Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-03-11 to 2020-12-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
deviations had no impact on the integrity of the study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
deviations had no impact on the integrity of the study
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
deviations had no impact on the integrity of the study
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
EC Number:
242-582-0
EC Name:
(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Cas Number:
18794-84-8
Molecular formula:
C15H24
IUPAC Name:
(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Test material form:
liquid
Details on test material:
Name of substance: (E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Batch/Lot #: 19RGT1210HP086
CAS# 18794-84-8
Physical Description: Colourless to pale-yellow liquid
Expiry date: 10 December 2022
Storage Conditions: In refrigerator (2-8°C)
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: Amyris, Inc. (5885 Hollis St, Suite 100, Emeryville, CA 94608, USA); Batch # 19RGT1210HP086

- Purity, including information on contaminants, isomers, etc.:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: test item stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data retained in the study records for Test Facility Study No. 20219872.

Test animals

Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
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: Not specified
- Age at study initiation: 6-7 weeks old at initiation of dosing
- Weight at study initiation: Males: 125 - 173 g; Females 102 - 138 g
- Fasting period before study: Not specified
- Housing: Up to 5 animals of the same sex and same dosing group together in Polycarbonate cages (Makrolon type IV, height 18 cm) containing sterilized sawdust as bedding material (Lignocel S 8 15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. During locomotor activity monitoring, 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 (SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water (e.g. ad libitum): Municipal tap water ad libitum
- Acclimation period: 13 days

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 21°C
- Humidity (%): 47 to 75%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light (except during designated procedures)

IN-LIFE DATES: From: 2020-06-17 To: 2020-09-30

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% Aqueous carboxymethyl cellulose with 1.25% Tween 80
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a suspension, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the 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): 0.5% Aqueous carboxymethyl cellulose with 1.25% Tween 80
Trial preparations were performed to select a 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. Raw data of these trials are retained by the Test Facility.
- Concentration in vehicle: 0, 20, 60, or 200 mgm/L for the control, 100, 300, and 1000 mg/Kg bw/day dose groups, respectively.
- Amount of vehicle (if gavage): 5 mL/Kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed using a validated analytical procedure (Test Facility Study No. 20219872). Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20219872) demonstrated that the test material is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20219872.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
once daily via oral gavage
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 - Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on results of a 14 day repeated dose toxicity study with oral exposure of (E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene in rats, Test Facility Study No. 20219871, and in an attempt to produce graded responses to the test material. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

- Rationale for animal assignment (if not random):
Animals were randomly assigned to groups at the discretion of the biotechnician. Males and females were randomized separately. Body weight variation did not exceed ± 20% of the sex mean.

- Fasting period before blood sampling for clinical biochemistry: Yes (overnight with a maximum of 24 hours)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily; from Day 1 onwards. Observed for mortality at least twice daily beginning upon arrival through termination (Except on days of receipt and necropsy where frequency was at least once daily).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly (from Week 1 and throughout the study, and on the day of necropsy)

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal weights measured weekly (from at least Day 1 and throughout the study).

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, from at least Day 1 and throughout the study)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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: Regular basis throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once during the pre-treatment period and during week 13 of the Dosing Period
- Dose groups that were examined: Pretreatment Period - All Study animals once (including spare animals); Dosing Period - All Group 1 and 4 Study animals during Week 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: All animals
- Parameters checked in table [No.3] were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: End of treatment
- Animals fasted: Yes (overnight with a maximum of 24 hours)
- How many animals: All animals

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the Dosing Period. The first 5 animals per sex per group during Week 12
- Dose groups that were examined: The first 5 animals per sex per group during Week 12
- Battery of functions tested: sensory activity / grip strength / motor activity / other: The following tests were performed:
- hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent).

- fore- and hind-limb grip strength were recorded as the mean of three measurements according to SOP DIE-H-125.

- locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.

