Distillates (petroleum), hydrodesulfurized middle

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study ongoing - Draft report received - see justification for more details

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

As of 19th April 2023 the study report is at the draft stage and will not be finialised in time for the update required to meet the ECHA deadline. All available information to date is included in this ESR, which will be updated once the final report becomes available. All results reported have not been QA checked and are not finalised - these should be considered provisional results only. For this reason, no summary tables are currently included in the ESR, these will be added when the report is finalised and updates to the text sections will be made as appropriate.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Gas oils (petroleum), hydrodesulfurized

EC Number:

265-182-8

EC Name:

Gas oils (petroleum), hydrodesulfurized

Cas Number:

64742-79-6

IUPAC Name:

Gas oils (petroleum), hydrodesulfurized

Test material form:

liquid

Details on test material:

Concawe sample ref S712
Clear light green liquid

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is the standard laboratory rodent species used for toxicity assessment and is also recommended by various regulatory authorities.
The Wistar rat was selected due to the large amount of background data available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology Pvt. Ltd. Plot 4B, MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078 (Licensed breeder of Charles River)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at start of treatement: 12 weeks
- Weight at study initiation: 360 to 421g (males) and 245 to 285g (females). At the start of treatment, the body weight range of the animals did not exceed 20% of mean boday weight in each group and sex
- Fasting period before study:
- Housing:
One or two rats of same sex were housed in a single cage. Animals in Groups 1 to 4 were housed in pairs. Due to the odd number of animals in the recovery groups, the last animal in the recovery group of each sex was housed individually and the remaining were housed in pairs
Standard solid floor transparent polysulfone cages (Size: approximately L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for drinking water in polycarbonate bottle with stainless steel sipper tube and feed hopper facilities for powder diet.
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): as libitum
- Acclimation period: five days

DETAILS OF FOOD AND WATER QUALITY:
Test item mixed in Altromin Rat/Mice Maintenance powder diet manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was provided ad libitum to animals in the treatment groups
Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Limited., Mumbai 400 001, India was provided ad libitum to rats
in polycarbonate bottles with stainless steel sipper tubes.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 - 24.2 C
- Humidity (%): 63-65%
- Air changes (per hr): 12.4-13.0
- Photoperiod (hrs dark / hrs light): 12 light/12 dark

IN-LIFE DATES: From: 16th November 2022 To: 20th March 2023

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The test item was administered orally as admixture with diet. Doses were expressed as parts per million (ppm) in diet and as the target mg/kg body weight/day; the ppm was adjusted periodically to maintain a stable mg/kg/day exposure. The oral route was chosen because this route is a possible route of human exposure for the test item. Human exposure is most likely via the dermal route, but oral administration may allow assessment of potential systemic effects.

Vehicle:

other: standard feed

Details on oral exposure:

The following procedure was followed when 6 kg of experimental diet was prepared.
The required quantities of test item were weighed in a labelled glass beaker and mixed with 0.5 kg of basal diet in a stainless-steel drum for two minutes manually and then added in portions to the remaining bulk diet (9.5 or 13.5 kg) and mixed in a stainless-steel ribbon blender for 20 minutes. For animals in the control group, 6 kg of the basal diet was mixed in a clean stainless steel ribbon blender for 20 minutes.

The feed was mixed once or twice weekly and used within the stability period. The experimental fortified diet was stored in the polyethylene bags within labelled stainless-steel drums in the experimental room.

The dietary concentrations were adjusted at 2-weekly intervals based on body weight and food consumption of previous week and presumed body weight gain and food consumption.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diet was analysed for stability, homogeneity and concentration:
Stability
The stability of the test item was established under Eurofins Advinus Study No.: G25612 at 500 and 20,000 ppm fortification level in diet. Based on the results the test item was found to be stable for 15 days at room temperature in an sealed container, and for 7
days in an open container.

