Distillates (petroleum), hydrotreated middle

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and 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
- Housing:
Pre-mating - two rats of same sex per cage
Mating - two rats (one male: one female of the same group) per cage
Post-mating - presumed pregnant females (pregnancy confirmed by vaginal plug or presence of sperm in vaginal smear) were housed individually until sacrifice; males housed with former cage partners
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 - 24.1 C
- Humidity (%): 64-67%
- Air changes (per hr): 12.6
- Photoperiod (hrs dark / hrs light): 12 light/12 dark

IN-LIFE DATES: From: 8th April 2022 To: 7th July 2022

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:

Following procedure was followed when 6 kg of experimental diet was prepared.
The required quantities of test item were weighed in the labeled 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 (5.5 kg) and mixed in stainless steel ribbon blender for 20 minutes. For animals in control group, 6 kg of the basal diet was mixed in 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.

Males:
Concentrations in the diet for the first two weeks of the study were calculated using body weights and feed consumption measured during the pre-treatment period. Subsequently, concentrations in the diet were adjusted on Days 14, 28 and 42, based on the preceding week body weights and food consumption. During the mating period, males had access to female diet.

Females:
Concentrations in the diet for the first two weeks of the study were calculated using body weights and feed consumption measured during the pre-treatment period. Subsequently, concentrations in the diet were adjusted on Day 14 based on the preceding week body weights and food consumption.
In order to avoid overexposure, during gestation and lactation periods, dietary concentrations were calculated twice during gestation (GD 0 and GD 14) and twice on lactation (LD 0 and 7) as detailed below:
1. On GD 0, the concentrations were calculated from the GD 0 body weight and food consumption measured during pre-mating period on Day 28 from the respective groups.
2. On GD 14, the concentrations were calculated by adding 60g to the GD 14 body weight and food consumption from the respective groups.
3. On LD 0, the constant body weight of 350g for G2 and G3, 330g for G4 and constant food weight of 35g were used to calculate the concentrations for respective groups.
4. On LD 7, the concentrations were calculated from the LD 7 body weight and food consumption measured from the respective groups.

The left-over formulated diet from all preparation were sent for incineration.

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.: G23247 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 month 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 measured concentration was within +/- 20% of the nominal concentration and the % RSD was

Duration of treatment / exposure:

Males: 46 days (including 4 weeks prior to mating, mating and post-mating period
Femalse 4 weeks prior to mating and continued through mating, pregnancy and up to Lactation Day (LD) 13.

Frequency of treatment:

Diet was available ad libitum.

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Actual doses (mg/kg/day): Males mean 95 (range 84-103); females (pre-mating) mean 100 (range 99-102); females (gestation) mean 101 (range 89-111); females (lactation) mean 127 (range 112-135)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Actual doses (mg/kg/day): Males mean 284 (range 254-309); females (pre-mating) mean 302 (range 280-324); females (gestation) mean 310 (range 285-333); females (lactation) mean 375 (range 332-443)

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

Males mean 707 (range 660-759); females (pre-mating) mean 748 (range 665-809); females (gestation) mean 812 (range 795-835); females (lactation) mean 977 (range 852-1075)

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:
The dose levels were selected for this study in consultation between Sponsor and testing laboratory based on the results of a 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats performed as a range-finding and palatability study. (The study duration was increased to 21 days after initiation, to allow the animals to overcome initial palatability issues with the diet).
- Fasting period before blood sampling for clinical biochemistry: animals fasted overnight at end of treatment period

The test item was admixed with the powder diet at a target dose level of 100, 300 and 750 mg/kg/day for low, mid and high dose groups respectively and administered orally. Each group in the experiment comprised of 10 male and 10 female rats.

The males were administered orally through diet for six weeks (46 days), up to and including the day before scheduled sacrifice (this included four weeks prior to mating, during the mating period and approximately, one week post mating).

Females were administered orally through diet throughout the treatment period (64 ± 2 days reflecting the variable time to conception excluding female those sacrificed as not littered or dead before LD 14). This included four weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy (22-24 days) and 13 days after delivery, up to and including the day before scheduled sacrifice.