IMMUNOLOGY: No

OTHER:

- Estrous Stage Determination: on the day of necropsy, a vaginal smear was taken to determine the stage of estrus from all females. Estrous stage was evaluated by examining the vaginal cytology of samples obtained by vaginal smear procedures.
Sacrifice and pathology:
A necropsy was conducted for animals that died on study, and specified tissues were saved. Animals surviving until scheduled euthanasia were weighed and euthanized using isoflurane, followed by exsanguination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy. Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

GROSS PATHOLOGY: Yes (see table 4)

Organs identified in table 4 were weighed at necropsy for all scheduled euthanasia animals. 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 ratio (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes (see table 5)

Representative samples of the tissues identified in table 5 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated. Tissues identified in table 5 (except animal identification, nasal body cavity, bone marrow smears, clitoral gland, harderian gland, lacrimal gland, preputial gland, sublingual and parotid salivary glands, larynx, tibial nerve, and tongue) were embedded in paraffin (Klinipath, Duiven, The Netherlands), sectioned, mounted on glass slides, and stained with hematoxylin and eosin (Klinipath, Duiven, The Netherlands).
Statistics:
For information on statistics, please see the section 'Any other information on materials and methods incl. tables' below.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant clinical signs were observed during the daily detailed clinical observations and no findings were noted during the arena observations.

Salivation was seen after dosing in animals of all dose groups with the highest incidence at 300 and 1000 mg/Kg/day. This was not considered to be toxicologically relevant, taking into account the transient nature and minor severity of the effect and its time of occurrence (i.e. immediately after dosing). This sign was considered to be a physiological response related to taste of the test material rather than a sign of systemic toxicity.

Hunched posture and piloerection were observed on Day 55 in 1/10 males and 2/10 females in the 1000 mg/Kg/day dose group. Erected fur was also observed incidentally in one male in the 100 mg/Kg/day dose group. Based on the incidence of these clinical signs, this was not considered to be toxicologically relevant.

Other clinical signs (abnormal breathing sounds, fur loss or thin covered fur, scabs, protruding eyeballs and discharge from the eye) observed during the dosing period 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 and did not show any apparent dose related trend. At the incidence observed, these were considered to be unrelated to treatment with the test material.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control female rat (Animal No. 50) died during blood sampling. This was considered to be a technical error. Deaths at blood sampling are considered to be of incidental nature. No treatment-related mortality was observed during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A slightly reduced body weight gain was noted for males in the 300 and 1000 mg/Kg/day dose groups from the third week of treatment onwards, which resulted in absolute body weights of 9.5% and -7.7% at end of treatment, respectively, when compared to control. Based on the magnitude of the change and absence of a clear dose-response relationship, this was not considered to be adverse. No effect on body weight (gain) was noted for females. Although absolute body weight appeared lower for females at 300 mg/kg/day, body weight gain was comparable to control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption in treated animals was similar to the control group through the study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related ophthalmology findings were observed through the study period.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted in hematological or coagulation parameters through the study period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A lower concentration of triglycerides was noted for males at 300 and 1000 mg/Kg/day (0.62x and 0.44x of control, respectively). Based on the direction of the change and in absence of any correlated clinical pathology or microscopic findings, this was considered to be non-adverse.

A decreased concentration of total bilirubin (0.83x, 0.75x and 0.76x of control) was noted for females at 100, 300 and 1000 mg/Kg/day, respectively. In addition, increased potassium (1.08x of control) and phospholipid (1.17x of control) concentrations were noted for females at 1000 mg/Kg/day. Based on the magnitude of the change and/or as bilirubin values remained within the historical control range , these were not considered to be toxicologically relevant.
Endocrine findings:
no effects observed
Description (incidence and severity):
T3, T4, and TSH levels in male and female rats remained unaffected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Reduced hind grip strength (0.68x and 0.69x of control, respectively) was noted for males at 300 and 1000 mg/Kg/day. In addition, a reduced number of total movements and ambulations (0.65x and 0.78x of control, respectively) were observed for males at 1000 mg/Kg/day. Since these results were not supported by clinical observations and had no supportive morphological correlated in the examined neuronal tissues, these findings were not considered to represent adverse neurobehavioural effects.