Homogeneity and concentration
These analyses were carried out on Day 1 and during the 2nd (Day 42) and 3rd (Day 70) months of the treatment period. Six samples were taken from diet, 2 each from top, middle and bottom layers. Analysis was conducted as described in the study no. cited above. Diets were considered acceptable when the mean of me asured concentration was within +/- 20% of the nominal concentration and the % RSD was

Duration of treatment / exposure:

90 days

Frequency of treatment:

Diet was available ad libitum.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on results of OECD 422 study; 100 mg/kg/day was chosen as the low dose and would not be expected to cause any toxic effects. The dose of 1000 mg/kg/day was expected to cause a slight reduction in bodyweight and induce some systemic affects. The mid dose of 300 mg/kg/day was selected to provide information on dose response relationship for toxicity
- Fasting period before blood sampling for clinical biochemistry:
- Rationale for selecting satellite groups: Control and high dose to investigate the reversibility of any findigns
- Post-exposure recovery period in satellite groups: 28 days
- Dose range finding studies: OECD 422 study

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
twice daily/once daily

All rats were observed for morbidity and mortality twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs of toxicity, the observation for morbidity and mortality was carried out once in the morning during weekend and holidays.
All rats were observed for clinical signs once daily during the treatment period.
On the days of scheduled detailed clinical examination (once weekly - see below), clinical signs were included as a part of detailed clinical observations except on Day 1; detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals.
On the day of euthanasia of rats, observations for clinical signs were recorded once in the morning prior to scheduled euthanasia.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
weekly

Detailed clinical examination was done prior to the treatment on Day 0 and at weekly intervals thereafter (±1 day) during course of the in-life for all parental rats.
During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, 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) or bizarre behaviour (e.g., self-mutilation, walking backwards).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded initially on Day 1 of treatment and at weekly intervals thereafter (+/- 1 day) for all groups of rats during treatment and recovery periods. Fasted body weight was recorded prior to scheduled sacrifice.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of treatment and at the end of the treatment/recovery periods by a trained veterinarian.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery periods; approximately 3.0 mL of blood was collected by retro-orbital plexus puncture method under isoflurane anaesthesia (shared with clinical chemistry, haematology).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals:
all animals
- See table in Section "Any other informaiton on materials and methods incl. tables" for details of parameters checked.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery periods; approximately 3.0 mL of blood was collected by retro-orbital plexus puncture method under isoflurane anaesthesia (shared with clinical chemistry, haematology).
- Animals fasted: Yes
- How many animals:
all animals
- See table in Section "Any other informaiton on materials and methods incl. tables" for details of parameters checked.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: at the end of the treatment and recovery periods; approximately 3.0 mL of blood was collected by retro-orbital plexus puncture method under isoflurane anaesthesia (shared with clinical chemistry, haematology)
- Animals fasted: Yes
- How many animals: all animals

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment and recovery periods
- Metabolism cages used for collection of urine: rats were placed overnight in specially fabricated cages with urine collection tubes
- Animals fasted: Yes, water allowed ad libitum
- See table in Section "Any other informaiton on materials and methods incl. tables" for details of parameters checked.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: towards the end of the treatment period for main groups (Day 88) and during recovery period for recovery groups (Day 115).
- Dose groups that were examined: all groups
- Battery of functions tested:

Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions.
Observations during Removal of Animal from Home Cage and Handling (ease of removal from home cage, handling reactivity, palpebral closure, eye examination, piloerection, lacrimation, salivation, skin/fur examination, perineum wetness, respiration, muscle tone and extensor thrust response)
Open Field Observation (gait, posture, tremors, mobility score, arousal level, clonic or tonic movements, stereotypic behaviour, bizarre behaviour, urination, defecation, rearing and abnormal vocalizations)
Sensory activity (approach response, touch response, click response, tail-pinch response, pupil response, aerial righting reflex)
Motor activity was measured using an automated animal activity measuring system (Make: Columbus Instruments) for 30 minutes
Grip strength Hind limb and fore limb grip performance was tested using dual grip strength meter (Model: Columbus Instruments)
Landing Hind limb Foot splay was assessed by dropping the rat on to a horizontal surface of the table top from a short height and the distance between the hind feet upon landing was measured

OTHER:
Oestrous Cycle
Vaginal smear was examined in all female rats and the stage of oestrous cycle was recorded daily for at least two weeks prior to necropsy to evaluate the oestrous cycle length and normality. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the treatment and recovery periods after overnight fasting (water allowed). Terminal body weights were recorded for all animals immediately prior to sacrifice and were euthanized by exsanguination under isoflurane anesthesia.

