose level (mg/kg bw/day)

Number of animals

Body weight change (g) during days post coitum

 

3 to 5

5 to 6

6 to 7

7 to 8

8 to 11

11 to 14

14 to 17

17 to 20

0 (control)

24

Mean

10

1

5

5

20

20

30

40

sd

6

4

4

3

5

5

7

8

100

24

Mean

9

3

4

5

20

19

29

39

sd

5

5

3

3

4

4

6

8

300

24

Mean

9

3

3

5

19

19

30

40

sd

4

5

3

3

4

5

6

8

1000

24

Mean

9

2

5

4

19

18

27

39

sd

3

2

3

3

6

5

5

7

Dose level (mg/kg bw/day)

Number of animals

Body weight change (g) from day 5post coitum

 

6

7

8

11

14

17

20

0 (control)

24

Mean

1

6

11

31

50

80

120

sd

4

5

6

8

10

15

20

100

24

Mean

3

7

12

33

52

80

119

sd

5

6

7

9

11

15

19

300

24

Mean

3

6

11

30

50

79

119

sd

5

5

6

7

10

15

21

1000

24

Mean

2

7

11

30

47

74

113

sd

2

4

5

7

10

13

17

Table 5 - Gravid Uterus weight and adjusted body weight and body weight change during gestation – group mean values

Dose level (mg/kg bw/day)

Number of animals

 

Body weight (g) on dayspost coitum

Body weight change (g) during dayspost coitum

Gravid Ulterus weight (g)

Adjusted body weight (g) day 20

Adjusted body weight (g) change 5-20

5

20

5-20

0 (control)

24

Mean

250

370

120

73.99

296

46

sd

26

37

20

12.94

31

14

100

24

Mean

252

371

119

72.93

298

46

sd

19

31

19

11.68

24

13

300

24

Mean

250

369

119

78.00

291

41

sd

19

30

21

13.71

23

15

1000

24

Mean

250

363

113

75.08

287

38

sd

20

28

17

9.66

22

12

Table 6 – Food consumption during gestation – group mean values

Dose level (mg/kg bw/day)

Number of animals

Food consumption (g/rat/day) between dayspost coitum

 

3 to 5

5 to 8

8 to 11

11 to 14

14 to 17

17 to 20

0 (control)

24    

Mean

23

21

22

23

24

25

sd

3

3

3

3

3

3

100

24

Mean

23

21

22

23

24

24

sd

2

3

2

3

3

3

300

24

Mean

23

21

22

23

24

24

sd

2

2

2

2

2

3

1000

24

Mean

23

21

22

23

24

23

sd

2

2

2

3

3

3

 

● = n-23 Days 5 to 8

∆ = n=23 Days 14 to 17

□ = n=23 Days 3 to 5

Table 7 – Necropsy Findings – group incidences

 

 

Dose Level (mg/kg bw/day)

0 (control)

100

300

1000

Number of animals examined

24

24

24

24

Generalised fur loss

0

0

0

1

Mass om left mammary gland (20mm x 20mm)

0

0

1

0

No abnormalities detected

0

24

23

23

 


Table 8 – Litter data – Group mean values

Dose level (mg/kg bw/day)

Number of litter

 

Number of Corpora Lutea

Number Implants

Number of Embryonic Deaths

Implantation Loss %

Number of Live Implants

% Male Foetuses

Mean Male Foetal Weight (g)

Mean Female Foetal Weight (g)

Mean Foetal Weight (g)

Mean Placental Weight (g)

Litter Weight (g)

Total Placental Weight (g)

Early

Late

Total

Pre

Post

Male

Female

Total

0 (control)

