Registration Dossier

Administrative data

Description of key information

An assessment of sub-acute and sub-chronic toxicity.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
other: Read across to equivalent substance.
Remarks:
A full justification for the read across applied for this substance is contained within Section 1.4, reference " Explanation of NovaSpec Base Oil - FINAL".
Adequacy of study:
key study
Study period:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This literature paper is classified as K2, reliable with restrictions as it was conducted to a recognised guideline similar to guideline study OECD TG 408, on a known structural analogue, and is a well documented report.
Justification for type of information:
A full justification for the read across applied for this substance is contained within Section 1.4, reference " Explanation of NovaSpec Base Oil - FINAL".
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
not applicable
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
other: F344 and Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS - Source: Charles River Laboratories, Kingston, New York - Age at study initiation: 6 weeks - Weight at study initiation: F344:84 to 104 grams; Sprague-Dawley: 132 to 166 grams - Housing: Individually - Diet (e.g. ad libitum): ad libitum - Water (e.g. ad libitum): ad libitum - Acclimation period: 14 days ENVIRONMENTAL CONDITIONS - Temperature (°C): 20 to 24 - Humidity (%): 40% to 70% - Air changes (per hr): Not reported - Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light IN-LIFE DATES: Not reported
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: DIET PREPARATION - Rate of preparation of diet (frequency): Not reported - Mixing appropriate amounts with Purina certified rodent chow, Purina mills. - Storage temperature of food: Not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diet concentration verification was conducted on weeks 1, 5, 9, and 13. All concentrations were stated to be between 85% and 110% of the nominal concentration. There were also periodic measurements for stability and homogeneity.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Continuous in feed
Remarks:
Doses / Concentrations:0, 2000, 20,000 ppm (0, 0.2, 2% respectively) Basis:nominal in diet
No. of animals per sex per dose:
Fifteen animals per treatment group, an additional 10 F344 rats in the control and high-dose group were sacrificed on day 30 or 61
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Doses were selected based on previous publications. - Rationale for animal assignment (if not random): Not reported - Rationale for selecting satellite groups: Not reported
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes - Time schedule: daily - Cage side observation details were not provided. DETAILED CLINICAL OBSERVATIONS: No data BODY WEIGHT: Yes - Time schedule for examinations: weekly FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): - Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes - Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data FOOD EFFICIENCY: - Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No OPHTHALMOSCOPIC EXAMINATION: No HAEMATOLOGY: Yes - Time schedule for collection of blood: at scheduled sacrifice - Anaesthetic used for blood collection: Yes (methoxyflurane) - Animals fasted: Yes - How many animals: Ten per group - Parameters checked in table 1 were examined. CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: at scheduled sacrifice - Animals fasted: Yes - How many animals:Ten per group - Parameters checked in table 2 were examined. URINALYSIS: No NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes HISTOPATHOLOGY: Yes, although it is stated that an extensive list of tissues were preserved, it states that only the liver, mesenteric lymph nodes, and gross lesions were processed for histology.
Other examinations:
The heart, liver, kidneys, ovaries, and spleen were weighed.
Statistics:
For parametric data, a one way analysis of variance with F distribution followed by a Dunnett's test were used. A standard linear regression analysis was also performed. For nonparametric data, a Kruskal-Wallis test followed by Dunn's summed rank test were used. A Jonckheere's test for monotonic trends was used to test for a dose response.
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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: There were no clinical signs in either rat strain at any dose level during the study. There were no effects on mortality. BODY WEIGHT AND WEIGHT GAIN: Apart from a transient decrease in body weight for one day at one dose level in one strain of rat, there were no effects on growth rate. FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): There were no effects on food consumption. There were no data on the compound intake. HAEMATOLOGY: Hematological effects were only noted in F344 rats and included an increase in white cell count. At day 30 this was observed in the 2.0% group, but by 90 days was seen in both dose groups in a dose-related manner with a significant increase observed in the high-dose group (21.1%, 24.5%, and 36.1% in control, 0.2% and 2.0% groups). CLINICAL CHEMISTRY: There were also marginal differences in some serum chemistry values of F 344 rats compared to controls, but these changes were not considered clinically significant. ORGAN WEIGHTS: The absolute and relative liver weights of the F344 rats in the 2% group were increased by 1.2- and 1.3-fold respectively and in the 0.2% group both absolute and relative liver weights were increased 1.1-fold. The mesenteric lymph nodes (absolute and relative) were increased in the 2% group by 1.4 to 3.7 fold depending on the time of sacrifice. There were no changes in organ weight in the Sprague-Dawley rat. HISTOPATHOLOGY: Histopathological changes in the livers of the treated F344 rats consisted of the occurrence of an increased incidence of microgranuloma. These were not present at day 30, but were present in more than 80% of the animals in the 2% dose group after 61 days. By 90 days microgranulomas were also observed in the 0.2% dose group (incidence not stated in the publication). Central necrosis and/or the presence of Langhan's type cells were seen in occasional microgranuloma. The incidence and severity were both dose and temporally-related as the appearance of granuloma occurred at 61 days in the high-dose group and was present in both dose groups at 92 days. Histiocytosis occurred in the mesenteric lymph nodes of the F 344 rats in the 2% dose group after 30 days. Microgranulomas were observed at 61 and 92 days in the 2% and also in the 0.2% groups at 92 days. The incidence and severity of the microgranulomas were increased in a dose related manner. Histopathological examination of the livers of the Sprague Dawley rats revealed that in contrast to the F 344 rats there were no microgranulomas in either dose group, but there was a slightly increased incidence of minimal multifocal chronic inflammation in the 2% dose group. No histological changes were observed in the mesenteric lymph node of the Sprague Dawley rats at either dose level. OTHER FINDINGS: There was a dose and strain related increase in mineral hydrocarbon in the liver and mesenteric lymph nodes.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 20 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: ≥ approx. 1600 mg/kg bw/day; overall effect in Sprague-Dawley rats
Key result
Dose descriptor:
LOEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: approx. 160 mg/kg bw/day; histopathology in F344 rats
Critical effects observed:
not specified
Conclusions:
the F344 rats and the LOAEL is 2000 ppm, based on the histopathology in the liver and mesenteric lymph nodes accompanied by changes in the weight of those organs. The NOAEL in the Sprague-Dawley rat is greater than or equal to 20,000 ppm, based on the lack of any toxic effects at the highest dose.However, in the paper “ORAL TOXICITY OF MINERAL OILS AND RELATED COMPOUNDS - A review” by Prof. Atte von Wright, University of Eastern Finland, a critical review of this data is undertaken via comparative assessment of other similar studies utilising Fischer 344 and other species. The following are evaluated:Baldwin et al. (1992) - 90 day feeding studies Smith et al. (1996) - 90 day feeding studiesTrimmer et al. (2004) - chronic (two year) oral toxicity studyIn the review of Miller et al. (1996), a discrepancy with the effects observed in Fischer 344 rats and the either non‐existent or relatively insignificant effects of mineral oils in other species and strains was pointed out. There are several historical studies performed with either paraffinic or naphthenic oils in beagle dogs, Sprague‐Dawley rats and Long‐Evans rats, in which no significant microscopic anomalies in liver, kidneys, spleen or mesenteric lymphnodes were detected and no mineral oil deposits in the tissues were observed (Shubik et al, 1962; Smith et al, 1995; McKee et al., 1987; Firriolo et al. 1995). The authors also point out that the lipogranulomas of the liver observed in human autopsies are considered clinically insignificant, while those observed in Fischer 344 rats can be associated with inflammatory and necrotic lesions. The authors further suggest that the observed effects reflect rat strain specific differences in the responses to mineral oils.Finally, the lymph nodal and hepatic lesions observed in rats orally exposed to mineral oils were further assessed in a special pathology workshop held in 2001 at the Fraunhofer Institute of Toxicology (Hannover, Germany) (Carlton et al. 2001). According to the final conclusion of the workshop: “The available data suggest that the granumalotous lesions experimentally induced by MHC‐feeding, particularly in the liver of F344 rats, are exaggerated toxicological responses peculiar to rats.”Therefore, in accordance with the CLP Regulation (EC No 1272/2008), specifically section 3.9.2.9.9 which states that: “Thus it is feasible that a specific profile of toxicity occurs in repeat-dose animal studies at a dose/concentration below the guidance value, such as < 100 mg/kg bw/day by the oral route, however the nature of the effect, such as nephrotoxicity seen only in male rats of a particular strain known to be susceptible to this effect may result in the decision not to classify. “ It is proposed that classification and labelling is not considered attributable to these effects. On the basis of the effects noted in the study, it is considered that these are species specific. The effects noted appear to be specific to the Fischer strain and this is adequately proven.
Executive summary:

