Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 December 2014 - 30 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to the OECD 408 guideline and GLP compliance. Fully adequate for assessment.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
read-across: supporting information
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
02 December 2014 - 30 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to the OECD 408 guideline and GLP compliance. Fully adequate for assessment.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
other: Independent analysis
Reason / purpose for cross-reference:
other: GLP Certificate
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Test Material : Octadecene
- CAS 27070-58-2
- Chemical name: C18 Isomerized Olefin
– Physical State/Appearance : Clear colourless liquid
- Purity : 94.1%- Label : C18 Isomerised Olefin Lot 747273
– Batch Number : 747273- Date Received : 03 March 2014
- Storage Conditions : Ambient temperature, in the dark and under nitrogen
- Expiry Date : 10 Feb 2016

Octadecene used in this study was a typical production sample, according to the details included in the boundary composition in IUCLID section 1.2. Octadecene was chosen in the Higher Olefins category testing strategy because it represents a substance with high di-sub content (category range 0.3 – 94%) and high tri-sub content (category range 1 - 65%). Please see the testing strategy attached in section 13 for further details.
Species:
rat
Strain:
other: Wistar Han™:RccHan™:WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: six to eight weeks old
- Weight at study initiation: Males: 200 to 232g, Females: 141 to 175g
- Housing: The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum): A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK - free access
- Water (e.g. ad libitum): free access
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hr light/12 hr dark

Route of administration:
oral: gavage
Vehicle:
other: arachis oil BP
Details on oral exposure:
The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.

The volume of test and control item administered to each animal was based on the most recent
scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item was prepared in Arachis oil BP solution. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results of the analytical determination showed that the formulations were stable for at least 18 days. Formulations were prepared and stored at approximately 4 °C in the dark.
The results indicate that the prepared formulations were within 90% to 102% of the nominal concentration confirming the suitability and accuracy of the formulation procedure.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a fourteen day dose range finding study in the rat (Harlan Study Number: 41403655). In this study, a high dosage of 1000 mg/kg bw/day was well tolerated, therefore, the dose levels 0 (Control), 100, 300 and 1000 mg/kg bw/day were selected for the OECD 408 study.

- The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. - Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.
Observations and examinations performed and frequency:
Clinical Observations:
- Signs of toxicity
- Ill-health or behavioural change immediately before dosing

Functional Observations:
- Prior to the start of treatment and at weekly intervals thereafter

Behavioural Assessment
- Functional Performance Tests: Motor Activity, Forelimb/Hindlimb Grip Strength
- Sensory Reactivity

Body Weight
- Individual body weights were recorded on Day 1 (prior to dosing), at weekly intervals thereafter and at terminal kill

Food Consumption
- Weekly intervals throughout the study

Water Consumption
- Daily by visual inspection of the water bottles

Ophthalmoscopic Examination
- Pre-treatment and before termination of treatment (during Week 12)

Laboratory Investigations
- End of the study
- Haematology
- Blood Chemistry

Sacrifice and pathology:
Sacrifice: All animals were terminated by intravenous overdose of a suitable barbiturate agent followed by exsanguination.

Pathology
- Necropsy: all animals were subjected to a full external and internal examination.
- Organ Weights (Adrenals, Ovaries, Brain Spleen, Epididymides, Testes, Heart, Thymus, Kidneys, Uterus, Liver)
- Histopathology: Samples of selected tissues were removed from all animals and preserved.
- Tissues from all control and high dose group animals were examined microscopically.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the Provantis TM Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate
covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data.

If no dose response was found but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the MannWhitney U test (non-parametric). Probability values (p) are presented as follows:

p<0.01 **
p<0.05 *
p>0.05 (not significant)


Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment at 1000 mg/kg bw/day was associated with increased post-dosing salivation for both sexes. Similar increased post-dosing salivation was also observed for one male and four females at 300 mg/kg bw/day.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of treatment on body weight performance for either sex at 100, 300 or 1000 mg/kg bw/day.

Overall body weight gain for treated animals was slightly superior to control and the occasional statistically significant differences in body weight gain for treated animals were considered to reflect normal biological variation and were unrelated to treatment.

Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on food consumption for either sex at 100, 300 or 1000 mg/kg bw/day.

Food efficiency:
no effects observed
Description (incidence and severity):
There was no effect of treatment on food conversion efficiency for either sex at 100, 300 or 1000 mg/kg bw/day.

Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on water consumption for either sex at dosages of 100, 300 or 1000 mg/kg bw/day.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmic examination of the eyes from rats receiving 1000 mg/kg bw/day did not indicate any effect of treatment.

Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was considered to be no effect of treatment on hematology parameters for either sex at 100, 300 or 1000 mg/kg bw/day.

