Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 December 2014 - 30 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according 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-reference
Reason / purpose:
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:
10 December 2014 - 30 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according 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:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
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. certificate)
Specific details on test material used for the study:
Identification: Hydrocarbons, C12-30, olefin-rich, ethylene polymn by product CAS 68911-05-7
Alternative Identification:ENORDET 0341
Physical State/Appearance:Clear colorless liquid
Purity:100%
Batch Number:DT1719380A
Date Received: 09 April 2014
Storage Conditions: Ambient temperature, in the dark under Nitrogen.
Expiry Date: December 2018

Hydrocarbons, C12-30, olefin-rich, ethylene polymn by product, CAS# 68911-05-7 used in this study was a typical production sample, according to the details included in the boundary composition in IUCLID section 1.2. Hydrocarbons, C12-30, olefin-rich, ethylene polymn by product were chosen in the Higher Olefins category testing strategy because it represents a substance with high high di- sub content (category Range 0.3 – 94%) and high tetra-sub content (category Range 0 – 8%), even / odd carbon number. 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 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: 195 to 233g, Females: 159 to 192g
- 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: 7 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

Experimental Starting Date: 10 December 2014
Experimental Completion Date: 30 November 2015

Justification for specie selection: The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

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 26 days. Formulations were prepared and stored at approximately 4 °C in the dark.
The results indicate that the prepared formulations were within 96% to 108% 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: 41403659. In this study, a high dosage of 1000 mg/kg bw/day was well tolerated, therefore, the dose 0 (Control), 100, 300 and 1000 mg/kg bw/day have been selected for the OECD 408 study.

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
- Hematology
- 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. All tissues from control and 1000 mg/kg/day groups 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):
There were no clinical signs observed that indicated any effect of treatment at dosages of 100, 300 or 1000 mg/kg bw/day.

Two females treated with 1000 mg/kg bw/day showed increased salivation post-dosing on Day 55 and for one of these females on Day 56 also. Increased post-dosing salivation is frequently observed when animals are dosed via the oral gavage route and is generally considered to reflect distaste or slight irritancy of the dosing formulations rather than any systemic effect of treatment.

One male treated with 100 mg/kg bw/day had red/brown staining around the snout on Day 91. In the absence of a similar effect at the high dosage group this finding was considered to be incidental.
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 gain for either sex at dosages of 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 observed 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, treatment-related
Description (incidence and severity):
For males at 1000 mg/kg bw/day, mean corpuscular hemoglobin concentration was statistically significantly lower than control. All of the individual values were within historical control range and in the absence of any associated changes the intergroup difference was considered of no toxicological significance.

For females at 1000 mg/kg bw/day, erythrocyte count was statistically significantly lower than control; however only two individual values for these treated animals was below the historical control range. In the absence of any associated changes the intergroup difference was considered of no toxicological significance.

For females at 100 mg/kg bw/day, mean corpuscular hemoglobin concentration was statistically significantly lower than control. All of the individual values were within historical control range and in the absence of a true dose related response, the intergroup difference was considered of no toxicological significance.
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, lower albumin, total protein and albumin/globulin ratio attained statistical significance when compared with control. The majority of individual values were within the historical control range and in the absence of any histopathological correlates, the intergroup differences were considered to be incidental and unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
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.

Sensory Reactivity Assessments:
There were no differences observed in the scores for sensory reactivity for either sex during the study.


Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was considered to be no effect of treatment on the organ weights measured for either sex at 100, 300 or 1000 mg/kg bw/day.

At all dosages, absolute and body weight relative ovary weights were statistically significantly lower than control. All of the individual values were within historical control range and in the absence of a true dose related response or any histopathological correlates, the intergroup differences were considered of no toxicological significance.




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:
Clinical Observations
- No treatment-related effects
Increased salivation post-dosing on (Days 55 and 56) was observed in 2 females treated with 1000 mg/kg bw/day. This effects was considered related to the oral gavage route and reflect distaste or slight irritancy of the dosing formulations rather than any systemic effect of treatment.
Red/brown staining around the snout was observed in only one animal. This finding was considered to be incidental.

Body Weight
- There was no effect of treatment on body weight gain for either sex at dosages of 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
- 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 observed 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.

Functional Observations
-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.

Sensory Reactivity Assessments:
There were no differences observed in the scores for sensory reactivity for either sex during the study.