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals, and no effect on grip strength or motor activity was noted for females. 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):
The mean absolute liver weights were higher in males and females at 1000 mg/Kg/day. The mean relative liver weights were increased in males at 100, 300 and 1000 mg/Kg/day and in females at 300 and 1000 mg/Kg/day. The mean relative thyroid and kidney weights were increased in males and females at 300 and 1000 mg/Kg/day. The absolute testis weights were increased in males at 1000 mg/Kg/day. The relative testis and epididymis weights were increased in males at 300 and 1000 mg/Kg/day.

The terminal body weights of the males were lower in males at 300 and 1000 mg/Kg/day compared to controls. The decrease was more pronounced in males at 300 mg/Kg/day (0.85x of control) than in males at 1000 mg/Kg/day (0.90x control). This lack of a dose-effect relationship makes it difficult to interpret the organ weights in males.

Absolute liver and testis weights in the males and relative epididymis weight in the males and thyroid weight in the females were all within the range of historical controls for this strain and age.

Statistically significant higher testis weight (absolute and relative to body weight) in males at 300 and 1000 mg/Kg/day was mainly due to a low mean weight in the control group, as Animal No 4 showed considerable testicular atrophy. Although relative testis weight at 300 and 1000 mg/Kg/day exceeded the HCD range (Mean: 0.91; P5-P95: 0.757-1.093 (n= 128)), the lack of a dose-effect relationship for male body weight makes interpretation of relative testis weight difficult. In addition, in the absence of histopathological correlates, the relevance of increased absolute and relative testis weight is unclear.

Probable test item-related organ weight changes were the increased absolute and relative liver weights in males (all dose levels) and females at 300 and 1000 mg/kg/day and the increased relative kidneys weights in females at 300 and 1000 mg/kg/day. In both organs, there were no microscopic correlates, and therefore, these weight changes were considered adaptive and non-adverse. Additionally, the relative kidney weights in the females were within historical control range.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross necropsy did not reveal any remarkable findings. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test material-related microscopic findings after treatment were noted in the stomach of the 100, 300 and 1000 mg/Kg/day group males and females. Diffuse epithelial hyperplasia and hyperkeratosis in the non-glandular stomach were seen at minimal to mild degree in 6/10 males and in 5/10 females at 100 mg/Kg/day, 4/9 males and 6/10 females (one of which showed hyperkeratosis only) at 300 mg/Kg/day and 10/10 males and 10/10 females at 1000 mg/Kg/day. These are regarded to be local treatment-related alterations, resulting from the irritating properties of the test material and at the recorded severities, considered to be non-adverse.

In the adrenal glands minimal mononuclear infiltrates were observed in 5/10 females at 100 mg/Kg/day, 4/10 females at 300 mg/Kg/day and in 4/10 females at 1000 mg/Kg/day, but also in 1/10 control females. Because of a lack of a dose-effect relationship this finding, was not considered treatment-related.
Histopathological findings: neoplastic:
not examined
Details on results:
Dose Formulation Analyses

Accuracy
The concentrations analyzed in the formulations of Groups 2 (100 mg/Kg bw/day), 3 (300 mg/Kg bw/day) and 4 (1000 mg/Kg bw/day)were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).

For the formulation of Group 3 prepared for use in Week 13, the mean accuracy was above the target concentration (i.e.118% of target). As this occurred for one group on a single occasion, and since the deviation from the acceptance criteria was minimal and animals received at least 300 mg/kg/day, this deviation was considered not to have had a significant impact on the toxicological evaluation. No test item was detected in the Group 1 formulations.