HISTOPATHOLOGY: Yes
Histopathological examination was carried out on the preserved tissues/organs from the vehicle control (G1) and high dose group animals (G4). Histopathological examination of the testes also included a qualitative assessment of stages of spermatogenesis. In addition, all gross lesions from all the animals were examined microscopically.

The following tissues were suspected as target organs at high dose and thus examined in the lower dose groups and recovery groups.
Males: Liver, kidneys, thyroid gland and adrenal gland.
Females: Liver and thyroid gland.

See table in Section "Any other informaiton on materials and methods incl. tables" for details of gross
observations and tissues collected.


A peer review of microscopic slides was performed by an independent consultant. The individual animal microscopic findings and all other pertinent data was provided to the peer review pathologist. Based on the information provided, the peer reviewer selected the number of animals and tissues to be reviewed. At the time of peer review, additional data, namely, in-life findings, clinical pathology, organ weights and gross pathology data was provided to the reviewer. The findings were documented and are included in the raw data file. The details of peer review process and final diagnosis arrived at by consensus between the primary and peer review pathologists was presented in a peer review certificate and signed by both pathologists and this certificate is included in the main report at the time of finalization. The peer review certificate was issued after the pathology report has been signed.

Optional endpoint(s):

Optional endpoints:
Sperm motility was evaluated manually using the sperm samples collected from the right vas deferens, immediately after necropsy.
For morphological evaluation of sperm, smears were made using the semen samples collected from right vas deferens immediately after necropsy from all male rats, fixed with acetone for further evaluation by a manual method. At least 200 spermatozoa per sample were classified.
For manual enumeration of sperm count, the right epididymis from all males was collected and frozen (approximately at -20°C).
Sperm motility was evaluated using a microscope for the total motility and progressive motility of all males. Sperm morphology and epididymal sperm count was performed for rats of control and high dose males (main groups).

Statistics:

Data was captured using the ProvantisTM laboratory information management system (LIMS).
Most parameters were evaluated using the Levene’s Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When the data was found to be homogeneous and of normal distribution, the data was analysed by analysis of variance (ANOVA). When the data was found to be nonhomogeneous or of non-normal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA was found to be significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.

For two groups, the comparison of mean data between treatment and control groups was performed using student’s t-test

Descriptive statistics Mean, SD, Percentages and individual numbers are presented by Treatment group and Day.

All hypothesis testing was carried out at the 5% (2-sided) significance level.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights and body weight gains were unaffected throughout treatment in males and females up to 1000 mg/kg/day. There was a slight trend for lower body weights in males given 1000 mg/kg/day but this never attained consistent statistical significance (except for total weight gain between days 0 to 90 for females).
These statistical variations were considered to be incidental as the changes were not dose proportional and/or consistent.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A reduction in food consumption was observed on few occasions among all the treated groups in both sexes; at the beginning of the study this may have reflected a palatability adaptation to the diet. The reduced food consumption did not notably alter the body weight and weight gains and was therefore considered not related to treatment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No test item related hematological changes were noted in females at any of the dose levels tested.

At 1000 mg/kg/day in males, decreased red blood cell count, hemoglobin and hematocrit and an increased percentage of reticulocytes were noted. Further, higher total leukocyte, neutrophil and lymphocyte counts were noted in males. However, these changes did not show any microscopic correlates in the hemopoietic organs examined and hence, were considered as non-adverse effect of test item administration. All these changes were reversible at the end of 28-day recovery period except the minimal increase in reticulocytes.
All other statistical significances observed in haematology parameters were considered incidental in nature, as the changes were of minimal magnitude and/or not dose progressive.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