At all dosages, mean corpuscular hemoglobin concentration for both sexes was lower than control; with the exception of females at 100 mg/kg bw/day, all differences from control attained statistical significance. However, only individual values for two males at 100 mg/kg bw/day and one male at 300 mg/kg bw/day exceeded the historical control range, while this historical range was exceeded by the majority of control males. For females only one control female exceeded the historical control range and all individual values for treated females were within the historical control range.

For females at 1000 mg/kg bw/day, mean hemoglobin level were also statistically significantly lower than control; however only one individual value for these treated animals was below the historical control range while this historical range was exceeded by four control females.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was considered to be no effect of treatment on blood chemistry parameters for either sex at 100, 300 or 1000 mg/kg bw/day.

For males at 1000 mg/kg bw/day, higher albumin and calcium levels attained statistical significance when compared with control but all individual values were within the historical control range; additionally for albumin there was no accompanying statistically significant difference from control for total protein or albumin/globulin ratio. Lower bile acid levels for males at this dosage also attained statistical significance when compared with control. While
individual values for half of these treated animals exceeded the control range, the majority of control values also exceeded this historical range. These differences from control in blood parameters, in the absence of any histopathological correlates were considered to be incidental and unrelated to treatment.

Additionally, for females at 1000 mg/kg bw/day, lower urea and creatinine levels also attained statistical significance when compared with control but all individual values for treated animals were within the historical control range while approximately half of the control urea values exceeded this historical range. For females at all dosage levels, lower bile acid values attained statistical significance when compared to controls but there was no dose response relationship.
Higher inorganic phosphorus levels for females at 300 mg/kg bw/day and lower glucose levels for females at 100 mg/kg bw/day also attained statistical significance when compared with control but there were no similar findings at the high dosage of 1000 mg/kg bw/day.
Overall these differences in urea, bile acid, creatinine and glucose levels were considered to be incidental and unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral Assessments:
Weekly assessment of the animals in a standard arena did not reveal any obvious adverse effects of treatment at dosages of 100, 300 or 1000 mg/kg bw/day.

Functional Performance Tests:
Assessment of functional performance using grip strength and motor activity did not indicate any obvious effects of treatment for either at dosages of 100, 300 or 1000 mg/kg bw/day.

For males, higher values for hind limb grip strength during the third testing occasion attained statistical significance at all dosages, when compared with controls. Additionally, fore limb grip strength for males at 300 mg/kg bw/day was also statistically significantly higher than control during the first testing occasion. Given the lack of any dosage relationship, these observed differences in performance from control were considered to reflect normal biological variation and were unrelated to treatment.


Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no statistically significant differences from control in absolute and body weight relative organ weights for either sex at 100, 300 or 1000 mg/kg bw/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor distribution of findings observed at terminal necropsy indicated any obvious effect of treatment 100, 300 or 1000 mg/kg bw/day.


Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor distribution of findings observed during microscopic examination of the tissues from animals that received 1000 mg/kg bw/day indicated any effect of treatment.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Mortality
- No unscheduled death

Clinical Observations
- 1000 mg/kg bw/day: male and female rats showed increased salivation.
- 300 mg/kg bw/day: Increased salivation was observed in one male and four females. This effect is not considered related to the treatment.
- 100 mg/kg bw/day: no effects

Functional Observations
- Hind limb grip strength fore limb grip strength were observed in males treated with 300 mg/kg bw/day. The lack of dose-response relationship suggested
that these observations reflected normal biological variation and were unrelated to treatment.
- No treatment-related effects were observed in the behavioural and sensory reactivity parameters.

Body Weight
- There was no effect of treatment on body weight performance for either sex at 100, 300 or 1000 mg/kg bw/day.
Overall body weight gain for treated animals was slightly superior to control and the occasional statistically significant differences in body weight gain for treatedanimals were considered to reflect normal biological variation and were unrelated to treatment.

Food Consumption
- There was no effect of treatment on food consumption for either sex at 100, 300 or 1000 mg/kg bw/day.

Water Consumption
- There was no effect of treatment on water consumption for either sex at dosages of 100, 300 or 1000 mg/kg bw/day.

Ophthalmoscopic Examination
- Ophthalmic examination of the eyes from rats receiving 1000 mg/kg bw/day did not indicate any effect of treatment.

Haematological findings
-There was considered to be no effect of treatment on hematology parameters for either sex at 100, 300 or 1000 mg/kg bw/day.

Blood Chemistry
- There was considered to be no effect of treatment on blood chemistry parameters for either sex at 100, 300 or 1000 mg/kg bw/day.

Pathology
Necropsy and Histopatology
- Neither the type, incidence nor distribution of findings observed during microscopic examination of the tissues from animals that received 1000 mg/kg bw/day indicated any effect of treatment.