A full justification for the read across applied for this substance is contained within Section 1.4, reference " Explanation of NovaSpec Base Oil - FINAL". This document contains a full explanation of the manufacturing route for the substance and discussion on why this substance should be considered as a UVCB white oil analogous to CAS 8042-47-5. Physical properties of the crude reaction product and the different product grades (or distillate fractions) derive from the general chemical structure characteristics (linear-branched alkanes with characteristic branching length and position). Specific unique chemical structures are not isolated in any process step and due to the tens of thousands of isomers and the high degree and variable nature branching and chain length, the base oil bulk properties are the result of the average structure characteristics. The unique viscosity grades each contain all the same typical chemical structures and predominantly overlapping Molecular Weight distributions. The unique viscosity and volatility characteristics of each viscosity grade derive from the boiling point distribution, a result of the short-path distillation (wiped film evaporation). This is identical to the production of petroleum derived white oils.

In order to avoid the requirement to further test a substance that is a derived white oil on animals, a weight of evidence approach is utilised to provide evidence of lack of repeated dose toxicity. This is via the utilisation of various literature papers.

In this subchronic study, groups of 15 female rats of two strains (F344 and Sprague-Dawley) were fed diets containing 0, 2000 or 20,000 (0, 0.2 or 2.0% respectively) P15(H) white oil. Ten animals were sacrificed after 90 days for toxicological evaluation and the remaining 5 rats were sacrificed after 91 days for a tissue analysis for mineral hydrocarbon content. In addition 20 extra female F-344 rats were fed diet containing 0 or 2% white oil. Ten of these animals were sacrificed after 30 days treatment and the remaining ten animals after 61 days treatment. These extra animals were included to follow the time course of the development of any lesions that developed. All animals were observed daily and body weights and food consumption were recorded weekly. At each scheduled sacrifice blood samples were taken for a wide range of haematological and serum chemistry determinations.

There were no clinical signs in either rat strain at any dose level during the study. There were no effects on mortality. Apart from a transient decrease in body weight for one day at one dose level in one strain of rat, there were no effects on growth rate. There were no effects on food consumption. There were no data on the compound intake. Hematological effects were only noted in F344 rats and included an increase in white cell count. At day 30 this was observed in the 2.0% group, but by 90 days was seen in both dose groups in a dose-related manner with a significant increase observed in the high-dose group (21.1%, 24.5%, and 36.1% in control, 0.2% and 2.0% groups). There were also marginal differences in some serum chemistry values of F 344 rats compared to controls, but these changes were not considered clinically significant. The absolute and relative liver weights of the F344 rats in the 2% group were increased by 1.2- and 1.3-fold respectively and in the 0.2% group both absolute and relative liver weights were increased 1.1-fold. The mesenteric lymph nodes (absolute and relative) were increased in the 2% group by 1.4 to 3.7 fold depending on the time of sacrifice. There were no changes in organ weight in the Sprague-Dawley rat. Histopathological changes in the livers of the treated F344 rats consisted of the occurrence of an increased incidence of microgranuloma. These were not present at day 30, but were present in more than 80% of the animals in the 2% dose group after 61 days. By 90 days microgranulomas were also observed in the 0.2% dose group (incidence not stated in the publication). Central necrosis and/or the presence of Langhan's type cells were seen in occasional microgranuloma. The incidence and severity were both dose and temporally-related as the appearance of granuloma occurred at 61 days in the high-dose group and was present in both dose groups at 92 days. Histiocytosis occurred in the mesenteric lymph nodes of the F 344 rats in the 2% dose group after 30 days. Microgranulomas were observed at 61 and 92 days in the 2% and also in the 0.2% groups at 92 days. The incidence and severity of the microgranulomas were increased in a dose related manner. Histopathological examination of the livers of the Sprague Dawley rats revealed that in contrast to the F 344 rats there were no microgranulomas in either dose group, but there was a slightly increased incidence of minimal multifocal chronic inflammation in the 2% dose group. No histological changes were observed in the mesenteric lymph node of the Sprague Dawley rats at either dose level. There was a dose and strain related increase in mineral hydrocarbon in the liver and mesenteric lymph nodes.

White mineral oil determined to be more toxic to F344 rats than to Sprague-Dawley rats. There was no NOAEL in the F344 rats and the LOAEL is 2000 ppm, based on the histopathology in the liver and mesenteric lymph nodes accompanied by changes in the weight of those organs. The NOAEL in the Sprague-Dawley rat is greater than or equal to 20,000 ppm, based on the lack of any toxic effects at the highest dose. However on the basis of the dose levels and associated effects, no classification is applicable.

However, in the paper “ORAL TOXICITY OF MINERAL OILS AND RELATED COMPOUNDS - A review” by Prof. Atte von Wright, University of Eastern Finland, a critical review of this data is undertaken via comparative assessment of other similar studies utilising Fischer 344 and other species. The following are evaluated:

 

Baldwin et al. (1992) - 90 day feeding studies

Smith et al. (1996) - 90 day feeding studies

Trimmer et al. (2004) - chronic (two year) oral toxicity study

 

In the review of Miller et al. (1996), a discrepancy with the effects observed in Fischer 344 rats and the either nonexistent or relatively insignificant effects of mineral oils in other species and strains was pointed out. There are several historical studies performed with either paraffinic or naphthenic oils in beagle dogs, SpragueDawley rats and LongEvans rats, in which no significant microscopic anomalies in liver, kidneys, spleen or mesenteric lymph nodes were detected and no mineral oil deposits in the tissues were observed (Shubik et al, 1962; Smith et al, 1995; McKee et al., 1987; Firriolo et al. 1995).