Positive control:

not applicable

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: see below
Individual body weights of males were recorded initially on Day 0 of treatment and at weekly intervals thereafter up to scheduled sacrifice.
Individual body weights of females were recorded initially on Day 1 of treatment and at weekly intervals thereafter till mating confirmation with males.
All dams were weighed on Gestation Days (GDs) 0, 4, 7, 11, 14, 18 and 20 and on LDs 0, 4, 7 and 13.

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period; approximately 4.0 mL of blood was collected by retro-orbital plexus
puncture method under isoflurane anaesthesia (shared with clinical chemistry, haematology and metabolomics).
- 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 period; approximately 4.0 mL of blood was collected by retro-orbital plexus
puncture method under isoflurane anaesthesia (shared with clinical chemistry, haematology and metabolomics).
- 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: see below
- Animals fasted: No
- How many animals: see below
All adults and pups were anesthetized with isoflurane and approximately 1.0 mL of blood was collected by retro-orbital plexus puncture and by the jugular vein with incised at the neck region, respectively. Serum was separated as per the following schedule for the determination of total T4 and TSH hormones:
• At least two available pups per litter on Lactation Day (LD) 4.
• All dams one day prior to sacrifice that is on LD 13.
• Two available pups per litter prior to sacrifice on Day LD13.
• All adult males three days prior to sacrifice (Day 47) that is on Day 44.
Pup blood was pooled by litter for thyroid hormone analyses.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment period
- 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: at termination i.e. towards the end of the treatment period for males (Day 43) and during lactation period for females (LD 13).
- Dose groups that were examined: all males and littered females
- 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:
For reproductive toxicity specific parameters, please see linked record in Section 7.8.1 of IUCLID

Sacrifice and pathology:

Scheduled necrospy
Males: After at least 6 weeks of treatment (Day 47).
Females killed at scheduled termination: Day 14 of lactation.
F1 Offspring: Selected offspring for thyroid hormone analysis – on LD 4.
Scheduled kill of Offspring: LD 13.

GROSS PATHOLOGY: Yes
All adult animals and pups were subjected to detailed necropsy and findings were recorded. The adult animals killed at term (dams on LD 14) were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia. All the surviving pups were necropsied on LD 13 and findings were recorded. Particular attention was paid to the external genitals which were examined for signs of altered development. Dead pups were examined for possible defects and/or cause of death.
HISTOPATHOLOGY: Yes
Tissues/organs collected from randomly selected 5 males and 5 littered females in the control and high dose groups were examined microscopically for histopathological changes. In addition, the reproductive organs and thyroid tissues from the remaining males and females in the control and 750 mg/kg/day
dose groups were examined microscopically for histopathological changes. Histopathological examination of the testes was included a qualitative assessment of stages of spermatogenesis. In addition, all gross lesions from all the animals were examined microscopically.

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

Optional endpoint(s):

For reproductive toxicity specific parameters, please see linked record in Section 7.8.1 of IUCLID

Other examinations:

Urine sample for PAH Metabolites Analysis
Urine sample was collected from all unfasted males from each group (including controls) towards the end of the study. The males had access to diet and water during
sampling. For urine sample collection, each rat was placed overnight in a specially fabricated cage (water and food allowed). After collection, the volume was measured, and samples were rapidly stored frozen and shipped to Sponsor designated site for analysis of PAH metabolites. The duration of the sampling time was recorded. The results of this analysis do not form part of this study report.

Blood collection for metabolomics
As part of the blood collection protocol for this study, some of the blood collectied was mixed with NaEDTA and sent off-site for metabolomics analyses. The results of these analyses do not form part of this study report.

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.
Data including mating and fertility indices were analysed using Chi-square test. When Chi-square was found to be significant, pairwise comparisons of treated
groups to the control group was made using a Fisher Exact test, to identify statistical difference in ProvantisTM built-in statistical tests

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed throughout the treatment period in either sex at any of the doses tested.
There were no abnormalities observed in the pups.