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

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

Table 6. Summary of Body Weight Gains (g)

 

 

Day(s) Relative to Start Date

1-8

8-15

15-22

22-29

29-36

36-43

43-50

50-57

57-64

64-71

71-78

78-85

85-91

Males

0 mg/Kg bw/day

Group 1

Mean

39.1

40.0

34.5

23.4

22.4

15.9

15.3

12.6

13.0

12.2

10.0

6.2

-14.5

SD

4.5

7.3

5.6

5.3

4.0

3.1

4.8

3.9

6.4

2.6

2.7

2.9

14

N

10

10

10

10

10

10

10

10

10

10

10

10

10

 

100 mg/Kg bw/day

Group 2

Mean

39.7

42.1

31.5

25.6

22.2

14.0

13.5

11.4

12.5

10.5

9.1

4.7

-9.0

SD

3.5

5.7

6.7

6.1

3.2

5.2

5.3

4.5

3.8

3.6

5.0

4.6

16.5

N

10

10

10

10

10

10

10

10

10

10

10

10

10

 

300 mg/Kg bw/day

Group 3

Mean

40.3

39.0

30.1

21.4

15.4*

10.3

11.4

9.3

7.3

8.6*

9.3

2.8

-8.7

SD

3.5

3.2

6.6

7.7

7.5

6.0

5.2

4.5

5.6

2.4

4.9

2.5

15.0

N

10

10

10

10

10

10

10

10

10

10

10

10

10

 

1000 mg/Kg bw/day

Group 4

Mean

40.3

42.2

31.6

23.9

16.2*

14.0

9.8

6.5**

12.0

6.9**

11.7

2.0

-15.9

SD

4.9

6.1

5.1

7.1

5.0

4.4

5.2

3.6

6.0

3.5

5.4

4.1

16.5

N

10

10

10

10

10

10

10

10

10

10

10

10

10

Females

 

 

1-8 (G)

8-15 (G)

15-22

(G1)

22-29

(G1)

29-36

(G1)

36-43

(G1)

43-50

(G1)

50-57

57-64

64-71

71-78

78-85

85-91

0 mg/Kg bw/day

Group 1

Mean

20.1

13.9

16.6

10.7

9.9

5.4

6.0

10.0

-2.3

4.2

7.8

0.2

-7.1

SD

3.5

4.2

7.6

5.7

3.8

5.3

3.9

5.5

4.4

4.5

4.9

4.6

20.3

N

10

10

10

10

10

10

10

10

10

10

10

10

10

 

100 mg/Kg bw/day

Group 2

Mean

12.8*

25.3**

14.9

10.6

8.1

10.3

3.7

4.4

3.3

5.0

6.6

-1.8

-13.5

SD

6.5

8.3

7.2

4.6

6.5

4.6

2.2

3.6

4.0

5.4

4.4

4.1

13.8

N

10

10

10

10

10

10

10

10

10

10

10

10

10

 

300 mg/Kg bw/day

Group 3

Mean

16.5

15.8

11.4

11.7

10.0

6.2

6.8

6.8

1.2

3.1

6.5

2.2

-4.7

SD

2.2

4.4

6.7

4.8

5.8

6.4

4.9

6.6

6.4

4.3

5.2

5.8

12.7

N

10

10

10

10

10

10

10

10

10

10

10

10

10

 

1000 mg/Kg bw/day

Group 4

Mean

21.0

16.1

12.8

11.3

8.4

7.2

6.3

4.8

-0.3

6.1

4.8

2.0

-14.1

SD

3.4

3.4

4.8

6.3

6.4

4.2

4.8

4.1

6.1

7.7

5.8

6.4

7.8

N

10

10

10

10

10

10

10

10

10

10

10

10

10

 [G] - Kruskal-Wallis & Dunn: * = p ≤ 0.05; ** = p ≤ 0.01

[G1] - Anova & Dunnett

Table 7. Summary of Motor Activity Results

 

0 mg/Kg bw/day

(Control – Group 1)

100 mg/Kg bw/day

(Group 2)

300 mg/Kg bw/day

(Group 3)

1000 mg/Kg bw/day

(Group 4)

Males

Total Movements

 

Mean

4563

4485

3730

2987*

N

5

5

5

5

Std Deviation

600

1018

1021

998

Ambulations

 

Mean

954

960

908

743

N

5

5

5

5

Std Deviation

210

245

344

352

Females

Total Movements

 

Mean

5091

5991

5381

4995

N

5

5

5

5

Std Deviation

1079

961

1411

2305

Ambulations

 