 

The authors also point out that the lipogranulomas of the liver observed in human autopsies are considered clinically insignificant, while those observed in Fischer 344 rats can be associated with inflammatory and necrotic lesions. The authors further suggest that the

observed effects reflect rat strain specific differences in the responses to mineral oils.

 

Finally, the lymph nodal and hepatic lesions observed in rats orally exposed to mineral oils were further assessed in a special pathology workshop held in 2001 at the Fraunhofer Institute of Toxicology (Hannover, Germany) (Carlton et al. 2001). According to the final conclusion of the workshop: “The available data suggest that the granumalotous lesions experimentally induced by MHCfeeding, particularly in the liver of F344 rats, are exaggerated toxicological responses peculiar to rats.”

Therefore, in accordance with the CLP Regulation (EC No 1272/2008), specifically section 3.9.2.9.9 which states that:

 

“Thus it is feasible that a specific profile of toxicity occurs in repeat-dose animal studies at a dose/concentration below the guidance value, such as < 100 mg/kg bw/day by the oral route, however the nature of the effect, such as nephrotoxicity seen only in male rats of a particular strain known to be susceptible to this effect may result in the decision not to classify. “

 

It is proposed that classification and labelling is not considered attributable to these effects.  On the basis of the effects noted in the study, it is considered that these are species specific. The effects noted appear to be specific to the Fischer strain and this is adequately proven.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
A full justification for the read across applied for this substance is contained within Section 1.4, reference " Explanation of NovaSpec Base Oil - FINAL".
Adequacy of study:
supporting study
Study period:
1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This literature paper is classified as K2, reliable with restrictions as it was conducted to a recognised guideline on a known structural analogue, and is a well documented report.
Justification for type of information:
A full justification for the read across applied for this substance is contained within Section 1.4, reference " Explanation of NovaSpec Base Oil - FINAL".
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
not specified
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
Fischer 344
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS - Source: Charles River, Manston, Kent - Age at study initiation: 6-8 weeks - Weight at study initiation: Not reported - Fasting period before study: no - Housing: Individual cages. - Diet (e.g. ad libitum): ad libitum - Water (e.g. ad libitum): ad libitum - Acclimation period: not reported ENVIRONMENTAL CONDITIONS - not reported
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Derived from naphthenic crudes of different viscosity specification. Oleum-treated White Oil was refined by oleum treatment following vacuum distillation of atmospheric reside. Hydro-treated White Oil was produced by hydrotreatment alone of the vacuum distillate of an atmospheric residue. DIET PREPARATION - Rate of preparation of diet (frequency): not reported - Mixing appropriate amounts with (Type of food): Powdered laboratory rat diet, LAD2 at concentrations raging from 10 to 2,000 ppm. - Storage temperature of food: Not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations and homogeneity of diets were always within 11% of nominal values. Analysis of dietary residues of hexane within 20 hours of mixing showed concentrations up to 251 ppm, which usually evaporated to below detectable limits (10 ppm) within 22 days.
Duration of treatment / exposure:
25 days
Frequency of treatment:
Ad libitum
Remarks:
Doses / Concentrations:500 and 10,000 ppm Basis:nominal in diet
No. of animals per sex per dose:
5 females per dose, per white oil
Control animals:
no
Details on study design:
Not reported
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: Weekly BODY WEIGHT: Yes - Time schedule for examinations: weekly FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): - Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No - Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No HAEMATOLOGY: Yes - Time schedule for collection of blood: terminal necropsy, tail vein blood was taken 7 days prior for while blood glucose analysis. - Anaesthetic used for blood collection: barbiturate anaesthesia. - Animals fasted: No - How many animals: all treated animals - Parameters checked were: cell volume, total erythroctye, leucocyte, and platelet count; hemoglobin concentration; mean corpuscular and mean platelet volume; mean corpuscular hemoglobin ; mean corpuscular hemoglobin concentration; and erythrocyte and platelet volume distribution width. Plasma variates were also analysed. CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: terminal necropsy - Animals fasted: No - How many animals: all animals URINALYSIS: No NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes HISTOPATHOLOGY: Yes
Other examinations:
In a 25 day feed study, 5 female Fischer-344 rats were given free access to diets containing 500 and 10,000 ppm of Oleum-treated White Oil or Hydrotreated White Oil. At terminal necropsy the livers and spleens of all rats were weighed and the livers, spleens and mesenteric lymph nodes were processed for routine histological examination. There were no haematological or serum chemistry evaluations in this study. Animals sacrificed after 25 days did not exhibit treatment-related histological changes in the mesenteric lymph nodes. No NOEL was reported, but was determined to be > 10,000 ppm based on the lack of treatment-related effects
Statistics:
Not reported
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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Animals sacrificed after 25 days did not exhibit treatment-related histological changes in the mesenteric lymph nodes. There were no mortalities and no adverse clinical signs associated with feeding either highly refined base oil. Any treatment-related effects evidenced by hematological, clinical chemical, and pathological changes were generally dose-related and the Oleum-treated highly refined base oil caused a greater pathological response than the Hydro-treated highly refined base oil
Key result
Dose descriptor:
NOEL
Effect level:
>= 10 000 ppm
Based on:
act. ingr.
Basis for effect level:
other: Noted absence of any treatment-related effects
Critical effects observed:
not specified
Conclusions:
Animals sacrificed after 25 days did not exhibit treatment-related histological changes in the mesenteric lymph nodes. There were no mortalities and no adverse clinical signs associated with feeding either highly refined base oil. Any treatment-related effects evidenced by hematological, clinical chemical, and pathological changes were generally dose-related and the Oleum-treated highly refined base oil caused a greater pathological response than the Hydro-treated highly refined base oil.
Executive summary:

A full justification for the read across applied for this substance is contained within Section 1.4, reference " Explanation of NovaSpec Base Oil - FINAL". This document contains a full explanation of the manufacturing route for the substance and discussion on why this substance should be considered as a UVCB white oil analogous to CAS 8042-47-5. Physical properties of the crude reaction product and the different product grades (or distillate fractions) derive from the general chemical structure characteristics (linear-branched alkanes with characteristic branching length and position). Specific unique chemical structures are not isolated in any process step and due to the tens of thousands of isomers and the high degree and variable nature branching and chain length, the base oil bulk properties are the result of the average structure characteristics. The unique viscosity grades each contain all the same typical chemical structures and predominantly overlapping Molecular Weight distributions. The unique viscosity and volatility characteristics of each viscosity grade derive from the boiling point distribution, a result of the short-path distillation (wiped film evaporation). This is identical to the production of petroleum derived white oils.

In order to avoid the requirement to further test a substance that is a derived white oil on animals, a weight of evidence approach is utilised to provide evidence of lack of repeated dose toxicity. This is via the utilisation of various literature papers.

