Paraffin waxes and Hydrocarbon waxes, oxidized, lithium salts

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 2016 - January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hydrocarbon waxes (petroleum), oxidized

EC Number:

265-205-1

EC Name:

Hydrocarbon waxes (petroleum), oxidized

Cas Number:

64743-00-6

Molecular formula:

too complex

IUPAC Name:

Hydrocarbon waxes (petroleum), oxidized

Test material form:

solid: flakes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: A28-13-C2043 and A28-14-C2004
- Expiration date of the lot/batch: 01 Oct 2023 and 05 June 2024
- Purity test date: See certificate of analysis

- Purity test date: See certificate of analysis

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature protected from light under nitrogen without contact with iron (ferrum)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietroal Natisone (UD), Italy.
- Age at study initiation: 27-29 days
- Weight at study initiation: 84-102g
- Fasting period before study: no
- Housing: 5 of one sex in clear polysulphone solid bottomed cages, nesting material was provided inside suitable bedding bags (nesting material was changed at least twice a week)
- Diet: commercially available laboratory rodent diet (4 RF 21, Mucedoly S.r.l., Settimo Milanese (MI), Italy) ad libitum
- Water: drinking water ad libitum
Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2015-15-07 (allocation of animals) To: 2095-21-19 (last day of necropsy)

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: powdered rodent diet

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly intervals
- Mixing appropriate amounts with (Type of food): The test item A28 was formulated, using powdered diet, by initial preparation of a pre-mix followed by dilution with further quantities of diet and mixing. The formulations were prepared separately for each group

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated by the Analytical Chemistry Department at RTC according to RTC SOPs (external LAUS Study No. VB14100602G926).
In RTC Study No. A1763, a 8 hour and 10 day stability at room temperature was assessed in the range from 100 to 20000 ppm.

Duration of treatment / exposure:

13 consecutive weeks followed by a recovery period of 4 weeks (control and high dose group), untreated diet was given to animals of the recovery phase during the recovery period

Frequency of treatment:

all animals in each group had access to their appropriate diets 7 days a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

actual dose received males: 109mg/kg bw/day (see table 2 below)
acutal dose received females: 112 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

actual dose received males: 327 mg/kg bw/day (see table 2 below)
actual dose received females: 308 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

actual dose received males: 1060 mg/kg bw/day (see table 2 below)
actual dose received females: 1047 mg/kg bw/day

No. of animals per sex per dose:

10, 5 additional animals per sex for the satellite groups (control and high dose group)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels of 20, 100 and 500 mg/kg/day were selected in consultation with the Sponsor based on information from a preliminary not GLP compliant study.
- Post-exposure recovery period in satellite groups: 4 weeks
- Study design of preliminary study: The toxicity and palatability of A28, when administered daily in powdered rodent diet to rats, was investigated over a period of 2 consecutive weeks. Three groups, each of 4 male and 4 female Sprague Dawley SD rats, received the test item in the diet (Mucedola, 4RF21) at the fixed inclusion levels of 200, 1000 and 5000 ppm (in terms of test item as supplied, corresponding to nominal dose levels of 20, 100 and 500 mg/kg bw/day) for 2 consecutive weeks. A fourth similarly constituted group received the untreated diet and acted as a control. The following investigations were performed: clinical signs, body weight, food consumption, organ weights and macroscopic observations at necropsy.
- Results of preliminary study: No mortality occurred and no clinical signs were seen during the study. No significant body weight differences were noted in treated animals when compared to controls. Food consumption was strongly reduced in the first day of the study in the high dose group but become comparable to the control group in the few subsequent days. In the low and mid-dose groups the food consumption of both sexes was comparable to the control group. The calculated mean daily achieved dosages for the 2 week period of treatment were 21, 108, 535 mg/kg body weight/day for the males and 23, 116 and 500 mg/kg body weight/day for the females, in terms of test item as supplied. A slight but not statistically significant increase was noted in the absolute and relative liver weights of the high dose animals. No other significant variations were observed in the weighed organs in all treatment groups. Treatment-related changes were observed in the liver of all males treated at 500 mg/kg/day. Such changes consisted of swollen or enlarged liver. Swollen liver was also observed in one control male. The remaining sporadic lesions, reported in control and treated animals of both sexes, were considered to be an expression of spontaneous and/or incidental pathology, seen in this species and age of untreated animals. On the basis of the results obtained in this preliminary study, it can be concluded that the test item, O-T-BUTYLPHENOL, when mixed with powdered rodent diet at the inclusion levels of 200, 1000 and 5000 ppm in terms of test item as supplied (corresponding to nominal dose levels of 20, 100 and 500 mg/kg bw/day), was well accepted and tolerated by Sprague Dawley SD rats.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (morality, clinical signs)
- Time schedule:
Mortality check: early each working day and again in the afternoon, at weekends and Public Holidays the second check were done at approx. mid-day
Clinical signs: daily, once before commencement of treatment and once during the study (performed at the same time interval each day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, once before commencement of treatment and once a week thereafter
- Neurotoxicity assessment: animals were examined in an open arena, observed parameters: changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereot ypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerec tion, unusual respiratory pattern), changes in fur, skin, eyes, mucous membranes, occurrences of se cretions and excretions.

