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Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Reference substance name:
1-Dodecene, Dimer
Cas Number:
62132-67-6
IUPAC Name:
1-Dodecene, Dimer
Test material form:
liquid
Details on test material:
Name of substance: 1-Dodecene, dimers
CAS# 62132-67-6
EC# 814-509-8
Batch # DBA0146456
Storage: At room temperature
Expiration date: 2023-MARCH-01
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

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

open allclose all
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

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.