24

Mean

13.7

12.3

0.2

0.3

0.5

10.2

3.9

6.5

5.3

11.8

54.7

4.22

4.04

4.13

0.52

48.22

6.15

sd

2.2

2.3

0.6

0.7

0.8

9.9

7.2

2.2

2.0

2.5

14.0

0.26

0.30

0.27

0.03

8.30

1.22

100

24

Mean

14.4

12.7

0.3

0.4

0.7

11.4

5.8

6.0

6.0

12.0

49.3

4.07

3.88

3.97

0.51

47.47

6.11

sd

2.3

1.8

0.6

0.7

0.9

7.9

7.4

2.1

1.7

2.1

13.4

0.29

0.29

0.28

0.05

7.79

1.06

300

24

Mean

15.5**

13.0

0.2

0.2

0.3

16.4

2.9

6.5

6.1

12.6

52.8

4.22

3.98

4.11

0.51

51.75

6.48

sd

1.4

1.9

0.4

0.6

0.9

11.9

8.1

2.0

2.5

2.3

17.0

0.19

0.17

0.18

0.05

9.49

1.35

1000

24

Mean

15.0

12.5

0.3

0.2

0.4

16.3

3.2

5.9

6.2

12.0

48.7

4.14

3.97

4.06

0.52

48.87

6.31

sd

1.8

1.5

0.7

0.4

0.7

10.4

5.5

1.9

1.8

1.5

13.9

0.30

0.20

0.21

0.05

6.01

0.92

Table 9 – Foetal External Findings – Group Incidences

 

 

Dose level (mg/kg bw/day)

0 (control)

100

300

1000

Number of foetuses (litters) examined

284 (24)

288 (24)

303 (24)

289 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Total Number Affected

1

1

0.4

4

3

1.3

1

1

0.2

6

6

2.4

Small

1

1

0.4

3

2

1.0

0

0

0.0

6

6

2.0

Subcutaneous Haemorrhage to back of head

0

0

0.0

0

0

0.0

1

1

0.2

0

0

0.0

Damaged tail

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.3

Atretic tail

0

0

0.0

1

1

0.3□

0

0

0.0

0

0

0.0

 

□= observations not recorded for one litter in error

Table 10 – Foetal Visceral Findings – Group Incidences

 

 

Dose level (mg/kg bw/day)

0 (control)

100

300

1000

Number of foetuses (litters) examined

149 (24)

150 (24)

157 (24)

149 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

HEAD

A – eye lens – ovoid

1

1

0.6

0

0

0.0

0

0

0.0

2

2

1.5

B – subcutaneous haemorrhage on head (including/excluding snout)

1

1

0.7

1

1

0.7

0

0

0.0

1

1

0.8

C – small cleft at front of palate

1

1

0.7

0

0

0.0

0

0

0.0

0

0

0.0

D – dilated brain ventricle (s)

1

1

0.7

2

2

1.4

0

0

0.0

4

4

2.7

E – bilateral brain ventricle (s)

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.5

F – small pituitary

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.5

NECK/THORAX

G – pericardial haemorrhage

1

1

0.5

0

0

0.0

0

0

0.0

1

1

0.7

H – undescended lobe (s) of thymus

9

8

6.0

9

3

5.7

3

3

1.8

4

4

2.9

I – subcutaneous oedema – neck region

1

1

0.5

0

0

0.0

0

0

0.0

0

0

0.0

J – small lobe of thyroid

1

1

0.7

0

0

0.0

0

0

0.0

0

0

0.0

K – persistent truncus arteriosus

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.5

L – enlarged right atrium

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.5

M – interventricular septal defect

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.5

N – no brachiocephalic trunk

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.5

ABDOMEN

O – small/no development of renal papilla(e)

20

10

12.6

26

15

18.0

29

11

18.0

21

11

14.6

P – kinked and/or dilated ureter (s)

13

7

8.2

18

12

12.3

26

9

16.3

16

9

11.2

Q – blood in abdomen

1

1

0.6

2

2

1.5

2

2

1.2

1

1

0.6

R – extralobulation of one liver lobe

2

2

1.3

3

3

2.3

1

1

0.6

0

0

0.0

S – increased renal pelvic cavitation

0

0

0.0

2

2

1.3

5

3

3.2

5

4

3.5

GENERAL

T – small foetus

0

0

0.0

0

0

0.0

0

0

0.0

2

2

1.5

U – vestigial tall

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.7

Total

35

18

23.4

39

19

27.0

34

13

21.0

28

14

19.3

 

NOTE: a foetus may appear in more than one category

 

Table 11 – Foetal Skeletal Development – Group Incidence

 

 

Dose level (mg/kg bw/day)

0 (control)