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, the weight of food consumed by each cage of rats was recorded
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, group mean daily intake per rat was calculated.
Calculation was carried out according to the following formula: achieved dosage (mg/kg/day) = ppm x mean daily food consumption (g) / mean period body weight (g)

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes, by means of an ophthalmoscope, and by a slitlamp microscope after the instillation of 0.5% Tropicamide
- Time schedule for examinations: prior to commencement of treatment, re-examination during week 13 of treatment
- Dose groups that were examined: prior treatment: all groups, re-examination: control and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy in week 13 of treatment and at the end of week 4 of the recovery period
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: 10 each sex from all groups during week 13, from all surviving animals of the recovery groups (control, high dose group)
- Parameters examined: haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count (neutrophils, lymphocytes, eosinophils, basophils, monocytes, large unstained cells), platelets, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy in week 13 of treatment and at the end of week 4 of the recovery period
- Animals fasted: Yes
- How many animals: 10 each sex from all groups during week 13, from all surviving animals of the recovery groups (control, high dose group), same animals as for haematology used
- Parameters examined: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, urea, creatinine, glucose, trigycerides, phosphorus, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G ratio, sodium, potassium, calcium, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: overnight prio to necropsy in week 13 of treatment and at the end of week 4 of the recovery period
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes and water bottles were removed, each animal received approx. 10 mL/kg bw of drinking water by gavage
- How many animals: 10 each sex from all groups during week 13, from all surviving animals of the recovery groups (control, high dose group), same animals as for haematology and clinical chemistry used
- Parameters examined: appearance, volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood, sediment (obtained from centrifugation at approx. 3000 rpm for 10 min, examined microscopically for: epithelial cells, leucocytes, erythrocytes, crystals, spermatozoa and precursors, other abnormal components)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during week 12 of treatment and once during week 4 of recovery
- Dose groups that were examined: all dose groups, all recovery groups
- Battery of functions tested: sensory activity (auditory, visual and propriocetive stimuli), grip strength, motor activity (measured over a period of 5 min/animal)

IMMUNOLOGY: No

OTHER: None

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1 below)
Samples of all tissues were fixed and preserved in 10% neutral buffered formalin (except eyes, testes and epididymides which were fixed in Modified Davidson's fluid and preserved in 70% ethanol)

HISTOPATHOLOGY: Yes (see table 1 below)
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin.
The examination was performed as detailed below:
- Tissues specified in table from all animals in the control and high dose groups killed at the end of the 13 weeks of treatment.
- All abnormalities in all main phase groups.

Statistics:

For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathology findings was carried out by means of a non-parametric Kolmogorov-Smirnov test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was slightly reduced (in some occasions also significantly, at statistical analysis) in the high dose males (-9% to -28%) from Week 5 to Week 13 of the treatment period. A slight reduction was also observed in the high dose females (-11% to -39%) at the end of the first week of study. These reductions were no longer present during the recovery period.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

I

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant fluctuations of some biochemical parameters were
recorded in treated animals, such as: decrease of glucose in males dosed at 300
in males receiving 1000 mg/kg/day (8%) and in females receiving 100 and
1000 mg/kg/day (11%), increase of bilirubin in females dosed at 300 and 1000
mg/kg/day (64% and 104%, respectively), increases of urea and creatinine in
females receiving 1000 mg/kg/day (13% and 12%, respectively) and increase
of albumin in all treated females (approximately 5%). Due to the low severity
and/or the absence of dose-relation, changes were not considered adverse

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A minor range of changes as seen in control and treated animals, having a relatively comparable incidence, or are characteristically seen in untreated Sprague Dawley rats of the same age.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest level tested

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 2: Achieved dosage

 

Expected dosage [mg/kg bw/day]	Achieved dosage weeks 1 - 13: mean ± SD [mg/kg bw/day]
	Males	Females
100	109 ±  1.1	112 ± 3.1
300	327 ± 8.9	308 ± 7.2
1000	1060 ± 78	1047 ± 30.0

Applicant's summary and conclusion

Conclusions:

No treatment-related changes, which could be considered adverse, were observed in male and female rats following dosing with A28 when administered in powdered rodent diet for 13 consecutive weeks at the approximate dosages of 100, 300 and 1000 mg/kg/day (corresponding


to mean achieved dose levels of 109, 327 and 1060 mg/kg/day for the males and 112, 308 and 1047 mg/ kg/day for the females). Therefore, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 1000 mg/kg/day.

Executive summary:

The oral toxicity of A28, when administered daily in powdered rodent diet to Sprague Dawley rats for 13 consecutive weeks at approximate dose levels of 100, 300 and 1000 mg/kg/day and recovery from any treatment-related effect during a period of 4 weeks, were investigated in this study.

No mortality occurred and no treatment-related clinical signs were observed during the study. No signs of toxic or neurotoxic effects, which could be considered adverse, were seen during the in vivo phase of the study. No lesions were recorded at ophthalmological examination. No changes of toxicological relevance were observed in body weight and food consumption. Statistically significant fluctuations of some clinical chemistry parameters were recorded in treated animals. All these changes were no longer observed at the end of recovery. Due to the low severity, the complete reversibility and/or the absence of dose-relation, all the above changes were not considered adverse. The absolute and/or relative weights of the liver and kidneys showed slight increases which, due to the low magnitude, complete reversibility and the absence of a support from the histopathological examination, were not considered adverse. No treatment-related findings were reported at post mortem macroscopic observations and histopathological examination.

In conclusion, no treatment-related changes, which could be considered adverse, were observed in male and female rats following dosing with A28, when administered in powdered rodent diet for 13 consecutive weeks at the approximate dosages of 100, 300 and 1000 mg/kg/day (corresponding to mean achieved dose levels of 109, 327 and 1060 mg/kg/day for the males and 112, 308 and 1047 mg/ kg/day for the females).

Therefore, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 1000 mg/kg/day.