Mortality:

no mortality observed

Description (incidence):

One female rat in the control group was found dead on LD1 (necropsy revealed the pericardium of the heart was adhered to the lung), and this was considered a spontaneous incident.
No mortalities were observed in the treated groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In males, the mean body weights and body weight gains were unaffected throughout treatment. There was a slight but not statistically significant reduction in bodyweight gain at 750 mg/kg/day in the first week of treatment and this corresponded to slightly reduced food consumption. Towards the end of treatment there was a transient but statistically significant reduction in bodyweight at 750 mg/kg/day, but at necropsy (Day 44) all groups were comparable to the controls.
In females there was a small, but statistically significant, reduction in bodyweight gain over the first 4 weeks of treatment in females given 750 mg/kg/day. This was not considered to be toxicologically relevant due to lack of consistency and further, the mean weekly body weights were comaparable to control.
Treatment had no effects on the body weights, body weight gain and food consumption measured during different intervals of the gestation period at all the tested doses when compared to the control group. Isolated occurrence of significantly lower body weight was observed on GD 11 at 750 mg/kg/day. However, the body weight gain is comparable to control during the same period. Hence, it was considered not related to treatment.
During lactation there was a decrease in absolute body weight and bodyweight gain in the 300 and 750 mg/kg/day groups, but overall bodyweight gain was not statistically different. Food consumption of Groups 3 and 4 was higher than anticipated and actual exposure to the test item was above the nominal dose level; this may have contributed to the slight decrease in bodyweight gain over
the lactation period.

See "Any other information on results" for bodyweight summary tables.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Generally, the mean food consumption was unaffected throughout treatment in males and during premating period in females at all the tested doses. A slight transient reduction in food consumption was observed during week 1. The sporadic incidences of lower/higher food consumption were considered incidental due to lack of consistency or dose progression.

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

Description (incidence and severity):

Increased total leucocyte count in association with increased lymphocytes/monocytes were noted in females at 300 and 750 mg/kg/day and considered as test item related. However, as no corresponding inflammatory changes were observed histologically at the highest dose group, this finding was considered not to be adverse.

See "Any other information on results" for haematology summary tables.

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Home Cage, Handling, Open field and Sensory Observations:
No treatment-related abnormalities were observed in any of the tested dose groups in both sexes.
Landing hind limb foot splay:
No treatment-related abnormalities were observed in any of the doses tested in both sexes.
Grip strength:
No significant changes were observed at all the tested doses in both sexes, except an incidental finding of marginally higher average grip strength values in males of 100 and 300 mg/kg/day.
Motor activity:
Isolated instances of statistically significant findings were observed in males at 750 mg/kg/day (distance travelled; interval 2 only) and females at 750 mg/kg/day (resting time; interval 3 only).
The changes were considered incidental as they were not dose proportional and/or consistent and the associated findings were not observed during home cage, handling, open field or sensory observations. In addition, the findings were not apparent in the remaining two intervals of data collection.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In adults, at 750 mg/kg/day, the minimal but statistically significant variations noted in weights of kidney (↑) in males, brain (↑) and spleen (↓) in females were not associated with any histological changes and thus considered as incidental findings.
Administration of test item resulted in higher liver weight in parental males and females at ≥ 300 mg/kg/day dose levels and was associated with hepatocyte hypertrophy, histologically.
For Liver weights see "Any other information on results" for summary tables.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

One male at 750 mg/kg/day had an enlarged liver (all lobes) that was associated with hepatocellular hypertrophy and was considered related to test item administration.
Incidental findings were noted in some animals across all groups; these were not considered related to test item treatment, but where there were microscopic correlates, these are presented in a table in "Any other information on results" for summary tables.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item related microscopic changes were noted in liver and thyroid in males and females.
Liver: Hepatocyte hypertrophy observed at ≥ 300 mg/kg/day dose in both sexes were considered as test item related. The severity and incidences were slightly
higher in females than the males. Hepatocellular hypertrophy was of minimal to mild severity, centrilobular in distribution and was considered as non-adverse adaptive response to test item in the absence of necrosis/degeneration in hepatocyte and in the liver enzyme profiles. This change was grossly associated with enlarged liver (single incidence in male at750 mg/kg/day) and increased weight.

Thyroid: Follicular cell hypertrophy was observed in males at ≥ 300 mg/kg/day with minimal to mild severity in males whereas in females at 750 mg/kg/day with minimal severity and considered as test item-related change. However, associated increased thyroid profile hormone was not noted in either sex. Thyroid follicular cell hypertrophy may be secondary to hepatocellular hypertrophy. The affected thyroid follicles were lined by cuboidal to columnar epithelium with central follicles tightly packed and smaller than normal follicle with depletion of secretion.

