1-Dodecene, dimers

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

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 guideline

Test guideline 1

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.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: 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

Test material information

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.

- 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

Details on route of administration:

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.

Vehicle:

corn oil

Remarks:

Sigma-Aldrich (Steinheim, Germany)

Details on oral 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

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 procedure (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 / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 - Control (corn oil)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2 - Low dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3 - Intermediate dose

Doses / concentrations 4

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

- Dose range finding studies:
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

Observations and examinations performed and frequency:

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.

OPHTHALMOSCOPIC EXAMINATION: No

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.

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)

URINALYSIS: No

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

IMMUNOLOGY: No

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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 4 and 5)

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.

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.

Statistics:

For information on statistics please see 'Any other information on materials and methods incl. tables'.

Results and discussion

Results of examinations

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.

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

Description (incidence and severity):

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.

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:

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

Effect levels

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

Target system / organ toxicity

Key result

Critical 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

Applicant's summary and conclusion

Conclusions:

Based on the lack of adverse treatment-related effects observed through the study period, the systemic 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.

 

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.

 

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 sacrificedin extremison Day 1 of lactation. The animal was found with a pale appearance and piloerection. Due to the deteriorating condition of this animal, it was sacrificedin 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.

 

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