An increase in total cholesterol, HDL and/or LDL cholesterol in males at ≥ 300 mg/kg/day and in females at 1000 mg/kg/day were considered as test item related. However, the lipid profile changes were not associated with any hepatocyte vacuolation indicative of lipid accumulation in liver and hence were considered as non adverse changes. All these changes were reversible at the end of the recovery period.
All other differences observed in biochemical parameters including the changes that reached statistical significance were considered incidental as changes lacked dose progression and/or were of minimal magnitude. In addition, there was no histopathological correlation.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Administration of test item resulted in higher liver weight (absolute and relative) in males at ≥ 100 mg/kg/day and in females at ≥ 300 mg/kg/day and was associated with hepatocyte hypertrophy, histologically.
Test item related higher kidney weight was noted in males at 1000 mg/kg/day and was associated with basophilic tubules and granular cast in tubules, histologically.
Test item related increased thyroid weight (relative to body weight) noted in males at ≥100 mg/kg/day was associated with hypertrophy of follicular epithelial cells, histologically.
At the end of recovery period, all these weight changes were completely reversible with the exception of the increased liver weight in males which continued to show a slight elevation at the end of the recovery period but had notably reduced since the withdrawal of treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related gross pathological changes observed in either sex at the end of the treatment and recovery periods.
All the gross alterations involving testes (enlarged; bilateral and flabby; unilateral); seminal vesicle/coagulating gland (small; unilateral) and uterus (dilated) were distributed randomly across the groups and were not considered as test item related. These lesions were subjected to microscopic evaluation and were considered to be incidental/spontaneous changes.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related gross pathological changes observed in either sex at the end of treatment and recovery period.
All the gross alterations involving testes (enlarged; bilateral and flabby; unilateral); seminal vesicle/coagulating gland (small; unilateral) and uterus (dilated) were distributed randomly across the groups and were not considered as test item related. These lesions were subjected to microscopic evaluation and were considered to be incidental/spontaneous changes.
In the liver, test item related centrilobular hypertrophy of the hepatocyte was noted in males ≥ 100 mg/kg/day and females at ≥ 300 mg/kg/day with a minimal to mild degree of severity. These changes were also associated with pigmented macrophages at 1000 mg/kg/day in a few rats in the centrilobular region. This liver histological change was considered a non-adverse adaptive response to the test item administration in the absence of significant changes in the liver enzyme profiles. All these changes at 1000 mg/kg/day were found to be reversible following withdrawal of treatment
Follicular epithelial hypertrophy with minimal to moderate degree of severity in the thyroid gland in males and females at ≥ 100 mg/kg/day was considered to be test item related. Hypertrophy of follicular epithelium wwith a diffuse pattern and an increase in height of epithelial cells with a decrease in eosinophilic secretion in the lumen. Further, epithelial cells appeared more basophilic in the thyroid follicles of male rats. However, the thyroid hormone profile was not affected. These histological changes were completely reversed at the end of the recovery period.
In kidneys, test item related incidences of tubular basophilia were noted in males at ≥ 100 mg/kg/day associated with hyaline pigment deposits in the tubular epithelium (pinkish pigment at 1000 mg/kg/day in males). Granular cast incidences in tubules at the cortico-medullary junction/outer medulla/cortex were noted at 1000 mg/kg/day and were considered as treatment related. However, the presence of granular cast /basophilia in tubular epithelium was not accompanied by any degenerative changes or kidney specific biochemical toxicity markers that would indicate toxicity and hence, histological changes in kidney were considered treatment related non-adverse findings and were also reversible.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Reproductive parameters
There were no test item-related histopathological changes in the reproductive tissues of the male or female rats. The qualitative assessment of spermatogenesis in testes did not reveal any changes in the examined rats.
The sperm parameters were not affected by the test item treatment. The percentage of progressively motile sperms/total motility were not affected at any of the dose levels tested. The percentage of normal/abnormal sperms at 1000 mg/kg/day were comparable to the control data. Similarly, the epididymal sperm count (number of sperms per gram of cauda) was not affected by the test item administration.

Details on results:

All results reported have not been QA checked and are not finalised - these should be considered provisional results only. For this reason, no summary tables are currently included in the ESR, these will be added when the report is finalised and updates to the text sections will be made as appropriate.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Remarks on result:

other: Provisional result

Remarks:

Based on draft report only and subject to change

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

All results reported have not been QA checked and are not finalised - these should be considered provisional results only.  For this reason, no summary tables are currently included in the ESR, these will be added when the report is finalised and updates to the text sections will be made as appropriate.

Applicant's summary and conclusion

Conclusions:

NOAEL based on draft report only (provisional result and subject to change) 1000 mg/kg/day (nominal)