Mean

1381

1677

1573

1626

N

5

5

5

5

Std Deviation

419

514

315

841

* indicates a p-value <0.05, ** indicates a p-value <0.01

MEAN and STDEV values are calculated per group, from each animal's total Total Movements and Ambulations over all intervals

 

Table 8. Summary of Functional Tests

 

 

0 mg/Kg bw/day

(Control – Group 1)

100 mg/Kg bw/day

(Group 2)

300 mg/Kg bw/day

(Group 3)

1000 mg/Kg bw/day

(Group 4)

Males

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 Force

Gram

Median

854

865

667

722

Std Deviation

180

271

191

167

N

5

5

5

5

 

Grip Hind

Gram

Median

637

622

436**

437**

Std Deviation

69

61

89

79

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 Force

Gram

Median

861

745

823

758

Std Deviation

195

60

218

127

N

5

5

5

5

 

Grip Hind

Gram

Median

434

339

393

343

Std Deviation

91

48

56

50

N

5

5

5

5

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

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

 

Table 9. Summary of Select Clinical Chemistry Results

 

 

Reporting Biochemistry

Reporting Special Chemistry

TRIG

(mmol/L)

[G]

TPROT

(g/L)

[G]

ALB

(g/L)

[G]

TBIL

(µmol/L)

[G]

UREA

(mmol/L)

[G]

LDL

(mmol/L)

[G]

K

(mmol/L)

[G]

PHOS

(mmol/L)

[G]

T3

(ng/mL)

[G]

T4

(ng/mL)

[G]

TSH

(mU/L)

[G]

Males - Day 92 Relative to Start Date

0 mg/Kg bw/day

Group 1

Mean

0.950

65.25

38.78

1.91

6.16

0.310

4.03

1.693

0.247

39.54

0.1392

SD

0.248

1.77

1.05

0.31

0.41

0.044

0.16

0.151

0.049

5.58

0.0707

N

10

10

10

10

10

10

10

10

10

10

10

 

100 mg/Kg bw/day

Group 2

Mean

0.934

63.75

38.79

1.78

6.41

0.323

4.01

1.717

0.261

36.69

0.1319

SD

0.282

3.14

1.48

0.31

0.77

0.048

0.25

0.127

0.054

7.74

0.1280

N

10

10

10

10

10

10

10

10

10

10

10

tCtrl

0.98

0.98

1.00

0.93

1.04

1.04

0.99

1.01

1.06

0.93

0.95

 

300 mg/Kg bw/day

Group 3

Mean

0.592**

63.60

39.29

1.55

6.53

0.342

4.01

1.759

0.261

36.02

0.1025

SD

0.260

2.20

1.11

0.23

1.20

0.066

0.23

0.119

0.035

6.61

0.0921

N

10

10

10

10

10

10

10

10

10

10

10

tCtrl

0.62

0.97

1.01

0.81

1.06

1.10

0.99

1.04

1.06

0.91

0.74

 

1000 mg/Kg bw/day

Group 4

Mean

0.415**

64.89

40.09

1.74

6.14

0.295

3.94

1.781

0.261

35.85

0.2165

SD

0.108

2.13

1.16

0.26

1.33

0.034

0.18

0.186

0.053

7.52

0.1516

N

10

10

10

10

10

10

10

10

10

10

10

tCtrl

0.44

0.99

1.03

0.91

1.00

0.95

0.98

1.05

1.06

0.91

1.56

Females - Day 93 Relative to Start Date

0 mg/Kg bw/day

Group 1

Mean

0.554

66.29

40.40

2.54

6.25

0.244

3.47

1.466

0.346

21.46

0.0667

SD

0.169

3.80

2.57

0.38

0.44

0.090

0.19

0.182

0.071

5.00

0.0691

N

9

9

9

9

9

9

9

9

9

9

9

 

