Alkenes, C15-18 α-, sulfurized

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 May 2020 - 22 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

67762-55-4

Test material form:

liquid

Details on test material:

- Description: Dark brown, opaque liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Afton Chemical Corporation 500 Spring Street Richmond, VA 23219 United States (Sponsor)

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The Sprague Dawley rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 7 weeks old
- Weight at study initiation: between 227 and 302 g (males), between 169 and 214 g (females)
- Fasting period before study: at least 8 hours (no more than 24 hours) before sample collection for clinical chemistry and overnight before the scheduled necropsies.
- Housing: Group housing of 2–4 animals of the same sex and same dosing group, in solid-bottom cages containing appropriate bedding material (Bed-O- Cobs® or other suitable material)
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet 5002, ad libitum (except during designated periods of fasting and during the conduct of the functional observational battery (FOB) and motor activity (MA) assessments)
- Water (e.g. ad libitum): tap water, treated by reverse osmosis and ultraviolet irradiation, ad libitum (except during designated procedures)
- Acclimation period: at least 7 days

DETAILS OF FOOD AND WATER QUALITY: Results of analysis for nutritional components and environmental contaminants are provided by the supplier and are on file at the Testing Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study. Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility. It is considered that there are no known contaminants in the water that would interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 26°C (target)
- Humidity (%): 30% to 70% (target)
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 5 June 2020 To: 1 October 2020

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of exposure was selected because this is a potential route of human exposure.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The prepared formulations were not adjusted for purity. The frequency of preparation was at least weekly.

VEHICLE
- Concentration in vehicle: 0, 10, 30, and 100 mg/mL
- Dose volume: 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for concentration and homogeneity analyses. Dose analysis results were verified prior to dose administration at each sampling interval. Analyses were performed by a gas chromatography method with flame ionization detection using a validated analytical procedure. Acceptance Criteria for concentration were mean sample concentration within 100% ± 15% of theoretical concentration; Individual sample concentration of ± 20%. Acceptance Criteria for homogeneity were relative standard deviation (RSD) of concentrations of smaller or equal 10% for each group.
The Sponsor provided data to demonstrate that the test substance was stable in the vehicle when prepared under the same conditions at concentrations bracketing those used in the present study and were homogeneous for at least 8 days at room temperature (D’Sa, 2020).

Duration of treatment / exposure:

90 consecutive days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 per sex per dose in the main study; 5 per sex in the vehicle control and the high dose group of the recovery study

Control animals:

yes, concurrent vehicle

Details on study design:

RECOVERY PHASE:
Recovery groups of 5 animals per sex in the vehicle control and high dose group were included to detect persistence or recovery over a period of 28 days after the last day of exposure. The first day of the recovery period was Day 91.

DOSE RATIONALE:
The dose levels were selected based on the results from a previously performed Combined 28-Day Repeated Dose Toxicity Study with the Reproductive/Developmental Toxicity Screening Test (OECD 422). The study was performed at WIL Research Europe B.V. (Project 500167), and the no-observed-adverse-effect level (NOAEL) for the study for systemic, reproductive, and developmental effects was 1000 mg/kg bw/day. The dose levels in this study were 100, 300, and 1000 mg/kg bw/day in an attempt to produce graded responses to the test substance. It was anticipated that the high-dose level would show drug-specific effects but not produce an incidence of fatalities that would prevent a meaningful evaluation. The lower dose levels were selected at intervals that were predicted to be narrow enough to reveal any dose-related trends. Though priority was given to detecting a dose-related trend, it was expected that the low-dose level would be a NOAEL.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 1–3 hours post-dosing, and at least once daily on non-dosing/recovery days.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On Day 1 (prior to dosing), weekly (± 2 days) during the study period, and on the day of the scheduled necropsy.

BODY WEIGHT: Yes
- Time schedule: On Day 1 (prior to dosing), weekly (± 2 days) during the study period, on the day prior to the scheduled necropsy, and on the day of the scheduled necropsy.

FOOD CONSUMPTION: Yes
- Time schedule: Weekly (± 2 days) throughout the study period, beginning on Day 1.

ESTROUS CYCLE DETERMINATION: Yes
- Time schedule: On the day of the scheduled necropsy, vaginal lavages were performed to determine the stage of estrous of all female animals in the main and recovery study.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once during the pretreatment period (included unused replacement animals), near the end of the dosing period (Day 89).
- Dose groups that were examined: All main and recovery study animals.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB): Yes
- Time schedule for examinations: During the second to last or final week of test substance administration and during the final week of the recovery period.
- Dose groups that were examined: All main and recovery study animals.
- Parameters examined are listed in tables 8-13 of the study report (p. 24-25).

MOTOR ACTIVITY: Yes
- Time schedule for examinations: During the second to last or final week of test substance administration and during the final week of the recovery period
- Dose groups that were examined: All main and recovery study animals.
- Procedure: Testing of treatment groups was balanced across the day of testing and executed by appropriately trained technicians. The test session was 60 minutes, consisted of twelve 5-minute intervals, and was reported in six 10-minute intervals. Motor activity evaluated total and ambulatory activity counts obtained for the 60-minute test session.
All motor activity sessions were performed in a sound-attenuated room equipped with a white-noise generation system to minimize environmental variations in the test conditions.