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects reported

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

no

Bodyweight Summary Tables

Male
Group & Sex	G1M	G2M	G3M	G4M
Dose (mg/kg/day)	0	100	300	750
Bodyweight (g) Day 0	392	390	395	389
Bodyweight (g) Day 7	429	427	431	413
Bodyweight (g) Day 28	462	472	470	459
Bodyweight (g) Day 44	484	495	485	484
Absolute Gain Days 0-44	92	105	90	95
Total % Weight Gain Days 0 -44	24	27	23	24

Female (pre-mating)
Group & Sex	G1F	G2F	G3F	G4F
Dose (mg/kg/day)	0	100	300	750
Bodyweight (g) Day 0	257	262	261	262
Bodyweight (g) Day 28	283	297	285	274
Absolute Gain Days 0-28	25.95	35.21	23.85	12.04*
Total % Weight Gain Days 0-28	10.14	13.38	9.16	4.64*
* = p < 0.05

Female (gestation)
Group & Sex	G1F	G2F	G3F	G4F
Dose (mg/kg/day)	0	100	300	750
Number of animals	9	10	10	10
GD0	283	298	287	275
GD20	421	444	424	298
Absolute Gain GD0-GD20	137	146	137	123

Female (lactation)
Group & Sex	G1F	G2F	G3F	G4F
Dose (mg/kg/day)	0	100	300	750
Number of animals	9	10	9	10
LD0	338.55	347.29	327.1	315.45
LD4	352.05	355.28	333.16*	330.57*
LD7	360.41	363.74	334.47*	331.55*
LD13	364.79	367.74	340.33*	325.95*
Absolute gain LD0-13	26.24	20.45	13.23	10.5
* = p < 0.05 as compared to G1F at timepoint

Maternal bodyweight gains and actual dose levels (g) during lactation period

Nominal dose (mg/kg/day)	300	300	750	750
Lactation Day Number	Actual dose (mg/kg/day	Bodyweight gain (g)	Actual dose (mg/kg/day	Bodyweight gain (g)
0-4	332.07	6.06	851.55	15.12
4-7	442.56	1.31	1003.76	0.98
7-13	349.72	5.87	1075.49	-5.6

Haematology

Test item related changes in haematological parameters of different groups have been listed below

Group	G1M	G2M	G3M	G4M	G1F	G2F	G3F	G4F
Dose (mg/kg/day)	0	100	300	750	0	100	300	750
No. of rats	10	10	10	10	8	10	9	9
WBC Absolute Mean (10^9/L)	8.7	9.71	9.24	8.71	8.6	8.52	10.45	12.34
WBC % diff		↑ 12%	↑ 6%	↑ 0.1%		↓ 1%	↑ 21%	↑ 43%
Lymphocyte Absolute Mean (109/L)	6.08	6.83	6.66	5.25	4.37	4.64	5.61	7.82
Lymphocyte % diff		↑ 12%	↑ 10%	↓ 14%		↑ 6%	↑ 28%	↑ 79%
Monocyte Absolute Mean (109/L)	0.27	0.3	0.24	0.33	0.33	0.87	0.35	0.46
Monocyte % diff		↑ 11%	↓ 13%	↑ 21%		↑ 167%	↑ 7%	↑ 41%

* = p < 0.05, Findings considered related to treatment in bold

Liver weights (absolute and relative)

Group	G1M	G2M	G3M	G4M	G1F	G2F	G3F	G4F
Dose (mg/kg/day)	0	100	300	750	0	100	300	750
No. of rats	10	10	10	10	8	10	9	9
Absolute weight (Mean)	12.16	12.12	13.44*	13.75*	11.74	12.07	12.79	13.08
Absolute weight (% diff)	-	-	↑ 10%	↑ 13%	-	↑ 3%	↑ 9%	↑ 11%
Relative to Bwt (Mean)	2.65	2.56	2.88*	3.03*	3.65	3.75	4.12*	4.43*
Relative to Bwt (% diff)	-	↓ 3%	↑ 12%	↑ 13%	-	↑ 4%	↑ 9%	↑ 11%