100 mg/Kg bw/day

Group 2

Mean

0.553

69.69*

42.86*

2.11*

6.36

0.323*

3.56

1.392

0.403

26.88

0.1081

SD

0.206

2.76

1.90

0.30

0.74

0.067

0.24

0.202

0.094

6.46

0.0916

N

10

10

10

10

10

10

10

10

10

10

10

tCtrl

1.00

1.05

1.06

0.83

1.02

1.32

1.02

0.95

1.17

1.25

1.62

 

300 mg/Kg bw/day

Group 3

Mean

0.549

66.38

40.98

1.91**

6.53

0.338*

3.59

1.430

0.361

25.82

0.1666

SD

0.183

2.21

1.36

0.32

0.68

0.065

0.25

0.177

0.083

9.07

0.1583

N

10

10

10

10

10

10

10

10

10

10

10

tCtrl

0.99

1.00

1.01

0.75

1.04

1.38

1.04

0.98

1.04

1.20

2.50

 

1000 mg/Kg bw/day

Group 4

Mean

0.649

66.91

41.71

1.93**

8.04**

0.321

3.76*

1.720*

0.310

23.92

0.1128

SD

0.159

2.85

1.73

0.37

1.34

0.050

0.13

0.202

0.050

3.06

0.1180

N

10

10

10

10

10

10

10

10

10

10

10

tCtrl

1.17

1.01

1.03

0.76

1.29

1.31

1.08

1.17

0.90

1.11

1.69

[G] - Anova & Dunnett; * = p ≤ 0.05, ** = p ≤ 0.01

[G1] - Kruskal-Wallis & Dunn

Table 10. Summary of Select Absolute Organ Weights

 

 

0 mg/Kg bw/day

(Control – Group 1)

100 mg/Kg bw/day

(Group 2)

300 mg/Kg bw/day

(Group 3)

1000 mg/Kg bw/day

(Group 4)

Males

Terminal Body Weight (g) [G]

Mean

368.6

356.7

313.3**

333.3

SD

25.9

29.3

35.0

37.3

N

10

10

10

10

% Difference

-

-3.2

-15.0

-9.6

 

Gland, Pituitary Weight (g) [G1]

Mean

0.00943

0.00800*

0.00774**

0.00860

SD

0.00114

0.00036

0.00087

0.00173

N

10

10

10

10

% Difference

-

-15.16437

-17.92153

-80170

 

Liver Weight (g) [G]

Mean

8.4872

9.2085

9.0167

10.5020**

SD

0.6663

0.7145

1.2427

1.5282

N

10

10

10

10

% Difference

-

8.4987

6.2388

23.7393

 

Testis Weight (g) [G]

Mean

3.3613

3.4501

3.5236

3.8070*

SD

0.4889

0.2154

0.4184

0.2092

N

10

10

10

10

% Difference

-

2.6418

4.8285

13.2598

 

Thymus Weight (g) [G]

Mean

0.2998

0.2732

0.2321*

0.2177**

SD

0.0596

0.0358

0.0623

0.0537

N

10

10

10

10

% Difference

-

-8.8726

-22.5817

-27.3849

Females

Terminal Body Weight (g) [G]

Mean

216.6

219.5

200.5

207.7

SD

14.1

21.2

9.0

20.5

N

9

10

10

10

% Difference

-

1.4

-7.4

-4.1

 

Heart Weight (g) [G2]

Mean

0.7115

0.7192

0.6241*

0.6733

SD

0.0420

0.0648

0.0398

0.0850

N

10

10

10

10

% Difference

-

1.0822

-12.2839

-5.3689

 

Liver Weight (g) [G1]

Mean

5.3857

5.7459

5.6869

7.3440**

SD

0.5985

0.7184

0.5116

0.5594

N

10

10

10

10

% Difference

-

6.6887

5.5932

36.3619

[G] - Anova & Dunnett: ** = p ≤ 0.01

[G1] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

[G2] - Kruskal-Wallis & Dunn: * = p ≤ 0.05

 

Table 11. Summary of Select Organ Weights Relative to Body Weight

 

 

0 mg/Kg bw/day

(Control – Group 1)

100 mg/Kg bw/day

(Group 2)