In this sub-acute oral toxicity feeding study five female Fischer-344 rats were fed Oleum-treated White mineral oil and Hydro-treated highly refined base oil for 25 daysat dose levels 500 or 10,000 ppm. Animals sacrificed after 25 days did not exhibit treatment-related histological changes in the mesenteric lymph nodes. There were no mortalities and no adverse clinical signs associated with feeding either white oils. Any treatment-related effects evidenced by hematological, clinical chemical, and pathological changes were generally dose-related and the Oleum-treated highly refined base oil caused a greater pathological response than the Hydro-treated highly refined base oil.

A NOAEL was not reported, but was determined to be greater than or equal to 10,000 ppm (the highest dose tested) based on the lack of treatment-related effects.

This study received a Klimisch score of two and is classified as reliable with restriction because it is an acceptable and well-documented literature study report following basic scientific principles.

No classification is applicable.

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 October 2014 to 19 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed in accordance with OECD test guidelines in compliance with GLP and reported with a valid GLP certificate.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
The measured range of the temperature in the animal room was out of the targeted range (actual range: 20.1 – 25.1 °C, target range: 22±3°C) These deviations have no effect on the integrity or validity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Species and strain: Crl:WI rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony. Males and females originated from different units to avoid subsequent brother/sister matings.Hygienic level: Standard laboratory conditions during the study.Number of animals: 48 male, 48 female rats, 12 animals/sex/group, 4 groups (main animals); 10 male and 10 female rats, 5/sex/group (recovery group) and 12 males and 12 females rats in the positive control group for the micronucleus test).
Age of animals: Young adult rats, 10-11 weeks old at starting and 12-13 weeks at mating.
Body weight range: Males: 327 g – 478 g, Females: 207 g - 273 g;
Acclimation period: At least 5 days
Husbandry
Animal health: Only healthy animals were used for the test, as certified by the staff Veterinarian. Females were nulliparous and non-pregnant.
Room number: 508
Cage type: Type II and/or III polypropylene/polycarbonate
Bedding: Lignocel® and GRADE 5 type wooden chips were available to the animals.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.1 – 25.1 °C (target range 22±3°C)
Relative humidity: 31 – 64 % (target range 30-70%)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 5 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting)
.Food and water supply
Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.
Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36, Hungary). Food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Animal identification
Each parental animal (P Generation) was identified by a number unique within the study written with indelible ink on the tail and cross-referenced to the Animal Master File at CiToxLAB Hungary Ltd. The animal number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section.The boxes (cages) were marked by identity cards, with information at least about study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery. Boxes were arranged in such a way that possible effects due to cage placement were minimized.
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
Poly(ethylene glycol) 400+0.2% Polysorbate 80
Details on oral exposure:
The vehicle was selected by the Study Director in consultation with the Sponsor based on the trial formulations performed in the Central Dispensary of CiToxLAB Hungary Ltd.
Name: Poly(ethylene glycol) 400
Lot No.: BCBL5307V/BCBM8497V//BCBK 9981V
Manufacturer: Sigma-Aldrich Co.
Expiry Date: 31 January 2015/31 January 2016/January 2015
Storage: Room temperature
Name: 0.2% Polysorbate 80
Lot No.: BCBL9041V/BCBN3690V
Manufacturer: Sigma-Aldrich Co.
Expiry Date: 30 November 2014/31 July 2016
Storage: Room temperature under inert gas
Preparation of the vehicle
For the preparation of 100 g (approximately 100 mL) vehicle, 0.2 g Polysorbate 80 was weighed by analytical balance into 99.8 g PEG 400, and was stirred by a magnetic stirrer.The test item was formulated in the vehicle as a visibly stable homogenous formulation at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared for 7 days and kept in refrigerator pending dosage in the first 14 days of the study, and daily afterwards.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity was performed at the Test Site using a gas chromatographic method (internal ID at the test site: FPBSTUDY-113-MEAS) to determine the test item content.Top, middle and bottom duplicate samples were taken into 50 mL polypropylene centrifuge tubes from each concentrations at four occasions (Week 1, Week 3 two occasions, Week 3 and Week 6), one set to analyze (which was collected in duplicates) and one set as a back-up, if required for any confirmatory analyses from all dose groups. Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements. Further samples were taken in the same manner several times (from Day 20 until Day 27, from Day 29 until 36, and from Day 38 until Day 54 daily only from the high dose formulations for confirmatory purposes.Top, middle and bottom duplicate samples were taken into 50 mL polypropylene centrifuge tubes from test item formulations, one set to analyze and one set as a backup.Analysis of test item formulations was performed in the Fumoprep Kft. At the first instance, the first set of samples taken on Day 0, Day 17/18 and Day 37 were analyzed.For confirmatory purposes, back up samples were also measured later.The positive control material (cyclophosphamide) was dissolved in physiological saline (10 mg/mL) for the treatment. The solution was prepared just before the treatment. The test item solutions were given to assure the same dosing volumes in rat (2 mL/kg bw).No dose formulation analysis was performed from the positive control solutions.
Analytical Method
60 g/L stock solution
Approx. 600 mg Nova Spec base oil test item was weighed by analytical balance into 10 mL volumetric flask and filled up with heptane. The solution was mixed well. Preparation of the vehicle (0.2% Polysorbate in PEG)
Approx. 0.2g Polysorbate 80 was weighed by analytical balance into approx. 99.8 g PEG-400 and was mixed with magnetic stirrer.
QC sample preparation
500 mg/mL formula was prepared as follows: 250 mg of NovaSpec Base Oil was weighed with analytical precision into 50 mL centrifuge tube and 215 mg PEG-400 (0.2% Polysorbate) was added. The QC samples were prepared the samples.Preparation of the samples0.5-1.0 mL formula was received (weighed with analytical precision) in 50 mL centrifuge tubes. 10 mL n-Heptane was added at least 0.01 mg precision and 10 mL HPLC grade water was pipetted into the tube. The tubes were shaken manually and sonicate for 15 min. approx. 2 mL were centrifuged with 15000 rpm for 2 min. After the centrifuging the upper phase was diluted with n-Heptane into the dynamic range of the calibration (5x dilution for the 150 mg/mL and 10x dilution for the 500 mg/mL levels).
Chromatographic System
Column: HP-1; 5 m x 0.53 mm; 2.65 μm (CGC-01/03)
Mobile Phase: H2
Flow Rate: 15 mL/min
Split Ratio: 1:5
Detection: FID
Temperature: 160-300
Init Temp: 160°C (0 min) 10°C/min to 300°C (5 min)
Acquisition time: 20.00 min
Injection volume: 0.5 μL
Duration of treatment / exposure:
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period) and then euthanized and subjected to necropsy examination.Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (4 days post-partum dosing). The day of birth is defined as Day 0 post-partum.
Frequency of treatment:
Test item or vehicle control-treated groups Groups 1-4 Main animals were administered the dosing solutions daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe.
Remarks:
Doses / Concentrations:0, 100, 300, 1000 mg/kg bw/dayBasis:actual ingested
No. of animals per sex per dose:
Twelve male and 12 female Wistar rats/group (Main) were orally dosed with the test item
Additional 5 male and 5 female Wistar rats were assigned to a Control and High dose recovery Group
Control animals:
yes, concurrent vehicle
Details on study design:
Justification of species/strain: The rat is regarded as a suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for dose range finding study (study code: 14/328-220PE).Randomization: All adult animals were sorted according to body weight by computer and divided in to weight ranges. There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that the mean group weights were similar. The grouping was controlled by SPSS/PC software, according to the actual body weight verifying the homogeneity/variability between/within the groups and cages. Males and females were randomized separately.Rationale for dose selection and route of administration: The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work, including the results of a repeated dose range finding study in the rat (CiToxLAB study code 14/328-220PE). The aim was to induce toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The selected highest dose of 1000 mg/kg bw/day is a limit dose for this study type.The oral route was selected as it is a possible route of exposure to the test item in humans.
Positive control:
Positive control not required for this study type.
Observations and examinations performed and frequency:
Clinical observations and functional observation battery (FOB)
Main and Recovery animals: Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and end of the working day.General clinical observations were conducted once a day, during the treatment period in the afternoon at approximately the same time with minor variations as practical during the working day.Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded including onset, degree and duration as applicable.Detailed examinations were conducted once before the first exposure (to allow for within-subject comparisons), then weekly at the morning time. These observations were made outside the home cage in a standard arena, at similar times as practical.Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also assessed. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.Main animals, randomly selected 5 males and 5 females: Assessment of any potential test item related neurotoxicity was performed during the last exposure week on five selected main males and females.Selected animals were subjected to the functional observation battery, including quantitative assessment of grip strength (manual and instrumental), and measurement of landing foot splay and fore/hind limb grip strength. Qualitative and quantitative assessment of motor activity were measured.To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots for the hind limbs was measured.Fore/hind limb grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of the test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed at 3 times for each animal on each test day. The procedure was repeated with the hind limbs and the appropriate grip support. The results were tabulated with individual and mean data.Manual assessment of sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity were conducted and the general physical condition and behaviour of animals was tested. A modified Irwin test was performed.Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated using a scoring system, where score “0” is given when the behaviour or reaction of the animal is considered normal, and -1 or -2, or +1 and +2 is given if the response is less, or heightened than expected in an untreated animal.Quantitative assessment of motor activity was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany).Locomotor activity were monitored by placing each animal individually into an open field for 1 hour observation time, when DVD recording of movement was made.Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings are made to produce the appropriate parameters. Obtained data was evaluated for distance travelled in 5 minute segments in the control and high dose groups.Recovery animals: Neurotoxicity evaluation was similarly conducted in the Recovery animals towards the end of the recovery period.Body weight measurementAll adult animals were weighed with accuracy of 1 g for randomization purposes, then on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on post partum Days PPD0 (within 24 hours after parturition), PPD4 and before termination. Body weights of the female animals was additionally taken on gestational Days GD10 and 17 in order to give accurate treatment volumes, but these data was not evaluated statistically.Food consumption measurementAnimal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 and weekly afterwards.CLINICAL PATHOLOGYRandomly selected five males and females from the main animals, and all recovery animals.All animals selected for blood sampling were fasted (overnight period of food deprivation). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy. Three samples were taken from each animal: one for haematology (1.2 mL blood, in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (1.4 mL blood in tubes with sodium citrate as anticoagulant) and one to obtain serum (approximately 1 mL blood as practical, in tubes with no anticoagulant) for clinical chemistry.
Sacrifice and pathology:
PATHOLOGY
Terminal procedures and macroscopic evaluation
Gross necropsy was performed on each animal irrespective of the date of death.Terminally (one day after the last treatment), all animals were sacrificed under anaesthesia by exsanguination.The external appearance was then examined and the cranium, thoracic and abdominal cavities were opened. The appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.Organ weight measurementsAt the time of termination, body weight and weight of the following organs of all adult animals was determined:- With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroidsTestes and epididymides were weighed individually. Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) was calculated and reported.Tissue preservation and microscopic evaluation
The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerves were retained in modified Davidson’s fixative, testes with epididymides in Bouin’s solution, and all other organs in 10% buffered formalin solution.In addition, on completion of the macroscopic examination the following tissues and organs were retained from all animals.The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.For the adult animals, detailed histological examination were performed as follows:• on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group),• all macroscopic findings (abnormalities), except of minor order from all animals• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the control and high dose group and of all males that failed to sire and all females that failed to deliver healthy pups.Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Other examinations:
None specified in the study report.
Statistics:
The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest). The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. For a significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.
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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly higher than control absolute and relative adrenal weight group mean values were noted in male animals in the High dose group
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
DOSE FORMULATION ANALYSIS
Analysis of all dose formulations was performed on samples collected on Week 1, Week 3 two occasions, Week 3 and Week 6. The actual concentrations varied between 70% and 105% of the nominal in the Low dose group, 72% and 103% in the Mid dose group, and 79 and 105% in the High dose group. No test item was detected in the control samples. However there was a relatively high individual variations in homogeneity between formulations, the mean concentrations were 45.9 mg/mL (92% of the nominal, n=18, SD = 4.10) for the Low dose group, 139.6 mg/mL (93% of the nominal n=18, SD=12.36 for the Mid dose group, and 458.69 mg/mL (92% of the nominal, n=54, SD=26.95) in the High dose group, therefore considered to be suitable for the study purposes, providing the expected exposure of the animals.MortalityThere was no mortality during the study.