100

300

1000

Number of foetuses (litters) examined

135 (24)

138 (24)

146 (24)

140 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

Number of ribs

13/*

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

13/13

135

24

100.0

138

24

100.0

145

23

99.4

140

24

100.0

Number of ossified sternebrae

<4

1

1

0.8

1

1

0.7

0

0

0.0

3

3

1.8

4

31

11

20.7

26

9

17.9

21

10

13.0

9

7

6.6

>4

103

24

78.5

111

24

81.4

125

24

87.0

128

24

91.6

Number of post lumber vertebral centra

<7

1

1

0.5

4

4

2.8

3

2

2.0

4

3

3.0

≥7

134

24

99.5

134

24

97.2

143

24

98.0

136

24

97.0

Number of post lumber vertebral arches

<5

8

4

5.1

11

7

8.0

0

0

0.0

4

4

2.8

≥5

127

24

94.9

127

23

92.0

146

24

100.0

136

24

97.2

Number of metacarpals

<6

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

6

18

8

10.4

24

10

17.6124

11

5

7.2

13

8

9.4

≥6

117

24

89.6

114

23

82.4

135

24

92.8

126

24

90.0

Number if forelimb phalanges

≤2

120

24

88.0

129

24

93.6

130

24

89.0

124

24

88.2

>2

15

9

12.0

9

6

6.4

16

9

11.0

16

9

11.8

Number of hindlimb phalanges

≤2

135

24

100.0

138

24

100.0

145

24

99.3

139

24

99.3

≥2

0

0

0.0

0

0

0.0

1

1

0.7

1

1

0.7

Number of metatarsals

<6

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

6

1

1

0.5

1

1

0.7

0

0

0.0

1

1

0.7

>6

134

24

99.5

136

24

98.6

146

24

100.0

138

24

98.7

Fontanelle size

Small

10

4

6.2

15

4

9.0

13

2

7.7

11

4

6.6

Medium

123

24

92.8

116

22

85.4

127

23

88.7

124

24

89.7

Large

2

2

1.1

7

3

5.6

6

4

3.6

5

4

3.7

NOTE: a foetus may appear in more than one category

*= Rib damaged at evisceration

 

Table 12 – Foetal Skeletal Findings – Group Incidence

 

 

Dose level (mg/kg bw/day)

0 (control)

100

300

1000

Number of foetuses (litters) examined

135 (24)

138 (24)

146 (24)

140 (24)

NF

NL

%†

NF

NL

%†

NF

NL

%†

NF

NL

%†

HEAD/NECK

A – incomplete ossification of one cranial bone (variant)

51

22

38.1

46

21

33.0

65

22

44.7

60

23

44.0

B – incomplete ossification of more than one cranial bone (variant)

38

15

25.7

47

18

36.8

36

16

25.1

38

18

28.7

C – irregular ossification of one cranial bone (variant)

29

14

23.4

25

15

17.7

31

14

20.6

29

16

21.0

D – incomplete ossification of more than one cranial bone

5

4

3.7

2

2

1.4

1

1

1.0

3

3

2.0

xx – incomplete ossification of one facial bone

3

3

2.7

6

5

4.7

4

4

2.5

2

2

1.7

E – incomplete ossification of more than one facial bone

1

1

0.8

3

1

2.5

1

1

0.6

1

1

0.7

F – irregular ossification of one facial bone

1

1

0.5

2

2

1.3

0

0

0.0

0

0

0.0

AA – irregular ossification of more than one facial bone

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

G – no ossification of hyoid

8

5

4.7

12

6

9.7

11

6

6.8

9

6

4.7

H – incomplete ossification of hyoid

4

2

2.6

5

2

3.7

3

3

1.7

0

0

0.0

I – extra area of ossification between left parietal and inter parietal

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

J – hyoid - small

0

0

0.0

1

1

0.7

1

1

0.6

2

1

1.4

K – hyoid - bipartite

0

0

0.0

0

0

0.0

1

1

0.6

1

1

0.7

STERNEBRAE (1-4)