Organ Weights
- There were no statistically significant differences from control in absolute and body weight relative organ weights for either sex at 100, 300 or 1000 mg/kg bw/day.

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No significant effects observed at 1000 mg/kg/day
Critical effects observed:
not specified

Table 1. Group Mean Functional Performance Test Values

 

Group

 

Males

Females

Overall

Activity

Test 3

Hindlimb (g)

Overall

Activity

Test 3

Hindlimb (g)

Group 1

(0 – Control)

Mean

918.0

255.9

1062.2

323.9

S.D.

284.8

45.3

264.3

81.5

N

10

10

10

10

 

Group 2

(100 mg/Kg bw/day)

Mean

768.7

465.0 **

939.7

292.0

S.D.

206.2

151.6

309.7

102.0

N

10

10

10

10

 

Group 3

(300 mg/Kg bw/day)

Mean

943.9

495.3 **

975.0

278.3

S.D.

335.0

166.8

255.0

108.6

N

10

10

10

10

 

Group 4

(1000 mg/Kg bw/day)

Mean

808.1

313.7**

1045.6

400.5

S.D.

306.2

106.8

240.4

176.5

N

10

10

10

10

General Footnote: Unit = Time (seconds) for Motor Activity Assessments

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

 

Table 2. Group Mean Hematological Values - Males

Group

 

Hb (g/dL)

MCHC (g/dL)

Group 1

(0 – Control)

Mean

16.17

34.60

S.D.

0.89

0.65

N

10

10

 

Group 2

(100 mg/Kg bw/day)

Mean

16.57

34.09*

S.D.

0.57

0.59

N

10

10

 

Group 3

(300 mg/Kg bw/day)

Mean

16.32

34.01*

S.D.

0.73

0.58

N

10

10

 

Group 4

(1000 mg/Kg bw/day)

Mean

16.30

33.56**

S.D.

0.61

0.39

N

10

10

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

 

 

Table 3. Group Mean Blood Chemical Values - Males

 

Group

 

Albumin (g/dL)

Ca++

mmol/l

Bile Acid

µmol/l

Group 1

(0 – Control)

Mean

3.76

2.680

21.16

S.D.

0.24

0.083

10.50

N

10

10

10

 

 

Group 2

(100 mg/Kg bw/day)

Mean

3.93

2.720

14.40

S.D.

0.18

0.149

9.37

N

10

10

10

 

 

Group 3

(300 mg/Kg bw/day)

Mean

3.86

2.731

15.22

S.D.

0.21

0.115

9.42

N

10

10

10

 

 

Group 4

(1000 mg/Kg bw/day)

Mean

3.97*

2.801*

12.26*

S.D.

0.13

0.080

7.91

N

10

10

10

* Significantly different from control group p<0.05

 

 

Table 4. Group Mean Hematological Values - Females

Group

 

Hb (g/dL)

MCHC (g/dL)

Group 1

(0 – Control)

Mean

16.13

34.37

S.D.

0.68

0.81

N

10

10

 

Group 2

(100 mg/Kg bw/day)

Mean

15.94

34.16

S.D.

0.54

0.27

N

10

10

 

Group 3

(300 mg/Kg bw/day)

Mean

16.14 

33.97*

S.D.

0.93

0.30

N

10

10

 

Group 4

(1000 mg/Kg bw/day)

Mean

14.94**

33.89*

S.D.

1.42

0.34

N

10

10

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

 

 

Table 5. Group Mean Blood Chemical Values - Females

 

 

 

 

Group

 

Urea

mg/dl

Glucose

mg/dl

P

mmol/l

ALAT

IU/l

Creat

mg/dl

Bile Acid

µmol/l

Group 1

(0 – Control)

Mean

49.4

183.4

1.62

55.8

0.892

36.18

S.D.

8.9

30.6

0.35

10.3

0.134

19.69

N

10

10

10

10

10

10

 

 

 

 

 

Group 2

(100 mg/Kg bw/day)

Mean

47.6

152.2**

1.55

63.5

0.852

15.20*

S.D.

7.8

14.4

0.27

21.6

0.071

14.89

N

10

10

10

10

10

10

 

 

 

 

 

Group 3

(300 mg/Kg bw/day)

Mean

45.1

169.5

1.96*

59.2

0.894

25.32*

S.D.

12.0

16.1

0.31

19.4

0.238

18.37

N

10

10

10

10

10

10

 

 

 

 

 

Group 4

(1000 mg/Kg bw/day)

Mean

40.4*

170.5

1.56

73.7*

0.795*

17.72*

S.D.