HAEMATOLOGY AND COAGULATION: Yes
- Time schedule for collection of blood: On the day of the scheduled necropsy
- Anaesthetic used for blood collection: For hematology: Isoflurane (if necessary); For coagulation: No
- Animals fasted: No
- How many animals: All main and recovery study animals.
- Parameters examined are listed in tables 15 and 16 of the study report (p. 26-27).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of the scheduled necropsy
- Anaesthetic used for blood collection: Isoflurane (if necessary)
- Animals fasted: Yes, at least 8 hours (no more than 24 hours)
- How many animals: All main and recovery study animals.
- Parameters examined are listed in table 17 of the study report (p. 27).

URINALYSIS: Yes
- Time schedule for collection of urine: Overnight of the day of the scheduled necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- How many animals: All main and recovery study animals.
- Parameters examined are listed in table 18 of the study report (p. 27).

THYROID HORMONES: Yes
- Time of blood sample collection: On the day of terminal necropsy (6:00-9:00)
- Anaesthetic used for blood collection: Isoflurane (if necessary)
- Animals fasted: Not specified
- How many animals: All main and recovery study animals.
- Parameters checked: T3 and T4 hormone, and thyroid stimulating hormone (TSH)

Sacrifice and pathology:

ORGAN WEIGHTS: Yes (see table 21 of the study report (p.30))

GROSS PATHOLOGY: Yes (see table 22 of the study report (p.31))

HISTOPATHOLOGY: According to guideline

Statistics:

The following statistical methods were used to analyze the data:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and or 5% levels. The pairwise comparisons are of the exposed groups (Group 2, 3, and 4) against the control group (Group 1).

Analysis were performed only with 3 or more observations, according to the following statistical method:
Parametric/ Non-parametric (Provantis): Body Weight, Body Weight Gains, Food Consumption, Hematology Variables, Coagulation Variables, Clinical Chemistry Variables, Urinalysis Variables. Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
Parametric / Non-parametric (WTDMSTM and/or SAS): Functional Observational Battery Data, Motor Activity Data, TSH, T3 and T4. The groups were compared using an overall one-way ANOVA F-test. In case the overall F-test was significant, then pairwise comparisons was conducted using Dunnett’s test.

The motor activity ambulatory and total counts data was statistically analyzed. For the purpose of the statistical analysis, each session consisted of 6 levels of 10 consecutive minutes for the factor Time. For each session and each sex the motor activity data were statistically analyzed separately using a repeated measures analysis of variance model, with Group, Time and their interaction, as fixed factors.
The corrected Akaike’s Information Criterion were used in order to select the homoscedastic model with the following covariance structures: Compound Symmetry, Heterogeneous Compound Symmetry, First-Order Autoregressive, and Heterogeneous First-Order Autoregres.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related clinical observations. All clinical observations in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control group male died on Day 74. The animal had no gross or microscopic findings that could explain the cause of death. The microscopic changes in the lung (accumulation of plant material in airways) was not associated with any degenerative or inflammatory changes, and was considered an agonal finding.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Some statistically significant differences were observed when the control and test substance-treated groups were compared. These differences were attributed to biological variability and were not considered related to test substance administration due to lack of a dose-response trend.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Some statistically significant differences were observed when the control and test substance-treated groups were compared. These differences were not considered test substance-related, and were instead attributed to normal variation.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated groups. All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Regarding coagulation, test substance-related and statistically significantly prolonged mean activated partial thromboplastin time and prothrombin time were noted in the 1000 mg/kg bw/day group males on Day 91, compared to the control group. These differences were not present at the end of the recovery period. There were no other test substance-related effects on coagulation parameters.
Hematology parameters were unaffected by test substance administration.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical chemistry parameters were unaffected by test substance administration. Any differences, regardless of statistical significance, were not considered test substance-related based on their general overlap of individual mean values with the range of concurrent control, and/or were of a magnitude of change commonly observed in rats under similar study conditions.

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid hormone parameters (T3, T4, hormones and TSH) were unaffected by test substance administration.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis parameters were unaffected by test substance administration. Any differences in urine parameters were not considered test substance-related based on the lack of a dose response for that parameter and/or the general overlap of individual mean values with the concurrent control values.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly lower rearing (males) and higher time to first step (females) were observed in the 1000 mg/kg bw/day group compared to the control group at the Week 18 evaluation, but were not considered test substance-related.
There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the Week 13 or Week 18 evaluations regarding: home cage, handling, sensory, neuromuscular and physiological observations.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean liver (males and females) and thyroid/parathyroid gland (males only) weights were higher in the 1000 mg/kg bw/day group compared to the control group (see Table 1 in the section "Any other information on results incl. tables"). The higher mean liver (males and females) and thyroid/parathryoid gland (males only) organ weight changes were minimal and did not have any microscopic correlates.
No other test substance-related organ weight changes were noted. There were isolated organ weight values that were statistically different from their respective controls. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test substance-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and therefore, were considered unrelated to administration of the test substance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test substance-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test substance.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No test substance-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test substance.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

ESTROUS CYCLE:
Estrous cycle was unaffected by test substance administration.