*: statistically significant change at p < 0.05

Changes marked ion bold considered related to test item administration

Incidental gross pathology changes and correlates

Organ	Doses affected (mg/kg/day)	Sex	Gross finding	Microscopic correlates
Liver	0	1 male	Red discolouration; papillary process	Hepatocellular Necrosis/haemorrhages
Kidney	0	1 male	Pelvis dilatation, unilateral	Confirmed histologically
Kidney	750	1 male	Pelvis dilatation, unilateral	Confirmed histologically
Testes	300	1 male	Flabby, bilateral	Degeneration, seminiferous tubules; bilateral
Epididymides	300	1 male	white focus; cauda; unilateral	sperm granuloma
Heart	0	1 female	Adhesion; pericardium	Pericardium; Inflammation; multi-focal
Urinary bladder	0	1 female	Red discolouration bilateral	Hypertrophy; transitional epithelium/submucosal haemorrhages/infiltration
Uterus	0	1 female	Abcess horn multiple	Multiple abcess in implant site
Uterus	100	1 female	focal red discolouration	Decidual reaction
Tail	750	1 male	wound	Focus epidermal; Necrosis

Conclusions:

Distillates (petroleum), hydrotreated middle (also known as Other Gas Oils CAS 64742-46-7) was administered through diet to Wistar rats at the target dose levels of 100, 300 and 750 mg/kg/day for 4 weeks prior to mating, during mating, and post mating (males) or 4 weeks prior to mating, during mating and pregnancy until 13 days after delivery (females).

As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose of 750 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) for systemic, reproduction and developmental toxicity for the test item is determined to be 750 mg/kg/day corresponding achieved dose level of 707 mg/kg/day for males and 799 mg/kg/day for females (corresponding achieved dose level throughout the treatment period) under the test conditions and doses employed.

Non-adverse hypertrophy in the liver and follicular cell hypertrophy of the thyroid resulted in a No Observed Effect level (NOEL) of 100 mg/kg/day in both sexes. Equivalent to 95 (84-103) mg/kg/day in males and 104 (95-127) mg/kg/day in females.

Executive summary:

Distillates (petroleum), hydrotreated middle (also known as Other Gas Oils CAS 64742-46-7) was administered through diet to Wistar rats at the target dose levels of 100, 300 and 750 mg/kg/day for 4 weeks prior to mating, during mating, and post mating (males) or 4 weeks prior to mating, during mating and pregnancy until 13 days after delivery (females) and resulted in following findings:

There were no effects on clinical observations, or the parameters investigated in the Functional Observation Battery.

Treatment had no effects on mean body weight, body weight gains and food consumption throughout the treatment period in males or during pre-mating period in females. In males, there was a slight but not statistically significant reduction in bodyweight gain at 750 mg/kg/day in the first week of treatment and this corresponded to slightly reduced food consumption. Towards the end of treatment there was a transient but statistically significant reduction in bodyweight at 750 mg/kg/day, but at necropsy (Day 44) all groups were comparable to the controls.

There was a reduction in maternal bodyweight at 300 and 750 mg/kg/day during the lactation period. However, actual achieved dosages were higher than the nominal level during this period due to increased food consumption; it is not

clear if this may have contributed to the difference in bodyweight. There was no effect at 100 mg/kg/day.

The mean oestrous cycle length and number of cycles during pre-mating period, pre-coital time was unaffected by the treatment at all of the tested doses. The number of pregnancies, gestation length (average days to litter), mating and fertility indices of sires and dams were unaffected at all the tested doses.

Test item had no treatment-related effects on the mean litter size, mean viable litter size, percentage of male pups, the number of dead pups at first observation, number of live pups on LD4 and LD13 up to the highest dose

tested. No treatment-related changes were observed in the survival index of pups, mean body weight of male and female pups per litter, litter mean pup body weight at birth, ano-genital distance and ano-genital ratio. There was a decrease in mean pup weight from Day 7 of lactation at 300 and 750 mg/kg/day, this may be related to the reduced maternal bodyweight gain during this period.

The vaginal smear examined for all the animals prior to necropsy did not show any abnormality.

Test item related increased WBC and lymphocytes/monocytes were noted in females at 300 and 750 mg/kg/day. However, no corresponding inflammatory changes were observed histologically at the highest dose group and this finding

was considered not to be adverse.

There were no biologically significant changes noted in thyroid stimulating hormone (TSH) and thyroxine (T4) levels in post-natal Days 4 and 13 pups nor in adult male or females.