DD – no ossification of one sternebra

0

0

0.0

1

1

0.7

0

0

0.0

0

0

0.0

L – no ossification of more than one sternebra

1

1

0.8

0

0

0.0

0

0

0.0

1

1

0.7

M – incomplete ossification of one sternebra

2

2

1.4

5

2

4.0

4

3

2.6

4

4

2.8

yy – incomplete ossification of more than one sternebra

0

0

0.0

0

0

0.0

1

1

0.5

0

0

0.0

N – one sternebra – small

4

2

2.4

3

2

1.8

1

1

0.6

0

0

0.0

O – one sternebra – bipartite

3

2

1.8

0

0

0.0

0

0

0.0

0

0

0.0

P – more than one sternebra – semi bipartite

0

0

0.0

1

1

0.7

0

0

0.0

0

0

0.0

Q – more than one sternebra – hemicentric

1

1

0.5

0

0

0.0

0

0

0.0

0

0

0.0

R – one sternebra – hemicentric

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

S – one sternebra – asymmetric ossification

5

5

4.0

1

1

0.7

5

5

3.6

1

1

0.6

T – more than one sternebra – asymmetric ossification

1

1

0.7

0

0

0.0

2

2

1.2

1

1

0.7

U – more than one sternebra – small

1

1

0.5

1

1

0.7

0

0

0.0

0

0

0.0

V – one sternebra – semi bipartite

1

1

0.5

3

2

1.9

0

0

0.0

0

0

0.0

RIBS

W – bilateral/unilateral wavy rib(s)

0

0

0.0

1

1

0.5

0

0

0.0

1

1

0.8

X – bilateral/unilateral rudimentary 13thrib(s)

1

1

1.0

0

0

0.0

2

2

1.1

0

0

0.0

Y – bilateral/unilateral 13thrib(s) short

9

6

7.6

8

7

6.1

7

4

4.1

7

4

5.1

Z – extra area of ossification in cartilage of rib 3 right side

1

1

0.6

0

0

0.0

0

0

0.0

0

0

0.0

aa – more than one rib - thickened

0

0

0.0

1

1

0.6

0

0

0.0

1

1

0.8

VERTERBRAE

bb – one thoracic vertebral centre semi-bipartite (variant)

25

16

18.3

24

14

17.9

24

14

15.9

36

20

26.2

cc – more than one thoracic vertebral centrum semi-bipartite (variant)

18

7

13.0

18

11

15.1

17

11

12.5

20

12

14.1

dd – one thoracic vertebral bipartite

4

3

2.5

4

4

2.9

7

6

4.7

6

6

4.2

ee – more than one thoracic vertebral centrum bipartite

1

1

0.7

2

2

1.5

0

0

0.0

0

0

0.0

ff – one lumbar centre not ossified

1

1

0.8

1

1

0.8

0

0

0.0

1

1

0.7

gg – thoracic vertebral centre not ossified

0

0

0.0

1

1

0.6

0

0

0.0

1

1

0.7

hh – lumbar centre not ossified

0

0

0.0

1

1

0.6

0

0

0.0

0

0

0.0

ii – one thoracic vertebral centre – small

1

1

0.5

0

0

0.0

0

0

0.0

0

0

0.0

jj – one thoracic vertebral centre – asymmetric

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

ll – precocious ossification of more than one cervical centrum

4

1

3.3

1

1

0.6

5

4

3.7

3

1

2.5

mm – one lumbar vertebral centre – small

0

0

0.0

1

1

0.7

0

0

0.0

0

0

0.0

nn - one lumbar vertebral centre – bipartite

0

0

0.0

1

1

0.7

0

0

0.0

0

0

0.0

oo – more than one lumbar vertebral centre – semi bipartite

0

0

0.0

1

1

0.7

0

0

0.0

0

0

0.0

zz – more than one one lumbar vertebral centre – small

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.7

Other

qq – incomplete ossification of schia

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

rr – pubis (es) – small

1

1

0.5

0

0

0.0

0

0

0.0

1

1

0.6

ss - incomplete ossification of pubis (es)