Haematological findings
- For males at 1000 mg/kg bw/day, mean corpuscular hemoglobin concentration was statistically significantly lower than control. All of the individual values were within historical control range and in the absence of any associated changes the intergroup difference was considered of no toxicological significance.
For females at 1000 mg/kg bw/day, erythrocyte count was statistically significantly lower than control; however only two individual values for these treated animals was below the historical control range. In the absence of any associated changes the intergroup difference was considered of no toxicological significance.
For females at 100 mg/kg bw/day, mean corpuscular hemoglobin concentration was statistically significantly lower than control. All of the individual values were within historical control range and in the absence of a true dose related response, the intergroup difference was considered of no toxicological significance.

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.
For males at 1000 mg/kg bw/day, lower albumin, total protein and albumin/globulin ratio attained statistical significance when compared with control. The majority of individual values were within the historical control range and in the absence of any histopathological correlates, the intergroup differences were considered to be incidental and unrelated to treatment.

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

Organ Weights
- There was considered to be no effect of treatment on the organ weights measured for either sex at 100, 300 or 1000 mg/kg bw/day.
At all dosages, absolute and body weight relative ovary weights were statistically significantly lower than control. All of the individual values were within historical control range and in the absence of a true dose related response or any histopathological correlates, the intergroup differences were considered of no toxicological significance.




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

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

788.2

858.1

700.1

911.4

S.D.

246.9

188.2

315.6

212.1

N

10

10

10

10

 

Group 2

(100 mg/Kg bw/day)

Mean

921.2

947.7

712.1

810.7

S.D.

176.5

289.6

231.5

258.8

N

10

10

10

10

 

Group 3

 (300 mg/Kg bw/day)

Mean

689.2

935.2

795.2

849.6

S.D.

199.7

257.0

354.1

192.4

N

10

10

10

10

 

Group 4

(1000 mg/Kg bw/day)

Mean

764.9

903.4

622.8

947.5

S.D.

221.0

264.7

192.3

213.5

N

10

10

10

10

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

 

Table 2. Group Mean Hematological Values - Males

Group

 

Hb (g/dL)

MCHC (g/dL)

Group 1

(0 – Control)

Mean

16.97

35.14

S.D.

1.16

0.88

N

10

10

 

Group 2

(100 mg/Kg bw/day)

Mean

17.05

35.00

S.D.

0.61

0.75

N

10

10

 

Group 3

(300 mg/Kg bw/day)

Mean

17.37

34.82

S.D.

0.47

0.53

N

10

10

 

Group 4

(1000 mg/Kg bw/day)

Mean

16.41

34.31*

S.D.

1.14

0.52

N

10

10

* Significantly different from control group p<0.05

 

Table 3. Group Mean Blood Chemical Values - Males

Group

 

Tot. Prot.

g/dl

Albumin

g/dl

A/G

Ratio

Group 1 (0 – Control)

Mean

7.608

4.00

1.117

S.D.

0.865

0.33

0.083

N

10

10

10

 

Group 2 (100 mg/Kg bw/day)

Mean

7.507

3.89

1.093

S.D.

0.687

0.21

  0.125

N

10

10

10

 

Group 3 (300 mg/Kg bw/day)

Mean

7.287

3.89

1.153

S.D.

0.402

0.11

0.080

N

10

10

10

 

Group 4 (1000 mg/Kg bw/day)

Mean

6.792**

3.72*

1.214*

S.D.

0.436

0.23

  0.064

N

10

10

10

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

 

Table 4. Group Mean Blood Chemical Values - Females

Group

 

Glucose

mg/dl

Group 1

(0 – Control)

Mean

167.5

S.D.

22.1

N

10

 

Group 2

(100 mg/Kg bw/day)

Mean

161.4

S.D.

30.8

N

10

 

Group 3

(300 mg/Kg bw/day)

Mean

151.8

S.D.

18.7

N

10

 

Group 4

(1000 mg/Kg bw/day)

Mean

158.8

S.D.

21.7

N

10

* Significantly different from control group p<0.05

 

 

 

Table 5. 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)

1.90581

   2.12537

2.21380

2.07667

1.44691

1.40400

1.46426

1.55144

S.D.

0.14310

0.26211

0.18932

0.19827

0.11512

0.13162

0.16807

0.11943

N

10

10

10

10

10

10

10

10

 

Mean (%)

0.535

0.574

    0.562

0.541

0.636

0.599

0.613

0.629

S.D.

0.023

0.045

0.048

0.047

0.063

0.046

0.032

0.042

N

10

10

10

10

10

10

10

10

 

Liver

Mean (g)

11.0333

11.8556

12.5011

12.4400

7.89038

7.56497

7.89763

8.26136

S.D.