300 mg/Kg bw/day

(Group 3)

1000 mg/Kg bw/day

(Group 4)

Males

Brain (%) [G]

Mean

0.5333

0.55871

0.62406**

0.58793

SD

0.05558

0.03998

0.07306

0.06633

N

10

10

10

10

% Difference

-

4.75782

17.01068

10.23738

 

Epididymis (%) [G]

Mean

0.29696

0.30480

0.35938**

0.35698*

SD

0.04922

0.02984

0.04744

0.04609

N

10

10

10

10

% Difference

-

2.63900

21.01661

20.20861

 

Gland, Prostate (%) [G]

Mean

0.22501

0.22850

0.29062**

0.21512

SD

0.05547

0.02510

0.05462

0.02685

N

10

10

10

10

% Difference

-

1.55053

29.15612

-4.39577

 

Thyroid/Parathyroid (%) [G]

Mean

0.00464

0.00477

0.00555*

0.00584**

SD

0.00076

0.00054

0.00096

0.00064

N

10

10

10

10

% Difference

-

2.81953

19.47448

25.81330

 

Kidney (%) [G]

Mean

0.64029

0.69440

0.76015**

0.79505**

SD

0.05149

0.05647

0.07148

0.05425

N

10

10

10

10

% Difference

-

8.44964

18.71942

24.16947

 

Liver (%) [G]

Mean

2.30615

2.58932**

2.87221**

3.14264**

SD

0.14689

0.20374

0.11410

0.14085

N

10

10

10

10

% Difference

-

12.27855

24.54547

36.27187

 

Testis (%) [G]

Mean

0.91554

0.97134

1.13255**

1.15158**

SD

0.14081

0.08138

0.15411

0.10665

N

10

10

10

10

% Difference

-

6.09537

23.70293

25.78223

Females

Thyroid/Parathyroid (%) [G1]

Mean

0.00572

0.00578

0.00690*

0.00707*

SD

0.00132

0.00062

0.00065

0.00117

N

9

10

10

10

% Difference

-

1.05563

20.55996

23.61621

 

Kidney (%) [G1]

Mean

0.66804

0.66998

0.75160**

0.77721**

SD

0.04122

0.03990

0.05838

0.05564

N

9

10

10

10

% Difference

-

0.29061

12.50779

16.34084

 

Liver (%) [G1]

Mean

2.48546

2.61627

2.83659**

3.55197**

SD

0.19940

0.16890

0.22484

0.27310

N

9

10

10

10

% Difference

-

5.26338

14.12767

42.91043

For males: [G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01;[G1] - Kruskal-Wallis & Dunn

For females: [G] - Kruskal-Wallis & Dunn;[G1] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

 

Table 12. Summary of Microscopic Pathology (Select Organs)

 

Males

Females

0 mg/Kg bw/day

(Control – Group 1)

100 mg/Kg bw/day

(Group 2)

300 mg/Kg bw/day

(Group 3)

1000 mg/Kg bw/day

(Group 4)

0 mg/Kg bw/day

(Control – Group 1)

100 mg/Kg bw/day

(Group 2)

300 mg/Kg bw/day

(Group 3)

1000 mg/Kg bw/day

(Group 4)

Number of Animals

10

10

10

10

10

10

10

10

Gland, Adrenal

 

 Examined

10

0

1

10

10

10

10

10

 No Visible Lesions

10

-

1

10

9

5

7

4

 Vacuolation; zona glomerulosa

0

-

0

0

0

0

0

3

    ….Minimal

0

-

0

0

0

0

0

2

    .…Mild

0

-

0

0

0

0

0

1

Infiltration, mononuclear cell; cortical

0

-

0

0

1

5

3

4

   ….Minimal

0

-

0

0

1

5

3

4

Infiltration, mononuclear cell; medullary

0

-

0

0

0

0

1

0

   ….Minimal

0

-

0

0

0

0

1

0

 

Stomach

 