1

1

0.8

5

2

3.7

0

0

0.0

2

2

1.3

tt - incomplete ossification of ischrum/ischia

1

1

0.8

2

1

1.7

0

0

0.0

0

0

0.0

uu – precocious ossification in tail region distal to sacrum

0

0

0.0

0

0

0.0

1

1

0.6

0

0

0.0

vv – no ossification – pubes

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

EE – incomplete ossification of ilia

0

0

0.0

0

0

0.0

0

0

0.0

1

1

0.6

Total

117

24

87.2

120

24

88.2

129

24

88.5

121

24

87.7

 

NOTE: a foetus may appear in more than one category

Conclusions:
The oral administration of Pentadecane, 7-methylene mixed with 1-tetradecene, dimers and trimers, hydrogenated to pregnant rats by oral gavage during organogenesis at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any toxicologically significant effects at any dose level. No significant changes were detected in the offspring parameters measured the ‘No Observed Effect Level’ (NOEL) was therefore, considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was therefore considered to be 1000 mg/kg bw/day.
Executive summary:

The study was designated to investigate the effects of the test item on embryonic and foetal development following repeated administration by gavage to the pregnant female during the period of organogenesis.

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil BP) to serve as a control.

Clinical signs, body weight change and food consumptions were monitored during the study.

 

All surviving females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, foetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were preserved in Industrial Methylated Spirit (IMS) and then transferred into 100% glycerol and examined for skeletal development. The remaining half were preserved in Bouin’s solution and examined viscerally.

 

The results are as follows,

 

Mortality. There were no unscheduled deaths.

 

Clinical Observations. There were no toxicologically significant clinical observations detected in any treated females.

 

Body Weight. No treatment-related effects in body weight development were detected.

 

Food Consumption. No adverse effects were detected in food consumption.

 

Post Mortem Studies. No toxicologically significant macroscopic abnormalities were detected in treated females at terminal kill. No treatment-related effects were detected in the uterine parameters examined, in foetal viability or in growth and development.

 

Foetal Evaluation. No treatment-related effects were detected on skeletal development or in the type and incidence of skeletal or visceral findings.

 

The oral administration of Pentadecane, 7-methylene mixed with 1-tetradecene, dimers and trimers, hydrogenated to pregnant rats by oral gavage during organogenesis at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any toxicologically significant effects at any dose level. No significant changes were detected in the offspring parameters measured the ‘No Observed Effect Level’ (NOEL) was therefore, considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was therefore considered to be 1000 mg/kg bw/day.

Endpoint:
developmental toxicity
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
Further justification for waiving the second-species requirement for PAOs is included in the attached weight of evidence document.
Species:
rabbit
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Two key Guideline 414 (one substance specific and one read across) studies in rodents available for assessment.
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

A key OECD Guideline 414 pre-natal developmental toxicity study was conducted to evaluate the potential effects of the test material (1-Dodecene, dimers (CAS No. 62132-67-6)) on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the rat(Labcorp Early Development Laboratories Ltd., 2022).

 

The test material was administered to time-mated female Hans Wistar rats (22/dose) once daily via oral gavage in a corn oil vehicle at doses of 0, 100, 300, or 1000 mg/kg bw/day from Day 6 to 20 after mating. The control group received corn oil only. Clinical observations, body weight, and food consumption were recorded and animals were sacrificed on Day 21 after mating. Adult females were examined macroscopically at necropsy on Day 21 after mating, blood samples were taken for thyroid hormone analysis and the gravid uterus and thyroid weights were recorded. All fetuses were examined macroscopically at necropsy and the ano-genital distance measured. Fetal pathology investigations were undertaken on all fetuses by detailed internal visceral examination or skeletal examination. 

 

No mortality or adverse treatment-related clinical signs of toxicity were observed through the study period. Overall body weight gain (GD 6-21), adjusted body weight change (GD 6-21), gravid uterine weight, and overall food consumption was unaffected by treatment at 100, 300 or 1000 mg/kg/day. While overall food consumption was statistically significantly high at 300 or 1000 mg/kg/day, the differences were marginal (106% or 112% of Control, respectively) and therefore considered to be incidental rather than treatment-related. There was no effect of treatment observed on serum concentrations of T3, T4, and TSH and no treatment-related changes were observed in the thyroids (with parathyroids). Gross necropsy did not reveal any remarkable treatment-related findings and no microscopic effects were observed in the thyroids.