5.5

18.6

0.28

14.5

0.048

13.51

N

10

10

10

10

10

10

 

 

Table 6. Group Mean Organ Weights with Corresponding Relative (% of Body Weight) Organ Weights

 

 

Males

Females

0

Control

100

mg/Kg bw/day

300

mg/Kg bw/day

1000

mg/Kg bw/day

0

Control

100

mg/Kg bw/day

300

mg/Kg bw/day

1000

mg/Kg bw/day

Kidneys

Mean (g)

2.12673

2.19061

2.26685

2.20039

1.40115

1.50356

1.42171

1.52647

S.D.

0.38474

0.23642

0.28298

0.28953

0.14245

0.08800

0.10481

0.14570

N

10

10

10

10

10

10

10

10

 

Mean (%)

0.541

0.520

0.558

0.528

0.578

0.604

0.569

0.615

S.D.

0.047

0.039

0.047

0.033

0.045

0.028

0.034

0.042

N

10

10

10

10

10

10

10

10

 

Liver

Mean (g)

12.6118

13.6667

13.6800

13.0910

8.20024

8.40268

8.26177

8.60674

S.D.

2.28005

1.53962

1.78418

2.96040

0.88450

0.86053

0.59297

1.01931 

N

10

10

10

10

10

10

10

10

 

Mean (%)

3.207

3.239

3.357

3.113

3.382

3.370

3.306

3.463

S.D.

0.261

0.176

0.219

0.286

0.258

0.292

0.201

0.267 

N

10

10

10

10

10

10

10

10

 

Spleen

Mean (g)

0.69673

0.80295

0.72930

0.76011

0.53533

0.57430

0.57771

0.61159

S.D.

0.10764

0.10653

0.09619

0.13641

0.05239

0.06556

0.12066

0.14834

N

10

10

10

10

10

10

10

10

 

Mean (%)

0.178

0.191

0.179

0.182

0.221

0.230

0.232

0.247

S.D.

0.023

0.024

0.016

0.017

0.020

0.025

0.053

0.063

N

10

10

10

10

10

10

10

10

* Significantly different from control group p<0.05

 

** Significantly different from control group p<0.01

 

 

 

 

 

 

 

Table 7. Summary Incidence of Necropsy Findings - Males

 

Males

0

Control

100

mg/Kg bw/day

300

mg/Kg bw/day

1000

mg/Kg bw/day

Number of animals examined

10

10

10

10

Adrenals

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

10

10

10

9

Damaged On Removal; right

0

0

0

1

 

Kidneys

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

10

9

10

10

Increased Pelvic Space; right

0

1

0

0

Fluid Filled; right

0

1

0

0

 

Lungs (With Bronchi)

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

9

10

10

8

Discolouration; Red

1

0

0

2

 

Table 8. Summary Incidence of Necropsy Findings - Females

 

Females

0

Control

100

mg/Kg bw/day

300

mg/Kg bw/day

1000

mg/Kg bw/day

Number of animals examined

10

10

10

10

Brain (Including Cerebrum, Cerebellum And Pons)

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

9

10

10

10

Damaged On Removal; right

1

0

0

0

 

Lungs (With Bronchi)

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

6

9

9

9

Discolouration; red

4

1

1

1

 

Uterus (With Cervix)

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

10

10

9

10

Discolouration; Red

0

0

1

0

 

 

 

Conclusions:
Based on the results of this study, the No Observed Effect Level (NOEL) for systemic toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

The daily oral gavage administration of Octadecene CAS 27070-58-2 to rats for a period of ninety consecutive days at dosages of 100, 300 or 1000 mg/kg bw/day was well tolerated and was not associated with any obvious signs of systemic toxicity for animals of either sex.  At 1000 mg/kg bw/day and, to a much lesser extent, 300 mg/kg bw/day, treatment was associated with increased post-dosing salivation for both sexes but this was considered to reflect distaste or slight irritancy of the dosing formulations rather than any systemic effect of treatment.  There were no effects of treatment indicated by body weight performance, food and water consumption, food conversion efficiency, functional observations or ophthalmic examinations.  

Intergroup differences for hematology and blood chemistry parameters did not indicate any  clear effect of treatment and the few mean values that attained statistical significance when compared with control were considered to be incidental and of no toxicological significance.  There were no statistically significant differences apparent for organ weights and neither macroscopic nor subsequent microscopic examination of tissues revealed any treatment related effects.

Based on the results of this study, the No Observed Effect Level (NOEL) for systemic toxicity was considered to be 1000 mg/kg bw/day.