1.18487

1.28494

1.01173

1.65424

0.95250

0.83806

1.05317

0.86573

N

10

10

10

10

10

10

10

10

 

Mean (%)

3.093

3.199

    3.167

3.235

3.455

3.231

3.301

3.341

S.D.

0.224

0.142

0.151

0.364

0.302

0.323

0.248

0.188

N

10

10

10

10

10

10

10

10

 

Ovaries

Mean (g)

 

 

 

 

0.13797

0.12121*

0.11627**

0.11934**

S.D.

 

 

 

 

0.02525

0.01600

0.02304

0.01851

N

 

 

 

 

10

10

10

10

 

Mean (%)

 

 

 

 

0.061

0.052*

0.048**

0.048**

S.D.

 

 

 

 

0.012

0.006 

0.007

0.008

N

 

 

 

 

10

10

10

10

 

* Significantly different from control group p<0.05

 

** Significantly different from control group p<0.01

 

Table 5. 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

Lungs (With Bronchi)

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

8

9

10

9

Discolouration; red

2

1

0

1

 

Kidneys

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

10

9

10

10

Increased Pelvic Space; right

0

1

0

0

 

 

Table 6. 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

Lungs (With Bronchi)

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

8

8

9

10

Discolouration; Red

2

2

1

0

 

 

 

 

 

 

Conclusions:
Based on the results of this ninety day study, the No Observed Effect Level (NOEL) for systemic toxicity of Hydrocarbons, C12-30, olefin-rich, ethylene polymn by product CAS 68911-05-7 was considered to be 1000 mg/kg bw/day.
Executive summary:

The test material Hydrocarbons, C12-30, olefin-rich, ethylene polymn by product CAS 68911 -05 -7 was administrated orally to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day for 90 consecutive days.

The results demosntrated 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 treatment-related 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 of Hydrocarbons, C12-30, olefin-rich, ethylene polymn by product CAS 68911-05-7 was considered to be 1000 mg/kg bw/day.

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:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
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. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Clear colourless liquid
Details on test material:
- Test Material : Hydrocarbons, C12-30, olefin-rich, ethylene polymn by product
- CAS 68911-05-7
- Physical State/Appearance : Clear colourless liquid
- Purity: 100%
- Batch Number : DT1719380A
- Date Received : 09 April 2014
- Storage Conditions : Ambient temperature, in the dark and under nitrogen
- Expiry Date : December 2018
Specific details on test material used for the study:
Identification: Hydrocarbons, C12-30, olefin-rich, ethylene polymn by product CAS 68911-05-7
Alternative Identification:ENORDET 0341
Physical State/Appearance:Clear colorless liquid
Purity:100%
Batch Number:DT1719380A
Date Received: 09 April 2014
Storage Conditions: Ambient temperature, in the dark under Nitrogen.
Expiry Date: December 2018

Hydrocarbons, C12-30, olefin-rich, ethylene polymn by product, CAS# 68911-05-7 used in this study was a typical production sample, according to the details included in the boundary composition in IUCLID section 1.2. Hydrocarbons, C12-30, olefin-rich, ethylene polymn by product were chosen in the Higher Olefins category testing strategy because it represents a substance with high high di- sub content (category Range 0.3 – 94%) and high tetra-sub content (category Range 0 – 8%), even / odd carbon number. 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 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: 195 to 233g, Females: 159 to 192g
- 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: 7 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

Experimental Starting Date: 10 December 2014
Experimental Completion Date: 30 November 2015

Justification for specie selection: The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

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 26 days. Formulations were prepared and stored at approximately 4 °C in the dark.
The results indicate that the prepared formulations were within 96% to 108% 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: 41403659. In this study, a high dosage of 1000 mg/kg bw/day was well tolerated, therefore, the dose 0 (Control), 100, 300 and 1000 mg/kg bw/day have been selected for the OECD 408 study.

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
- Hematology
- 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. All tissues from control and 1000 mg/kg/day groups 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):
There were no clinical signs observed that indicated any effect of treatment at dosages of 100, 300 or 1000 mg/kg bw/day.

Two females treated with 1000 mg/kg bw/day showed increased salivation post-dosing on Day 55 and for one of these females on Day 56 also. Increased post-dosing salivation is frequently observed when animals are dosed via the oral gavage route and is generally considered to reflect distaste or slight irritancy of the dosing formulations rather than any systemic effect of treatment.