 

Implantation losses, the numbers of resorptions and live young and the ratio of male to female offspring was unaffected by treatment.

 

Placental, fetal and total litter weights were also unaffected by treatment and no effect of treatment was observed on fetal anogenital distance. No major or minor treatment-related fetal abnormalities were observed. Two female fetuses in one litter (Dam/Litter No. 67) at 1000 mg/kg/day exhibited abnormalities of the head (one fetus had Microphthalmia (left eye) and one fetus had Brachygnathia and other related cranial abnormalities (jaw, palate, ear and nose)). However, given that only a single litter was affected and in the absence of related aetiology to other malformations at 1000 mg/kg/day or lower, these observations were not considered treatment-related. An increase in the incidence of the minor visceral abnormality subdural brain haemorrhage compared to control animals was observed at 1000 mg/kg/day (six fetuses in five litters). This exceeded the Historical Control Data (HCD) range (fetuses; 0-3, litters; 0-3) but was considered to have occurred during the necropsy procedure and therefore, was unrelated to treatment with the test material.

 

Based on the lack of adverse treatment-related effects observed, the No Observed Effect Level (NOEL) of 1-Dodecene, dimers (CAS No. 62132-67-6) for maternal toxicity, embryo-fetal survival, growth and development in rats was determined to be 1000 mg/kg/day.

Additionally, the oral administration of Pentadecane, 7-methylene mixed with 1-tetradecene, dimers and trimers, hydrogenated to pregnant rats by oral gavage during organogenesis at dose levels of 100, 300 and 1000 mg/kg bw/day did not result in any toxicologically significant effects at any dose level. No significant changes were detected in the offspring parameters measured the ‘No Observed Effect Level’ (NOEL) was therefore, considered to be 1000 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was therefore considered to be 1000 mg/kg bw/day (Harlan Laboratories, 2012).

 

A supporting OECD Guideline 422 combined 28-Day repeated dose toxicity study with the Reproduction / Developmental Toxicity Screening Test was conducted to evaluate the potential toxic effects of the test material (1-Dodecene, dimers; CAS# 62132-67-6) when given orally to Wistar Han rats and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development(Charles River Laboratories Den Bosch BV, 2022).

 

The test material Wistar Han rats (10/sex/dose) were administered once daily via oral gavage in a corn oil vehicle at doses of 0, 100, 300, or 1000 mg/kg/day for a minimum of 28 days for males (including at least 2 weeks of treatment prior to mating and during the mating period up to and including the day before scheduled necropsy). Female rats were dosed 7 days a week for at least 14 days prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy.

 

No treatment-related changes were observed in the following developmental toxicity parameters investigated in this study: gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T3, T4 and TSH thyroid hormone levels, and macroscopic examination. However, a reduction (85% compared to 92% in control) in the post-implantation survival index was observed for females at 1000 mg/kg/day. While this was considered to be treatment-related, no effects were observed on litter size and in absence of any corroborative histopathological findings, this reduction was considered non adverse.

 

Based on the lack of adverse treatment-related effects observed through the study period, the systemic, reproductive, and developmental toxicity No Observed Adverse Effect Level (NOAEL) for 1-Dodecene, dimers was determined to be ≥1000 mg/kg/day in the rat.

Justification for classification or non-classification

Based on available data, 1-dodecene, dimer does not meet the criteria for classification as a reproductive or developmental toxicant under CLP EU Regulation 1272/2008 (GHS aligned).

No 2-generation reproductive toxicity data were available for dec-1-ene, dimers, hydrogenated. Two one-generation reproduction toxicity studies from 1-decene, homopolymer, hydrogenated and structural analogues related to dec-1 -ene, dimers, hydrogenated showed no effects on reproductive parameters. Although results from these studies showed no reproductive or developmental effects at the highest dose tested, and while the data in combination present a reasonable weight of evidence upon which to judge the reproductive and developmental toxicity of poly alpha olefins, these study results cannot meet the requirement for results from a complete developmental or two-generation reproductive study. Nonetheless, the results are considered adequate do not raise concern with regard to classification of dec-1-ene, dimers, hydrogenated as toxic for reproduction or a development toxicant under CLP EU Regulation 1272/2008 (GHS aligned).

Additional information