Reason / purpose for cross-reference:
other: Independent analysis
Reason / purpose for cross-reference:
other: GLP Certificate

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Octadecene
EC Number:
248-205-6
EC Name:
Octadecene
Cas Number:
27070-58-2
Molecular formula:
C18H36
IUPAC Name:
octadecene
Test material form:
other: Clear colourless liquid
Details on test material:
- Test Material : Octadecene
- CAS 27070-58-2
- Chemical name: C18 Isomerized Olefin
- Physical State/Appearance : Clear colourless liquid
- Purity : 94.1%
- Label : C18 Isomerised Olefin Lot 747273
- Batch Number : 747273
- Date Received : 03 March 2014
- Storage Conditions : Ambient temperature, in the dark and under nitrogen
- Expiry Date : 10 Feb 2016
Specific details on test material used for the study:
- Test Material : Octadecene
- CAS 27070-58-2
- Chemical name: C18 Isomerized Olefin
– Physical State/Appearance : Clear colourless liquid
- Purity : 94.1%- Label : C18 Isomerised Olefin Lot 747273
– Batch Number : 747273- Date Received : 03 March 2014
- Storage Conditions : Ambient temperature, in the dark and under nitrogen
- Expiry Date : 10 Feb 2016

Octadecene used in this study was a typical production sample, according to the details included in the boundary composition in IUCLID section 1.2. Octadecene was chosen in the Higher Olefins category testing strategy because it represents a substance with high di-sub content (category range 0.3 – 94%) and high tri-sub content (category range 1 - 65%). Please see the testing strategy attached in section 13 for further details.

Test animals

Species:
rat
Strain:
other: Wistar Han™:RccHan™:WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: six to eight weeks old
- Weight at study initiation: Males: 200 to 232g, Females: 141 to 175g
- Housing: The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum): A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK - free access
- Water (e.g. ad libitum): free access
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hr light/12 hr dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: arachis oil BP
Details on oral exposure:
The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.

The volume of test and control item administered to each animal was based on the most recent
scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item was prepared in Arachis oil BP solution. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results of the analytical determination showed that the formulations were stable for at least 18 days. Formulations were prepared and stored at approximately 4 °C in the dark.
The results indicate that the prepared formulations were within 90% to 102% of the nominal concentration confirming the suitability and accuracy of the formulation procedure.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a fourteen day dose range finding study in the rat (Harlan Study Number: 41403655). In this study, a high dosage of 1000 mg/kg bw/day was well tolerated, therefore, the dose levels 0 (Control), 100, 300 and 1000 mg/kg bw/day were selected for the OECD 408 study.

- The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. - Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.

Examinations

Observations and examinations performed and frequency:
Clinical Observations:
- Signs of toxicity
- Ill-health or behavioural change immediately before dosing

Functional Observations:
- Prior to the start of treatment and at weekly intervals thereafter

Behavioural Assessment
- Functional Performance Tests: Motor Activity, Forelimb/Hindlimb Grip Strength
- Sensory Reactivity

Body Weight
- Individual body weights were recorded on Day 1 (prior to dosing), at weekly intervals thereafter and at terminal kill

Food Consumption
- Weekly intervals throughout the study

Water Consumption
- Daily by visual inspection of the water bottles

Ophthalmoscopic Examination
- Pre-treatment and before termination of treatment (during Week 12)

Laboratory Investigations
- End of the study
- Haematology
- Blood Chemistry

Sacrifice and pathology:
Sacrifice: All animals were terminated by intravenous overdose of a suitable barbiturate agent followed by exsanguination.

Pathology
- Necropsy: all animals were subjected to a full external and internal examination.
- Organ Weights (Adrenals, Ovaries, Brain Spleen, Epididymides, Testes, Heart, Thymus, Kidneys, Uterus, Liver)
- Histopathology: Samples of selected tissues were removed from all animals and preserved.
- Tissues from all control and high dose group animals were examined microscopically.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the Provantis TM Tables and Statistics Module as detailed as follows:

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate
covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data.

If no dose response was found but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the MannWhitney U test (non-parametric). Probability values (p) are presented as follows:

p<0.01 **
p<0.05 *
p>0.05 (not significant)


Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment at 1000 mg/kg bw/day was associated with increased post-dosing salivation for both sexes. Similar increased post-dosing salivation was also observed for one male and four females at 300 mg/kg bw/day.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of treatment on body weight performance for either sex at 100, 300 or 1000 mg/kg bw/day.

Overall body weight gain for treated animals was slightly superior to control and the occasional statistically significant differences in body weight gain for treatedanimals were considered to reflect normal biological variation and were unrelated to treatment.

Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on food consumption for either sex at 100, 300 or 1000 mg/kg bw/day.

Food efficiency:
no effects observed
Description (incidence and severity):
There was no effect of treatment on food conversion efficiency for either sex at 100, 300 or 1000 mg/kg bw/day.

Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on water consumption for either sex at dosages of 100, 300 or 1000 mg/kg bw/day.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmic examination of the eyes from rats receiving 1000 mg/kg bw/day did not indicate any effect of treatment.

Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was considered to be no effect of treatment on hematology parameters for either sex at 100, 300 or 1000 mg/kg bw/day.

At all dosages, mean corpuscular hemoglobin concentration for both sexes was lower than control; with the exception of females at 100 mg/kg bw/day, all differences from control attained statistical significance. However, only individual values for two males at 100 mg/kg bw/day and one male at 300 mg/kg bw/day exceeded the historical control range, while this historical range was exceeded by the majority of control males. For females only one control female exceeded the historical control range and all individual values for treated females were within the historical control range.

For females at 1000 mg/kg bw/day, mean hemoglobin level were also statistically significantly lower than control; however only one individual value for these treated animals was below the historical control range while this historical range was exceeded by four control females.

Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was considered to be no effect of treatment on blood chemistry parameters for either sex at 100, 300 or 1000 mg/kg bw/day.

For males at 1000 mg/kg bw/day, higher albumin and calcium levels attained statistical significance when compared with control but all individual values were within the historical control range; additionally for albumin there was no accompanying statistically significant difference from control for total protein or albumin/globulin ratio. Lower bile acid levels for males at this dosage also attained statistical significance when compared with control. While
individual values for half of these treated animals exceeded the control range, the majority of control values also exceeded this historical range. These differences from control in blood parameters, in the absence of any histopathological correlates were considered to be incidental and unrelated to treatment.

Additionally, for females at 1000 mg/kg bw/day, lower urea and creatinine levels also attained statistical significance when compared with control but all individual values for treated animals were within the historical control range while approximately half of the control urea values exceeded this historical range. For females at all dosage levels, lower bile acid values attained statistical significance when compared to controls but there was no dose response relationship.
Higher inorganic phosphorus levels for females at 300 mg/kg bw/day and lower glucose levels for females at 100 mg/kg bw/day also attained statistical significance when compared with control but there were no similar findings at the high dosage of 1000 mg/kg bw/day.
Overall these differences in urea, bile acid, creatinine and glucose levels were considered to be incidental and unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral Assessments:
Weekly assessment of the animals in a standard arena did not reveal any obvious adverse effects of treatment at dosages of 100, 300 or 1000 mg/kg bw/day.

Functional Performance Tests:
Assessment of functional performance using grip strength and motor activity did not indicate any obvious effects of treatment for either at dosages of 100, 300 or 1000 mg/kg bw/day.

For males, higher values for hind limb grip strength during the third testing occasion attained statistical significance at all dosages, when compared with controls. Additionally, fore limb grip strength for males at 300 mg/kg bw/day was also statistically significantly higher than control during the first testing occasion. Given the lack of any dosage relationship, these observed differences in performance from control were considered to reflect normal biological variation and were unrelated to treatment.


Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no statistically significant differences from control in absolute and body weight relative organ weights for either sex at 100, 300 or 1000 mg/kg bw/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor distribution of findings observed at terminal necropsy indicated any obvious effect of treatment 100, 300 or 1000 mg/kg bw/day.


Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor distribution of findings observed during microscopic examination of the tissues from animals that received 1000 mg/kg bw/day indicated any effect of treatment.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Mortality
- No unscheduled death

Clinical Observations
- 1000 mg/kg bw/day: male and female rats showed increased salivation.
- 300 mg/kg bw/day: Increased salivation was observed in one male and four females. This effect is not considered related to the treatment.
- 100 mg/kg bw/day: no effects

Functional Observations
- Hind limb grip strength fore limb grip strength were observed in males treated with 300 mg/kg bw/day. The lack of dose-response relationship suggested
that these observations reflected normal biological variation and were unrelated to treatment.
- No treatment-related effects were observed in the behavioural and sensory reactivity parameters.

Body Weight
- There was no effect of treatment on body weight performance for either sex at 100, 300 or 1000 mg/kg bw/day.
Overall body weight gain for treated animals was slightly superior to control and the occasional statistically significant differences in body weight gain for treatedanimals were considered to reflect normal biological variation and were unrelated to treatment.

Food Consumption
- There was no effect of treatment on food consumption for either sex at 100, 300 or 1000 mg/kg bw/day.

Water Consumption
- There was no effect of treatment on water consumption for either sex at dosages of 100, 300 or 1000 mg/kg bw/day.

Ophthalmoscopic Examination
- Ophthalmic examination of the eyes from rats receiving 1000 mg/kg bw/day did not indicate any effect of treatment.

Haematological findings
-There was considered to be no effect of treatment on hematology parameters for either sex at 100, 300 or 1000 mg/kg bw/day.