One male treated with 100 mg/kg bw/day had red/brown staining around the snout on Day 91. In the absence of a similar effect at the high dosage group this finding was considered to be incidental.
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 gain for either sex at dosages of 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 observed 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, treatment-related
Description (incidence and severity):
For males at 1000 mg/kg bw/day, mean corpuscular hemoglobin concentration was statistically significantly lower than control. All of the individual values were within historical control range and in the absence of any associated changes the intergroup difference was considered of no toxicological significance.

For females at 1000 mg/kg bw/day, erythrocyte count was statistically significantly lower than control; however only two individual values for these treated animals was below the historical control range. In the absence of any associated changes the intergroup difference was considered of no toxicological significance.

For females at 100 mg/kg bw/day, mean corpuscular hemoglobin concentration was statistically significantly lower than control. All of the individual values were within historical control range and in the absence of a true dose related response, the intergroup difference was considered of no toxicological significance.

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, lower albumin, total protein and albumin/globulin ratio attained statistical significance when compared with control. The majority of individual values were within the historical control range and in the absence of any histopathological correlates, the intergroup differences were considered to be incidental and unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
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.

Sensory Reactivity Assessments:
There were no differences observed in the scores for sensory reactivity for either sex during the study.


Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was considered to be no effect of treatment on the organ weights measured for either sex at 100, 300 or 1000 mg/kg bw/day.

At all dosages, absolute and body weight relative ovary weights were statistically significantly lower than control. All of the individual values were within historical control range and in the absence of a true dose related response or any histopathological correlates, the intergroup differences were considered of no toxicological significance.




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.
Other effects:
not specified
Details on results:
Clinical Observations
- No treatment-related effects
Increased salivation post-dosing on (Days 55 and 56) was observed in 2 females treated with 1000 mg/kg bw/day. This effects was considered related to the oral gavage route and reflect distaste or slight irritancy of the dosing formulations rather than any systemic effect of treatment.
Red/brown staining around the snout was observed in only one animal. This finding was considered to be incidental.

Body Weight
- There was no effect of treatment on body weight gain for either sex at dosages of 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
- 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 observed 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.

Functional Observations
-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.

Sensory Reactivity Assessments:
There were no differences observed in the scores for sensory reactivity for either sex during the study.

Haematological findings
- For males at 1000 mg/kg bw/day, mean corpuscular hemoglobin concentration was statistically significantly lower than control. All of the individual values were within historical control range and in the absence of any associated changes the intergroup difference was considered of no toxicological significance.
For females at 1000 mg/kg bw/day, erythrocyte count was statistically significantly lower than control; however only two individual values for these treated animals was below the historical control range. In the absence of any associated changes the intergroup difference was considered of no toxicological significance.
For females at 100 mg/kg bw/day, mean corpuscular hemoglobin concentration was statistically significantly lower than control. All of the individual values were within historical control range and in the absence of a true dose related response, the intergroup difference was considered of no toxicological significance.

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.
For males at 1000 mg/kg bw/day, lower albumin, total protein and albumin/globulin ratio attained statistical significance when compared with control. The majority of individual values were within the historical control range and in the absence of any histopathological correlates, the intergroup differences were considered to be incidental and unrelated to treatment.

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

Organ Weights
- There was considered to be no effect of treatment on the organ weights measured for either sex at 100, 300 or 1000 mg/kg bw/day.
At all dosages, absolute and body weight relative ovary weights were statistically significantly lower than control. All of the individual values were within historical control range and in the absence of a true dose related response or any histopathological correlates, the intergroup differences were considered of no toxicological significance.

Effect levels

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

Target system / organ toxicity

Key result
Critical effects observed:
no

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

788.2

858.1

700.1

911.4

S.D.

246.9

188.2

315.6

212.1

N

10

10

10

10

 

Group 2

(100 mg/Kg bw/day)

Mean

921.2

947.7

712.1

810.7

S.D.

176.5

289.6

231.5

258.8

N

10

10

10

10

 

Group 3

 (300 mg/Kg bw/day)

Mean

689.2

935.2

795.2

849.6

S.D.

199.7

257.0

354.1

192.4

N

10

10

10

10

 

Group 4

(1000 mg/Kg bw/day)

Mean

764.9

903.4

622.8

947.5

S.D.

221.0

264.7

192.3

213.5

N

10

10

10

10

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

 

Table 2. Group Mean Hematological Values - Males

Group

 

Hb (g/dL)

MCHC (g/dL)

Group 1

(0 – Control)

Mean

16.97

35.14

S.D.