Clinical observation
No test item related adverse effects or systemic clinical signs were noted following daily administration of the test item by oral gavage.Red discharge from the eyes was observed in one male in the Low dose group (100 mg/kg bw/day) on Day 20. The vaginal prolapse was noted in one Low dose female from day 40 (progressed to the prolapse of uterus), one Mid dose female from Day 29 and a prolapse of uterus was seen in one High dose female from Day 38. This was accompanied by piloerection at Mid and High dose.All the signs detailed above are considered incidental with no toxicological relevance.

Neurological assessment
There were no treatment related effects.There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups.There was no effect of treatment noted during the assessment of foot splay, grip strength or motor activity. The total distance travelled was comparable to the control for both sexes in the high dose group. The statistically significantly higher than control total travelled distance group mean value observed in recovery females is considered incidental and not toxicologically meaningful.

Body weight and body weight gain
There were no test item related adverse effects noted on the body weight and body weight gain values following daily administration of the test item at dose levels up to and including 1000 mg/kg bw/day.The statistically significantly lower than control body weight gain group mean value was noted in recovery females from Day 35 until Day 42, but considered incidental with no toxicological relevance.

Food consumption
There were no test item related differences in the food consumption in test item treated groups when compared to the Control. Small but statistically significantly changes such us higher than control group mean value in High dose males (1000 mg/kg bw/day) from Day 21 until Day 27 and higher than Control group mean value in females from Day 14 until GD0 are considered incidental with no toxicological relevance.