Blood Chemistry
- There was considered to be no effect of treatment on blood chemistry parameters for either sex at 100, 300 or 1000 mg/kg bw/day.

Pathology
Necropsy and Histopatology
- Neither the type, incidence nor distribution of findings observed during microscopic examination of the tissues from animals that received 1000 mg/kg bw/day indicated any effect of treatment.

Organ Weights
- There were no statistically significant differences from control in absolute and body weight relative organ weights for either sex at 100, 300 or 1000 mg/kg bw/day.


Effect levels

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No significant effects observed at 1000 mg/kg/day

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Group Mean Functional Performance Test Values

 

Group

 

Males

Females

Overall

Activity

Test 3

Hindlimb (g)

Overall

Activity

Test 3

Hindlimb (g)

Group 1

(0 – Control)

Mean

918.0

255.9

1062.2

323.9

S.D.

284.8

45.3

264.3

81.5

N

10

10

10

10

 

Group 2

(100 mg/Kg bw/day)

Mean

768.7

465.0 **

939.7

292.0

S.D.

206.2

151.6

309.7

102.0

N

10

10

10

10

 

Group 3

(300 mg/Kg bw/day)

Mean

943.9

495.3 **

975.0

278.3

S.D.

335.0

166.8

255.0

108.6

N

10

10

10

10

 

Group 4

(1000 mg/Kg bw/day)

Mean

808.1

313.7**

1045.6

400.5

S.D.

306.2

106.8

240.4

176.5

N

10

10

10

10

General Footnote: Unit = Time (seconds) for Motor Activity Assessments

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

 

Table 2. Group Mean Hematological Values - Males

Group

 

Hb (g/dL)

MCHC (g/dL)

Group 1

(0 – Control)

Mean

16.17

34.60

S.D.

0.89

0.65

N

10

10

 

Group 2

(100 mg/Kg bw/day)

Mean

16.57

34.09*

S.D.

0.57

0.59

N

10

10

 

Group 3

(300 mg/Kg bw/day)

Mean

16.32

34.01*

S.D.

0.73

0.58

N

10

10

 

Group 4

(1000 mg/Kg bw/day)

Mean

16.30

33.56**

S.D.

0.61

0.39

N

10

10

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

 

 

Table 3. Group Mean Blood Chemical Values - Males

 

Group

 

Albumin (g/dL)

Ca++

mmol/l

Bile Acid

µmol/l

Group 1

(0 – Control)

Mean

3.76

2.680

21.16

S.D.

0.24

0.083

10.50

N

10

10

10

 

 

Group 2

(100 mg/Kg bw/day)

Mean

3.93

2.720

14.40

S.D.

0.18

0.149

9.37

N

10

10

10

 

 

Group 3

(300 mg/Kg bw/day)

Mean

3.86

2.731

15.22

S.D.

0.21

0.115

9.42

N

10

10

10

 

 

Group 4

(1000 mg/Kg bw/day)

Mean

3.97*

2.801*

12.26*

S.D.

0.13

0.080

7.91

N

10

10

10

* Significantly different from control group p<0.05

 

 

Table 4. Group Mean Hematological Values - Females

Group

 

Hb (g/dL)

MCHC (g/dL)

Group 1

(0 – Control)

Mean

16.13

34.37

S.D.

0.68

0.81

N

10

10

 

Group 2

(100 mg/Kg bw/day)

Mean

15.94

34.16

S.D.

0.54

0.27

N

10

10

 

Group 3

(300 mg/Kg bw/day)

Mean

16.14 

33.97*

S.D.

0.93

0.30

N

10

10

 

Group 4

(1000 mg/Kg bw/day)

Mean

14.94**

33.89*

S.D.

1.42

0.34

N

10

10

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

 

 

Table 5. Group Mean Blood Chemical Values - Females

 

 

 

 

Group

 

Urea

mg/dl

Glucose

mg/dl

P

mmol/l

ALAT

IU/l

Creat

mg/dl

Bile Acid

µmol/l

Group 1

(0 – Control)

Mean

49.4

183.4

1.62

55.8

0.892

36.18

S.D.

8.9

30.6

0.35

10.3

0.134

19.69

N

10

10

10

10

10

10

 

 

 

 

 

Group 2

(100 mg/Kg bw/day)

Mean

47.6

152.2**

1.55

63.5

0.852

15.20*

S.D.

7.8

14.4

0.27

21.6

0.071

14.89

N

10

10

10

10

10

10

 

 

 

 

 

Group 3

(300 mg/Kg bw/day)

Mean

45.1

169.5

1.96*

59.2

0.894

25.32*

S.D.