MOTOR ACTIVITY:
Regarding motor activity patterns, combined ambulatory counts were statistically significantly lower for 300 mg/kg bw/day females at Week 13. However, this change was not considered test substance-related due to the lack of a dose response and no difference in total counts. There were no other statistically significant changes for the test substance-treated males and females when compared to the control group at the Week 13 and Week 18 evaluations.

RECOVERY ANIMALS (DAY 119):
Test substance-related higher mean liver and thyroid/parathryoid gland weights were not present at the end of recovery and there were no other test substance-related organ weight changes noted. There were isolated organ weight values that were statistically different from their respective controls. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and unrelated to administration of the test substance.
No test substance-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test substance.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test item related mortality or adverse findings

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Analysis of formulations:

No test item was detected in the control group formulation.

The mean accuracies for the concentrations in the formulations of the 100, 300 and 1000 mg/kg bw/day groups (Week 1, Week 6 and Week 13 formulations) were between 106% to 112% and therefore in agreement with the target concentrations for suspensions (85% to 115%).

The coefficient of variation for the formulations of the 100 and 1000 mg/kg bw/day groups were between 1.7 and 2.7% and therefore the formulations were considered homogeneous (i.e. coefficient of variation ≤ 10%).

Mean liver and thyroid/parathyroid gland weights are presented in Table 1. See section "Organ weight findings including organ / body weight ratios" for description of the observations.

Table 1: Summary of Organ Weight Data – Terminal Euthanasia (Day 91)^a 

	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (mg/kg bw/day)	0	100	300	1000	0	100	300	1000
No. Animals per Group	10	10	10	10	10	10	10	10
Liver (No. Weighed)	(10)	(10)	(10)	(10)	(10)	(10)	(10)	(10)
Absolute value (g)	19.27	20.36	19.32	19.99	9.46	9.38	9.52	10.61*
% of body weight	2.92	3.13	3.14	3.26*	3.15	3.05	3.34	3.35
% of brain weight	842.22	899.74	842.22	885.69	453.32	450.14	468.69	518.81**
Thyroid/Parathyroid (No. Weighed)	(10)	(10)	(10)	(10)	(10)	(10)	(10)	(10)
Absolute value (g)	0.021	0.021	0.024	0.025	0.017	0.019	0.018	0.018
% of body weight	0.0033	0.033	0.0039	0.0041*	0.0055	0.0061	0.0063	0.0057
% of brain weight	0.93	0.94	1.03	1.10	0.79	0.90	0.88	0.87

^a Organ weight values rounded to the appropriate decimal place to account for the size of the organ (values presented to all decimal places in Provantis tables).

* = Statistically significantly different from the control group at ≤ 0.05 using Dunnett’s test. ** = Statistically significantly different from the control group at ≤ 0.01 using Dunnett’s test. BOLD = Alkenes, C15-18 alpha-, Sulfurized-related.

Applicant's summary and conclusion

Conclusions:

In a 90-day oral repeated dose toxicity study with rats, conducted according to OECD/EC guidelines and GLP principles, the NOAEL was found to be >1000 mg/kg bw/day, based on absence of adverse effects at the highest dose level tested.

Executive summary:

A 90-day oral repeated dose toxicity study was conducted with Alkenes, C15-18 alpha-, sulfurized (in corn oil) according to OECD/EC guidelines and in accordance with GLP principles. Male and female rats were exposed to 100, 300 and 1000 mg test item/kg bw/day via oral gavage. Chemical analysis of the formulations confirmed correct concentrations and stability.

One control group male died on Day 74. The animal had no gross or microscopic findings that could explain the cause of death. The microscopic changes in the lung (accumulation of plant material in airways) was not associated with any degenerative or inflammatory changes and was considered an agonal finding.

There were no test substance-related clinical observations or effects on body weight, food consumption, hematology, clinical chemistry, urinalysis, estrous cycle, thyroid hormones, or FOB and motor activity. There were no test substance-related ophthalmic, macroscopic, or microscopic findings.

Test substance-related prolonged activated partial thromboplastin time and prothrombin time were noted in the 1000 mg/kg bw/day group males on Day 91. These differences were not present at the end of the recovery period.

Test substance-related effects on organ weights included higher liver (males and females) and thyroid gland (males only) weights in the 1000 mg/kg bw/day group. These differences were not present at the end of the recovery period.

In conclusion, administration of Alkenes, C15-18 alpha-, sulfurized once daily by oral gavage to Crl:CD(SD) rats at dose levels of 100, 300, and 1000 mg/kg bw/day for 90 days was tolerated at all doses with no test-item related mortality or adverse findings. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg bw/day.