Administration of test item resulted in higher liver weight in parental males and females at ≥ 300 mg/kg/day dose levels and was associated with hepatocyte hypertrophy, histologically. The reproductive tissue evaluation did not reveal any test item related effects.

Microscopically, hepatocyte hypertrophy at ≥300 mg/kg/day males and females were accompanied by higher liver weight. Follicular cell hypertrophy in thyroid in males at ≥300 mg/kg/day and females at 750 mg/kg/day were noted may be secondary to the liver findings. This finding was considered as adaptive nonadverse effect of test item administration.

There were no test item related gross and microscopic changes in reproductive organs in both males and females.

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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Distillates (petroleum), hydrotreated middle

EC Number:

265-148-2

EC Name:

Distillates (petroleum), hydrotreated middle

Cas Number:

64742-46-7

Molecular formula:

Not applicable - UVCB

IUPAC Name:

Distillates (petroleum), hydrotreated middle

Test material form:

liquid

Details on test material:

Concawe sample ref S726
Clear light green liquid

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and 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
- Housing:
Pre-mating - two rats of same sex per cage
Mating - two rats (one male: one female of the same group) per cage
Post-mating - presumed pregnant females (pregnancy confirmed by vaginal plug or presence of
sperm in vaginal smear) were housed individually until sacrifice; males housed with former cage
partners
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 Spezialfut
ter 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 - 24.1 C
- Humidity (%): 64-67%
- Air changes (per hr): 12.6
- Photoperiod (hrs dark / hrs light): 12 light/12 dark

IN-LIFE DATES: From: 8th April 2022 To: 7th July 2022

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: standard feed

Details on exposure:

Details on oral exposure
Following procedure was followed when 6 kg of experimental diet was prepared.
The required quantities of test item were weighed in the labeled 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 (5.5 kg) and mixed in stainless steel ribbon blender for 20 minutes. For animals in control group, 6 kg of the basal diet was mixed in 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.

Males:
Concentrations in the diet for the first two weeks of the study were calculated using body weights and feed consumption measured during the pre-treatment period. Subsequently, concentrations in the diet were adjusted on Days 14, 28 and 42, based on the preceding week body weights and food consumption. During the mating period, males had access to female diet.

Females:
Concentrations in the diet for the first two weeks of the study were calculated using body weights and feed consumption measured during the pre-treatment period. Subsequently, concentrations in the diet were adjusted on Day 14 based on the preceding week body weights and food consumption. In order to avoid overexposure, during gestation and lactation periods, dietary concentrations were calculated twice during gestation (GD 0 and GD 14) and twice on lactation (LD 0 and 7) as detailed below:
1. On GD 0, the concentrations were calculated from the GD 0 body weight and food consumption measured during pre-mating period on Day 28 from the respective groups.
2. On GD 14, the concentrations were calculated by adding 60g to the GD 14 body weight and food consumption from the respective groups.
3. On LD 0, the constant body weight of 350g for G2 and G3, 330g for G4 and constant food weight of 35g were used to calculate the concentrations for respective groups.
4. On LD 7, the concentrations were calculated from the LD 7 body weight and food consumption measured from the respective groups.
The left-over formulated diet from all preparation were sent for incineration.

Details on mating procedure:

One female was placed with one male from the same group. Cohabitation was continued until there was evidence of sperm in the vaginal smear and /or
vaginal plug (all females were confirmed as mated within 6 days from the day of cohabitation).
The day of confirmed mating was designated as Gestation Day 0 (GD 0). The pre-coital time was calculated for each female.

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.: G23247 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 month 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 measured concentration was within +/- 20% of the nominal concentration and the % RSD was

Duration of treatment / exposure:

Males: 46 days (including 4 weeks prior to mating, mating and post-mating period
Femalse 4 weeks prior to mating and continued through mating, pregnancy and up to Lactation Day (LD) 13.

Frequency of treatment:

Diet was available ad libitum.