  Examined

10

10

9

10

10

10

10

10

  No Visible Lesions

10

4

5

0

10

4

5

2

  Not Examined: Not Present In Section

0

0

1

0

-

-

-

-

  Hyperplasia; epithelial, non-glandular

0

6

4

10

0

5

5

8

    ….Minimal

0

3

3

0

0

0

2

1

    ….Mild

0

3

1

10

0

5

3

7

Infiltration, mixed cell

0

1

0

0

0

1

0

0

    ….Minimal

0

1

0

0

0

1

0

0

Hyperkeratosis; non-glandular

0

6

4

10

0

5

6

8

   ….Minimal

0

3

3

0

0

0

2

1

   ….Mild

0

3

1

10

0

5

4

7

Infiltration, mononuclear cell

0

0

0

2

0

1

0

0

  ….Minimal

0

0

0

2

0

1

0

0

Applicant's summary and conclusion

Conclusions:
Administration of (E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene by once daily oral gavage was well tolerated in Wistar Han rats at levels of 100, 300 and 1000 mg/Kg/day. Based on the results observed, the No Observed Adverse Effect Level (NOAEL) for (E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene was determined to be at least 1000 mg/Kg/day.
Executive summary:

In a key OECD Guideline 408 oral sub-chronic repeated dose study, the potential toxicity of the test material ((E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene; CAS# 18794-84-8) was evaluated following daily oral gavage administration to Wistar Han rats (10/sex/dose) for a period of 90 days at 0, 100, 300, or 1000 mg/Kg bw/day. The control animals received the vehicle, 0.5% Aqueous carboxymethyl cellulose with 1.25% Tween 80 only. 

 

Animals were observed for mortality and clinical signs and functional observation tests, body weight measurements, food consumption determinations, and ophthalmologic evaluations were conducted. Clinical pathology parameters (hematology, coagulation and clinical chemistry), gross necropsy, organ weight determinations, and histopathologic examinations were also undertaken.

 

There were no unscheduled deaths, except for one control female (Animal No. 50) that died during blood sampling, which was considered to be an accidental death and not treatment-related.

 

A slightly reduced body weight gain was observed in males at 300 and 1000 mg/Kg/day from the third week of treatment onwards, which resulted in absolute body weights of 9.5% and -7.7% at end of treatment, respectively, when compared to the control animals. Based on the magnitude of the change and absence of a clear dose-response relationship, this was considered to be non-adverse.

 

At the end of the treatment period, reduced hind grip strength was observed in males at 300 and 1000 mg/Kg/day. In addition, a reduced number of total movements and ambulations were observed for males at 1000 mg/Kg/day. These effects were considered not to represent an adverse effect on neurobehaviour as the results were not supported by clinical observations and had no supportive morphological correlates in the examined neuronal tissues.

 

A lower concentration of triglycerides was observed in male rats in the 300 and 1000 mg/Kg/day dose groups. In the absence of any correlated microscopic findings, this was considered to be non-adverse.

 

Following necropsy, treatment-related higher liver weights (absolute and/or relative to body weight) were present in males starting at 100 mg/Kg/day and in females starting at 300 mg/Kg/day. Higher relative kidney weights were recorded in females in the 300 and 1000 mg/Kg/day dose groups. There were no microscopic correlates, and therefore, these weight changes were considered to be non-adverse. At histopathological examination, diffuse epithelial hyperplasia and hyperkeratosis in the non-glandular stomach were observed at minimal to mild degree in males and females starting at 100 mg/Kg/day. These were regarded as local treatment-related alterations, resulting from the irritating properties of the test material and at the recorded severities, considered to be non-adverse.

 

No toxicologically significant changes were observed in any of the remaining parameters investigated (i.e. clinical appearance, ophthalmoscopy, food consumption, hematology, coagulation, thyroid hormones (T3, T4, & TSH), and macroscopic examination).

 

Administration of (E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene by once daily oral gavage was well tolerated in Wistar Han rats at levels of 100, 300 and 1000 mg/Kg/day. Based on the results observed, the No Observed Adverse Effect Level (NOAEL) for (E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene was determined to be at least 1000 mg/Kg/day.