12.0

16.1

0.31

19.4

0.238

18.37

N

10

10

10

10

10

10

 

 

 

 

 

Group 4

(1000 mg/Kg bw/day)

Mean

40.4*

170.5

1.56

73.7*

0.795*

17.72*

S.D.

5.5

18.6

0.28

14.5

0.048

13.51

N

10

10

10

10

10

10

 * Significantly different from control group p<0.05 

** Significantly different from control group p<0.01

 

Table 6. Group Mean Organ Weights with Corresponding Relative (% of Body Weight) Organ Weights

 

 

Males

Females

0

Control

100

mg/Kg bw/day

300

mg/Kg bw/day

1000

mg/Kg bw/day

0

Control

100

mg/Kg bw/day

300

mg/Kg bw/day

1000

mg/Kg bw/day

Kidneys

Mean (g)

2.12673

2.19061

2.26685

2.20039

1.40115

1.50356

1.42171

1.52647

S.D.

0.38474

0.23642

0.28298

0.28953

0.14245

0.08800

0.10481

0.14570

N

10

10

10

10

10

10

10

10

 

Mean (%)

0.541

0.520

0.558

0.528

0.578

0.604

0.569

0.615

S.D.

0.047

0.039

0.047

0.033

0.045

0.028

0.034

0.042

N

10

10

10

10

10

10

10

10

 

Liver

Mean (g)

12.6118

13.6667

13.6800

13.0910

8.20024

8.40268

8.26177

8.60674

S.D.

2.28005

1.53962

1.78418

2.96040

0.88450

0.86053

0.59297

1.01931 

N

10

10

10

10

10

10

10

10

 

Mean (%)

3.207

3.239

3.357

3.113

3.382

3.370

3.306

3.463

S.D.

0.261

0.176

0.219

0.286

0.258

0.292

0.201

0.267 

N

10

10

10

10

10

10

10

10

 

Spleen

Mean (g)

0.69673

0.80295

0.72930

0.76011

0.53533

0.57430

0.57771

0.61159

S.D.

0.10764

0.10653

0.09619

0.13641

0.05239

0.06556

0.12066

0.14834

N

10

10

10

10

10

10

10

10

 

Mean (%)

0.178

0.191

0.179

0.182

0.221

0.230

0.232

0.247

S.D.

0.023

0.024

0.016

0.017

0.020

0.025

0.053

0.063

N

10

10

10

10

10

10

10

10

 

Table 7. Summary Incidence of Necropsy Findings - Males

 

Males

0

Control

100

mg/Kg bw/day

300

mg/Kg bw/day

1000

mg/Kg bw/day

Number of animals examined

10

10

10

10

Adrenals

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

10

10

10

9

Damaged On Removal; right

0

0

0

1

 

Kidneys

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

10

9

10

10

Increased Pelvic Space; right

0

1

0

0

Fluid Filled; right

0

1

0

0

 

Lungs (With Bronchi)

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

9

10

10

8

Discolouration; Red

1

0

0

2

 

Table 8. Summary Incidence of Necropsy Findings - Females

 

Females

0

Control

100

mg/Kg bw/day

300

mg/Kg bw/day

1000

mg/Kg bw/day

Number of animals examined

10

10

10

10

Brain (Including Cerebrum, Cerebellum And Pons)

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

9

10

10

10

Damaged On Removal; right

1

0

0

0

 

Lungs (With Bronchi)

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

6

9

9

9

Discolouration; red

4

1

1

1

 

Uterus (With Cervix)

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

10

10

9

10

Discolouration; Red

0

0

1

0

 

 

 

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the No Observed Effect Level (NOEL) for systemic toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

The daily oral gavage administration of Octadecene CAS 27070-58-2 to rats for a period of ninety consecutive days at dosages of 100, 300 or 1000 mg/kg bw/day was well tolerated and was not associated with any obvious signs of systemic toxicity for animals of either sex.  At 1000 mg/kg bw/day and, to a much lesser extent, 300 mg/kg bw/day, treatment was associated with increased post-dosing salivation for both sexes but this was considered to reflect distaste or slight irritancy of the dosing formulations rather than any systemic effect of treatment.  There were no effects of treatment indicated by body weight performance, food and water consumption, food conversion efficiency, functional observations or ophthalmic examinations.  

Intergroup differences for hematology and blood chemistry parameters did not indicate any  clear effect of treatment and the few mean values that attained statistical significance when compared with control were considered to be incidental and of no toxicological significance.  There were no statistically significant differences apparent for organ weights and neither macroscopic nor subsequent microscopic examination of tissues revealed any treatment related effects.

Based on the results of this study, the No Observed Effect Level (NOEL) for systemic toxicity was considered to be 1000 mg/kg bw/day.

Categories Display