Clinical pathology
Haematology
There were no test item related differences. Statistically significant differences in large unstained cells group mean value in the Low dose females (100 mg/kg bw/day) was considered incidental. Statistically significantly higher (p<0.05, +60%) than Control monocyte percent (MONO%) group mean value was measured in the High dose group in females (1000 mg/kg bw/day). However, it is ascribed to lower control values rather than to the effect of treatment (historical control mean of CiToxLAB Hungary Ltd. Is 3.129 %). The actual control mean is 2.020 %, and the High dose mean is 3.240 %, which are similar to the historical mean.

Clinical chemistry
There were no test item related differences.In males, statistically significantly lower than control albumin (p≤0.05, -6%; p≤0.01, -8%) and total protein (p≤0.05, -3%; p≤0.01, -5%) group mean values were noted in the Mid and High dose group. The group mean values in the Mid and High dose groups were comparable with the historical control mean of CiToxLAB Hungary Ltd. while the study control values were higher. Therefore the changes were ascribed to a relatively higher study control rather than a test item related effect. (study control is 31.56 g/L for albumin, the historical control mean is 29.80 g/L, while the mid and low dose mean values are 29.78 g/L and 30.70 g/L respectively. For the total protein the study control is 59.44 g/L, while the historical mean is 56.50 g/L, and the group mean in the mid and high dose groups are 57.54 g/L and 57.96 g/L respectively).UrinalysisThere were no test item related differences.

PATHOLOGY
EVALUATION AND ORGAN WEIGHTS

Macroscopic Findings
No test item related macroscopic findings were observed up to the dose level of 1000 mg/kg bw/day.Pale discoloration/pelvic dilation of the kidney, enlargement of the thyroids/parathyroids, enlarged right testis, red discoloration of the lungs, dilatation/enlargement of the uterus or spleen, red discoloration of the glandular stomach, were observed without meaningful incidence and were considered to be incidental or a common background.Microscopic FindingsNo test item related microscopic changes were noted at a dose level of 1000 mg/kg bw/day.Minimal focal myocardial degeneration in 2/5 Control males, minimal focal interstitial cell infiltrate in 2/5 Control and 1/5 High dose males were seen in the heart. Minimal chronic progressive nephropathy in 1/7 High dose male, minimal unilateral interstitial inflammation in 1/5 Control male, minimal unilateral pyelitis in 1/5 Control female, mild unilateral pelvic dilatation in 2/7 High dose males, minimal focal unilateral tubular basophilia in 1/8 High dose male were observed in the kidney. Diffuse moderate edema of the lungs in 1/6 Control female and mild periportal fibrosis of the liver in 1/5 Control female were also noted. In the spleen, minimal to moderate extramedullary hematopoiesis was recorded in 3/5 Control and 4/5 High dose males and 2/5 Control, 3/3 Mid dose and 5/5 High dose females. Minimal focal erosion of the glandular stomach in 1/5 High dose female, moderate tubular dilatation of the right testis in 2/2 Mid Dose males, moderate uterine dilatation in 1/3 Mid dose female, and pyometra in 1/2 Low dose female were detected.These changes were incidental or considered to be a common background observation.Organ weightThere were no treatment related adverse effects on organ weights.Statistically significantly higher than control absolute (p≤0.01, +21%) and relative (p≤0.01, +19% related to body weight and p≤0.01, +20% related to brain weight) adrenal weight group mean values were noted in male animals in the High dose group (1000 mg/kg bw/day). The causative role of the test item cannot be excluded, however, the majority of individual results were within the historical control range of CiToxLAB Hungary Ltd., and no association with clinical pathology or pathology effects was noted. These are therefore considered as a not adverse.Other occasional statistically significant differences were not dose-related and therefore considered incidental and of no toxicological significance.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: changes in adrenal absolute and relative weights.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: changes in adrenal absolute and relative weights.
Critical effects observed:
not specified

Actual concentrations in the low and mid dose groups

Date of sampling

Nominal concentration (mg/mL)

Actual concentration (mg/mL)

% of the nominal

Nominal concentration (mg/mL)

Actual concentration (mg/mL)

% of the nominal

22 October 2014

50

47.7

95

150

144.0

96

43.6

87

131.9

88

43.2

86

126.6

84

05 November 2014

45.9

92

140.5

94

45.9

92

137.0

91

46.3

93

143.1

95

08 November 2014

49.7

99

145.1

97

46.6

93

141.8

95

47.8

96

144.6

96

28 November 2014

48.8

98

147.4

98

52.3

105

147.1

98

48.1

96

144.6

96

22 October 2014*

50.1

100

152.9

102

41.4

83

118.3

79

48.5

97

153.0

102

28 November 2014*

40.7

81

107.4

72

44.6

89

154.7

103

34.8

70

132.5

88

*Back up samples

 

Actual concentrations in the high dose group

Date of sampling

Nominal concentration (mg/mL)

Actual concentration (mg/mL)

% of the nominal

Date of sampling

Nominal concentration (mg/mL)

Actual concentration (mg/mL)

% of the nominal

October 22 2014

500

394.0

79

07 December 2014

500

466.4

93

500

422.1

84

500

453.1

91

500

425.0

85

500

449.0

90

05 November 2014

500

438.9

88

500

460.6

92

500

413.2

83

500

459.3

92

500

426.2

85

500

453.8

91

08 November 2014

500

448.1

90

500

395.4

79

500

457.4

91

500

407.8

82

500

416.9

83

500

430.6

86

28 November 2014

500

481.4

96

500

427.3

85

500

469.4

94

08 December 2014

500

453.9

91

500

442.6

89

500

446.4

89

06 December 2014

500

468.7

94

500

467.8

94

500

465.8

93

500

471.0

94

500

475.4

95

500

464.1

93

500

486.4

97

500

473.9

95

500

483.8

97

500

477.9

96

500

482.8

97

500

472.7

95

500

472.5

95

500

478.0

96

500

478.2

96

500

474.5

95

500

457.0

91

500

480.8

96

500

463.6

93

500

486.9

97

500

476.4

95

22 October 2014

500

484.6

97

500

473.6

95

500

504.2

101

07 December 2014

500

433.3

87

500

467.0

93

500

439.9

88

28 November 2014

500

505.4

101

 

500

522.2

105

500

442.2

88

 

Summary of Organ Weight data – Male – Main (Table 1 of 2)

 

 

 

Body weight

Adrenal Glands

Brain

Heart

Kidneys

Liver

Prostate Gland

Spleen

Thymus

Thyroid/ thyroid

Seminal Vesicles

Epididym (left)

Epididym (right)

Epididym Det.

 

 

 

G

G

G

G

G

G

G

G

G

G

G

G

G

G

Group

Sex

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 CG

M

Mean

448.6

0.0715

2.195

1.373

3.032

12.383

0.934

0.916

0.432

0.0235

2.398

0.706

0.695

1.40083

S.D.

35.1

0.0107

0.120

0.122

0.393

1.561

0.196

0.137

0.072

0.0068

0.462

0.067

0.057

0.11743

N

12

12

12

12

12

12

12

12

12

12

12

12

12

12

2 LDG

M

Mean

460.5

0.0778

2.196

1.377

3.036

13.168

0.921

0.903

0.505*

0.0249

2.333

0.737

0.764*

1.50083

S.D.