1.16

0.88

N

10

10

 

Group 2

(100 mg/Kg bw/day)

Mean

17.05

35.00

S.D.

0.61

0.75

N

10

10

 

Group 3

(300 mg/Kg bw/day)

Mean

17.37

34.82

S.D.

0.47

0.53

N

10

10

 

Group 4

(1000 mg/Kg bw/day)

Mean

16.41

34.31*

S.D.

1.14

0.52

N

10

10

* Significantly different from control group p<0.05

 

Table 3. Group Mean Blood Chemical Values - Males

Group

 

Tot. Prot.

g/dl

Albumin

g/dl

A/G

Ratio

Group 1 (0 – Control)

Mean

7.608

4.00

1.117

S.D.

0.865

0.33

0.083

N

10

10

10

 

Group 2 (100 mg/Kg bw/day)

Mean

7.507

3.89

1.093

S.D.

0.687

0.21

  0.125

N

10

10

10

 

Group 3 (300 mg/Kg bw/day)

Mean

7.287

3.89

1.153

S.D.

0.402

0.11

0.080

N

10

10

10

 

Group 4 (1000 mg/Kg bw/day)

Mean

6.792**

3.72*

1.214*

S.D.

0.436

0.23

  0.064

N

10

10

10

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

 

Table 4. Group Mean Blood Chemical Values - Females

Group

 

Glucose

mg/dl

Group 1

(0 – Control)

Mean

167.5

S.D.

22.1

N

10

 

Group 2

(100 mg/Kg bw/day)

Mean

161.4

S.D.

30.8

N

10

 

Group 3

(300 mg/Kg bw/day)

Mean

151.8

S.D.

18.7

N

10

 

Group 4

(1000 mg/Kg bw/day)

Mean

158.8

S.D.

21.7

N

10

* Significantly different from control group p<0.05

 

 

 

Table 5. 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)

1.90581

   2.12537

2.21380

2.07667

1.44691

1.40400

1.46426

1.55144

S.D.

0.14310

0.26211

0.18932

0.19827

0.11512

0.13162

0.16807

0.11943

N

10

10

10

10

10

10

10

10

 

Mean (%)

0.535

0.574

    0.562

0.541

0.636

0.599

0.613

0.629

S.D.

0.023

0.045

0.048

0.047

0.063

0.046

0.032

0.042

N

10

10

10

10

10

10

10

10

 

Liver

Mean (g)

11.0333

11.8556

12.5011

12.4400

7.89038

7.56497

7.89763

8.26136

S.D.

1.18487

1.28494

1.01173

1.65424

0.95250

0.83806

1.05317

0.86573

N

10

10

10

10

10

10

10

10

 

Mean (%)

3.093

3.199

    3.167

3.235

3.455

3.231

3.301

3.341

S.D.

0.224

0.142

0.151

0.364

0.302

0.323

0.248

0.188

N

10

10

10

10

10

10

10

10

 

Ovaries

Mean (g)

 

 

 

 

0.13797

0.12121*

0.11627**

0.11934**

S.D.

 

 

 

 

0.02525

0.01600

0.02304

0.01851

N

 

 

 

 

10

10

10

10

 

Mean (%)

 

 

 

 

0.061

0.052*

0.048**

0.048**

S.D.

 

 

 

 

0.012

0.006 

0.007

0.008

N

 

 

 

 

10

10

10

10

 

* Significantly different from control group p<0.05

 

** Significantly different from control group p<0.01

 

Table 5. 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

Lungs (With Bronchi)

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

8

9

10

9

Discolouration; red

2

1

0

1

 

Kidneys

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

10

9

10

10

Increased Pelvic Space; right

0

1

0

0

 

 

Table 6. 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

Lungs (With Bronchi)

 

Submitted

(10)

(10)

(10)

(10)

No Visible Lesions

8

8

9

10

Discolouration; Red

2

2

1

0

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
Based on the results of this ninety day study, the No Observed Effect Level (NOEL) for systemic toxicity of Hydrocarbons, C12-30, olefin-rich, ethylene polymn by product CAS 68911-05-7 was considered to be 1000 mg/kg bw/day.
Executive summary:

The test material Hydrocarbons, C12-30, olefin-rich, ethylene polymn by product CAS 68911 -05 -7 was administrated orally to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day for 90 consecutive days.

The results demosntrated 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 treatment-related 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 of Hydrocarbons, C12-30, olefin-rich, ethylene polymn by product CAS 68911-05-7 was considered to be 1000 mg/kg bw/day.