Details on study schedule:

- Age at mating of the mated animals in the study: 16 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Actual doses (mg/kg/day): Males mean 95 (range 84-103); females (pre-mating) mean 100 (range 99-102); females (gestation) mean 101 (range 89-111); females (lactation) mean 127 (range112-135)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Actual doses (mg/kg/day): Males mean 284 (range 254-309); females (pre-mating) mean 302 (range 280-324); females (gestation) mean 310 (range 285-333); females (lactation) mean 375 (range 332-443)

Doses / concentrations 3

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

Actual doses (mg/kg/day): Males mean 707 (range 660-759); females (pre-mating) mean 748 (range 665-809); females (gestation) mean 812 (range 795-835); females (lactation) mean 977 (range 852-1075)

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: The dose levels were selected for this study in consultation between Sponsor and testing laboratory based on the results of a 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats performed as a range-finding and palatability study. (The study duration was increased to 21 days after initiation, to allow the animals to overcome initial palatability issues with the diet).
- Fasting period before blood sampling for clinical biochemistry: animals fasted overnight at end of treatment period

The test item was admixed with the powder diet at a target dose level of 100, 300 and 750 mg/kg/day for low, mid and high dose groups respectively and administered orally. Each group in the experiment comprised of 10 male and 10 female rats.

The males were administered orally through diet for six weeks (46 days), up to and including the day before scheduled sacrifice (this included four weeks prior to mating, during the mating period and approximately, one week post mating).

Females were administered orally through diet throughout the treatment period (64 ± 2 days reflecting the variable time to conception excluding female those sacrificed as not littered or dead before LD 14). This included four weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy (22-24 days) and 13 days after delivery, up to and including the day before scheduled sacrifice.

Positive control:

not applicable

Examinations

Parental animals: Observations and examinations:

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: see below
Individual body weights of males were recorded initially on Day 0 of treatment and at weekly intervals thereafter up to scheduled sacrifice.
Individual body weights of females were recorded initially on Day 1 of treatment and at weekly intervals thereafter till mating confirmation with males.
All dams were weighed on Gestation Days (GDs) 0, 4, 7, 11, 14, 18 and 20 and on LDs 0, 4, 7 and 13.

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period; approximately 4.0 mL of blood was collected by retro-orbital plexus puncture method under isoflurane anaesthesia (shared with clinical chemistry, haematology and meta bolomics).
- 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 period; approximately 4.0 mL of blood was collected by retro-orbital plexus puncture method under isoflurane anaesthesia (shared with clinical chemistry, haematology and metabolomics).
- 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: see below
- Animals fasted: No
- How many animals: see below
All adults and pups were anesthetized with isoflurane and approximately 1.0 mL of blood was collected by retro-orbital plexus puncture and by the jugular vein with incised at the neck region, respectively. Serum was separated as per the following schedule for the determination of total T4 and TSH hormones:
• At least two available pups per litter on Lactation Day (LD) 4.
• All dams one day prior to sacrifice that is on LD 13.
• Two available pups per litter prior to sacrifice on Day LD13.
• All adult males three days prior to sacrifice (Day 47) that is on Day 44.
Pup blood was pooled by litter for thyroid hormone analyses.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment period
- 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: at termination i.e. towards the end of the treatment period for
males (Day 43) and during lactation period for females (LD 13).
- Dose groups that were examined: all males and littered females
- 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:
Results from some of the parameters listed above are contained in the linked record in Section 7.5.1 of IUCLID

Oestrous cyclicity (parental animals):

Vaginal smears were examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular 4-5 days cyclicity for inclusion onto the study. The vaginal smear was also examined daily two weeks prior to mating period until evidence of mating.

The time interval (in days) from the dioestrus of an oestrous cycle to the next dioestrus was considered as the oestrous cycle length of an animal.

Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Sperm parameters (parental animals):

Testes and Epididymus were taken and weighed.
Histopathological examination of the testes was included a qualitative assessment of stages of spermatogenesis.

Litter observations:

At birth, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities.

The number of pups born (litter size), sex and individual pup body weight of male and female pups on LDs 0 and 4 were recorded.

The ano-genital distance (AGD) of each pup was measured on LD 0 and pup body weight was recorded. Ano-genital distance ratio (AGR) was calculated by dividing the ano-genital distance from the cube root of body weight.