41.2

0.0083

0.037

0.141

0.349

1.937

0.195

0.167

0.074

0.0078

0.447

0.082

0.089

0.15658

N

12

12

12

12

12

12

12

12

12

12

12

12

12

12

3 MDG

M

Mean

463.2

0.0767

2.202

1.352

3.036

12.950

0.932

0.943

0.478

0.0249

2.294

0.709

0.733

1.44250

S.D.

32.5

0.0131

0.094

0.195

0.219

1.154

0.253

0.155

0.100

0.0042

0.271

0.054

0.073

0.11678

N

12

12

12

12

12

12

12

12

12

12

12

12

12

12

4 HDG

M

Mean

457.3

0.0862**

2.212

1.410

3.136

13.017

0.859

0.975

0.438

0.0238

2.342

0.675

0.682

1.35667

S.D.

39.1

0.0097

0.053

0.172

0.392

1.598

0.188

0.142

0.055

0.0050

0.464

0.063

0.069

0.12872

N

12

12

12

12

12

12

12

12

12

12

12

12

12

12

 

NS

DN

NS

NS

NS

NS

NS

NS

DN

NS

NS

NS

DN

NS

 

Summary of Organ Weight data – Male – Main (Table 2 of 2)

 

 

 

Testes (left)

Testes (right)

Testis D

 

 

 

G

G

G

Group

Sex

 

 

 

 

1 CG

M

Mean

1.979

1.952

3.93083

S.D.

0.187

0.151

0.32959

N

12

12

12

2 LDG

M

Mean

2.003

2.026

4.02833

S.D.

0.125

0.182

0.29489

N

12

12

12

3 MDG

M

Mean

1.858

2.054

3.91250

S.D.

0.181

0.361

0.44628

N

12

12

12

4 HDG

M

Mean

1.901

1.877

3.77750

S.D.

0.149

10.60

0.30508

N

12

12

12

 

NS

NS

NS

 

Group 1 CG – 0 mg/kg bw/day         Group 2 LDG – 100 mg/kg bw/day  Group 3 MDG – 300 mg/kg bw/day Group 4 HDG – 1000 mg/kg bw/day

Remarks:

NS = Not Significant            U = Mann-Whitney U-Test

* = p<0.05                            DN = Duncan’s Multiple Range Test

** = p<0.01

Conclusions:
The No Observed Effect Level was determined to be at 1000 mg/kg bw/day in females, and at 300 mg/kg bw/day in males, due to the changes in adrenal absolute and relative weights.
Executive summary:

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat according to OECD 422 and OECD guidance document 43 was performed in order to obtain information on the possible toxic effect of the test item following repeated (daily) administration by oral gavage to Wistar rats at 3 different dose levels.

 

The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work, with the aim of inducing toxic effects but ideally no death or suffering at the highest dose (a limit dose for this study type) and a NOAEL at the lowest dose.

Twelve Wistar rats per sex/group were assigned to 4 Main study groups.

Additional 5 Wistar rats per sex/group were assigned to a Control and High dose Recovery groups.

Recovery animals scheduled to follow-up observations were not mated but kept without treatment for at least 14 days after the first scheduled euthanasia of dams to detect possible delayed occurrence, or persistence of, or recovery from toxic effects.

The control group was treated with the vehicle only.

 

Analysis of all dose formulations was performed on samples collected on Week 1, Week 3 two occasions, Week 3 and Week 6. The actual concentrations varied between 70% and 105% of the nominal in the Low dose group, 72% and 103% in the Mid dose group, and 79 and 105% in the High dose group. No test item was detected in the control samples. However there was a relatively high individual variations in homogeneity between formulations, the mean concentrations were 45.9 mg/mL (92% of the nominal, n=18, SD = 4.10) for the Low dose group, 139.6 mg/mL (93% of the nominal n=18, SD=12.36 for the Mid dose group, and 458.69 mg/mL (92% of the nominal, n=54, SD=26.95) in the High dose group, therefore considered to be suitable for the study purposes, providing the expected exposure of the animals.

 

Parameters monitored during the study included signs of morbidity and mortality twice daily, observation of clinical signs, body weight and body weight gain, and food consumption. Five selected males and females from the main group, and all recovery animals were subjected to a clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment in five selected males and females from the main group, and in all recovery animals. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult Main animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND4. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals.

 

For the Main adult animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups.

There was no mortality during the study.

No test item related adverse effects or systemic clinical signs were noted following daily administration of the test item.

Neurological examinations did not reveal any test item related adverse effect.

No test item related adverse effects were noted on body weights or body weight gains.

No effects were observed on the food consumption.

There were no toxicologically relevant effects noted at haematology measurements.

Clinical chemistry measurements did not reveal any test item related adverse effect.

No adverse effects were noted at urinalysis.

No test item related effects were observed at necropsy.

Histopathology examinations on selected tissues did not reveal any test item related effects in the High dose group.

There were no toxicologically significant effects observed in organ weights, however statistically significantly higher than control (+21%) absolute and relative adrenal weight was noted in the High dose group. However, no corresponding effects in clinical pathology or histopathology were noted.

 

Conclusion

Administration of NovaSpec Base Oil in PEG 400 + Polysorbate 80 via oral gavage to Wistar rats at the dose levels of 100, 300 and 1000 mg/kg bw/day was not associated with any adverse effects in general toxicology parameters. At the dose level of 1000 mg/kg bw/day, elevation of adrenal absolute and relative weights was noted, however considered as not adverse in the lack of any corresponding clinical pathology or histopathology effect.

In conclusion, under the conditions of this study, the No Observed Effect Level was determined to be at 1000 mg/kg bw/day in females, and at 300 mg/kg bw/day in males, due to the changes in adrenal absolute and relative weights.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A full justification for the read across applied for this substance is contained within Section 1.4, reference " Explanation of NovaSpec Base Oil - FINAL". This document contains a full explanation of the manufacturing route for the substance and discussion on why this substance should be considered as a UVCB white oil analogous to CAS 8042-47-5. Physical properties of the crude reaction product and the different product grades (or distillate fractions) derive from the general chemical structure characteristics (linear-branched alkanes with characteristic branching length and position). Specific unique chemical structures are not isolated in any process step and due to the tens of thousands of isomers and the high degree and variable nature branching and chain length, the base oil bulk properties are the result of the average structure characteristics. The unique viscosity grades each contain all the same typical chemical structures and predominantly overlapping Molecular Weight distributions. The unique viscosity and volatility characteristics of each viscosity grade derive from the boiling point distribution, a result of the short-path distillation (wiped film evaporation). This is identical to the production of petroleum derived white oils.

In order to avoid the requirement to further test a substance that is a derived white oil on animals, a weight of evidence approach is utilised to provide evidence of lack of repeated dose toxicity. This is via the utilisation of various literature papers.

In this subchronic study, groups of 15 female rats of two strains (F344 and Sprague-Dawley) were fed diets containing 0, 2000 or 20,000 (0, 0.2 or 2.0% respectively) P15(H) white oil. Ten animals were sacrificed after 90 days for toxicological evaluation and the remaining 5 rats were sacrificed after 91 days for a tissue analysis for mineral hydrocarbon content. In addition 20 extra female F-344 rats were fed diet containing 0 or 2% white oil. Ten of these animals were sacrificed after 30 days treatment and the remaining ten animals after 61 days treatment. These extra animals were included to follow the time course of the development of any lesions that developed. All animals were observed daily and body weights and food consumption were recorded weekly. At each scheduled sacrifice blood samples were taken for a wide range of haematological and serum chemistry determinations.

There were no clinical signs in either rat strain at any dose level during the study. There were no effects on mortality. Apart from a transient decrease in body weight for one day at one dose level in one strain of rat, there were no effects on growth rate. There were no effects on food consumption. There were no data on the compound intake. Hematological effects were only noted in F344 rats and included an increase in white cell count. At day 30 this was observed in the 2.0% group, but by 90 days was seen in both dose groups in a dose-related manner with a significant increase observed in the high-dose group (21.1%, 24.5%, and 36.1% in control, 0.2% and 2.0% groups). There were also marginal differences in some serum chemistry values of F 344 rats compared to controls, but these changes were not considered clinically significant. The absolute and relative liver weights of the F344 rats in the 2% group were increased by 1.2- and 1.3-fold respectively and in the 0.2% group both absolute and relative liver weights were increased 1.1-fold. The mesenteric lymph nodes (absolute and relative) were increased in the 2% group by 1.4 to 3.7 fold depending on the time of sacrifice. There were no changes in organ weight in the Sprague-Dawley rat. Histopathological changes in the livers of the treated F344 rats consisted of the occurrence of an increased incidence of microgranuloma. These were not present at day 30, but were present in more than 80% of the animals in the 2% dose group after 61 days. By 90 days microgranulomas were also observed in the 0.2% dose group (incidence not stated in the publication). Central necrosis and/or the presence of Langhan's type cells were seen in occasional microgranuloma. The incidence and severity were both dose and temporally-related as the appearance of granuloma occurred at 61 days in the high-dose group and was present in both dose groups at 92 days. Histiocytosis occurred in the mesenteric lymph nodes of the F 344 rats in the 2% dose group after 30 days. Microgranulomas were observed at 61 and 92 days in the 2% and also in the 0.2% groups at 92 days. The incidence and severity of the microgranulomas were increased in a dose related manner. Histopathological examination of the livers of the Sprague Dawley rats revealed that in contrast to the F 344 rats there were no microgranulomas in either dose group, but there was a slightly increased incidence of minimal multifocal chronic inflammation in the 2% dose group. No histological changes were observed in the mesenteric lymph node of the Sprague Dawley rats at either dose level. There was a dose and strain related increase in mineral hydrocarbon in the liver and mesenteric lymph nodes.

White mineral oil was determined to be more toxic to F344 rats than to Sprague-Dawley rats. There was no NOAEL in the F344 rats and the LOAEL is 2000 ppm, based on the histopathology in the liver and mesenteric lymph nodes accompanied by changes in the weight of those organs. The NOAEL in the Sprague-Dawley rat is greater than or equal to 20,000 ppm, based on the lack of any toxic effects at the highest dose.

However, in the paper “ORAL TOXICITY OF MINERAL OILS AND RELATED COMPOUNDS - A review” by Prof. Atte von Wright, University of Eastern Finland, a critical review of this data is undertaken via comparative assessment of other similar studies utilising Fischer 344 and other species. The following are evaluated:

 Baldwin et al. (1992) - 90 day feeding studies

Smith et al. (1996) - 90 day feeding studies

Trimmer et al. (2004) - chronic (two year) oral toxicity study

 In the review of Miller et al. (1996), a discrepancy with the effects observed in Fischer 344 rats and the either nonexistent or relatively insignificant effects of mineral oils in other species and strains was pointed out. There are several historical studies performed with either paraffinic or naphthenic oils in beagle dogs, SpragueDawley rats and LongEvans rats, in which no significant microscopic anomalies in liver, kidneys, spleen or mesenteric lymph nodes were detected and no mineral oil deposits in the tissues were observed (Shubik et al, 1962; Smith et al, 1995; McKee et al., 1987; Firriolo et al. 1995).

The authors also point out that the lipogranulomas of the liver observed in human autopsies are considered clinically insignificant, while those observed in Fischer 344 rats can be associated with inflammatory and necrotic lesions. The authors further suggest that the observed effects reflect rat strain specific differences in the responses to mineral oils.

The lymph nodal and hepatic lesions observed in rats orally exposed to mineral oils were further assessed in a special pathology workshop held in 2001 at the Fraunhofer Institute of Toxicology (Hannover, Germany) (Carlton et al. 2001). According to the final conclusion of the workshop: “The available data suggest that the granumalotous lesions experimentally induced by MHCfeeding, particularly in the liver of F344 rats, are exaggerated toxicological responses peculiar to rats.”

 

Therefore, in accordance with the CLP Regulation (EC No 1272/2008), specifically section 3.9.2.9.9 which states that:

 “Thus it is feasible that a specific profile of toxicity occurs in repeat-dose animal studies at a dose/concentration below the guidance value, such as < 100 mg/kg bw/day by the oral route, however the nature of the effect, such as nephrotoxicity seen only in male rats of a particular strain known to be susceptible to this effect may result in the decision not to classify. “

Administration of NovaSpec Base Oil in PEG 400 + Polysorbate 80 via oral gavage to Wistar rats at the dose levels of 100, 300 and 1000 mg/kg bw/day was not associated with any effect on reproduction in the parent males and females, or on development of offspring. Also, no adverse effects were noted in general toxicology parameters. At the dose level of 1000 mg/kg bw/day, elevation of adrenal absolute and relative weights was noted, however considered as not adverse in the lack of any corresponding clinical pathology or histopathology effect.

 

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) of NovaSpec Base Oil for general toxicity, reproduction parameters, and for the development of offspring F1 generation, was considered to be 1000 mg/kg bw/day. The No Observed Effect Level was set at 1000 mg/kg bw/day in females, and at 300 mg/kg bw/day in males, due to the changes in adrenal absolute and relative weights.

 

It is proposed that classification and labelling is not considered attributable to these effects.  On the basis of the effects noted in the study, it is considered that these are species specific. The effects noted appear to be specific to the Fischer strain and this is adequately proven.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint: 

The 28-day oral toxicity study with repro/developmental screening is selected as the definitive study, as it was conducted on the substance itself. The results are directly comparable to the analogue substances quoted for read across purposes.

Justification for classification or non-classification

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint: 

The 28-day oral toxicity study with repro/developmental screening is selected as the definitive study, as it was conducted on the substance itself. The results are directly comparable to the analogue substances quoted for read across purposes. The results indicate that no classification is applicable on the basis of the dose levels.