On LD 4, after recording the body weight, the size of each litter was adjusted by eliminating extra pups by random selection in Provantis to yield, as nearly as possible, four pups per sex per litter. Partial adjustment was
done when the number of male or female pups prevents having four of each sex per litter. Pups were not eliminated when the litter size was below the culling target (8 pups/litter). Blood samples were collected from the available surplus pups of either sex, pooled, and used for determination of serum Thyroxine (T4) and Thyroid stimulating hormone (TSH) levels.

After standardization, the individual pup body weight was measured on LD13.

The number of nipples/areolae in male pups was counted on LD 13.

All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.

The litters were observed daily to note the number of alive, dead and cannibalized pups.

In addition to daily clinical observations, all pups were observed for any abnormal behaviour.

Fertility index for dams, sires as well as the pup survival index LD 4, 7 and 14 were calculated.

Postmortem examinations (parental animals):

Scheduled necrospy
Males: After at least 6 weeks of treatment (Day 47).
Females killed at scheduled termination: Day 14 of lactation.

GROSS PATHOLOGY: Yes
All adult animals and pups were subjected to detailed necropsy and findings were recorded. The adult animals killed at term (dams on LD 14) were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia.

HISTOPATHOLOGY: Yes
Tissues/organs collected from randomly selected 5 males and 5 littered females in the control and high dose groups were examined microscopically for histopathological changes. In addition, the reproductive organs and thyroid tissues from the remaining males and females in the control and 750 mg/kg/day dose groups were examined microscopically for histopathological changes. Histopathological examination of the testes was included a qualitative assessment of stages of spermatogenesis. In addition, all gross lesions from all the animals were examined microscopically.

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

Postmortem examinations (offspring):

Scheduled necrospy
F1 Offspring: Selected offspring for thyroid hormone analysis – on LD 4.
Scheduled kill of Offspring: LD 13.

All the surviving pups were necropsied on LD 13 and findings were recorded. Particular attention was paid to the external genitals which were examined for signs of altered development. Dead pups were examined for possible defects and/or cause of death.

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.

Data including mating and fertility indices were analysed using Chi-square test. When Chi-square was found to be significant, pairwise comparisons of treated groups to the control group was made using a Fisher Exact test, to identify statistical difference in ProvantisTM built-in statistical tests.

Reproductive indices:

Reproductive Performance Data of Parents
a. Male mating index (%) = (Number of males with evidence of mating)/(Number of males cohabited) x 100
b. Male fertility index (%) = (Number of males siring a litter/impregnated a female)(Number of males with evidence of mating) x 100
c. Female mating index (%) = (Number of females mated)/(Number of females cohabited) x 100
d. Female fertility index (%) = (Number of pregnant females)/(Number of females with evidence of mating) x 100
e. Mean number of implantations per group = (Total number of implantations)/(Total number of pregnant animals)
f. Post implantation loss (%) = (Number of implantations - Number of live pups)/(Number of implantations) x 100

Offspring viability indices:

Litter data
a. Mean litter size per group = (Total Number of pups born)/(Total Number of littered animals)
b. Mean viable litter size = (No. of viable pups)/(Total Number of littered animals)
c. Live birth index (%) = (No. of viable pups born (at first observation))/(Total no. of pups born (at first observation)) x 100
d. Day 4 survival index (%) = (Number of viable pups on lactation Day 4)/(Number of viable pups born) x 100
e. Sex Ratio/ Percentage of male offspring (%) = (No. of male pups born)/(Total no. of pups born) x 100
f. Ano-genital Distance Ratio (mm/g1/3) = (Ano-genital distance)/(Cube root of body weight)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed throughout the treatment period in either sex at any of the doses tested.
There were no abnormalities observed in the pups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

One female rat in the control group was found dead on LD1 (necropsy revealed the pericardium of the heart was adhered to the lung), and this was considered a spontaneous incident.
No mortalities were observed in the treated groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In males, the mean body weights and body weight gains were unaffected throughout treatment. There was a slight but not statistically significant reduction in bodyweight gain at 750 mg/kg/day in the first week of treatment and this corresponded to slightly reduced food consumption. Towards the end of treatment there was a transient but statistically significant reduction in bodyweight at 750 mg/kg/day, but at necropsy (Day 44) all groups were comparable to the controls.

In females there was a small, but statistically significant, reduction in bodyweight gain over the first 4 weeks of treatment in females given 750 mg/kg/day. This was not considered to be toxicologically relevant due to lack of consistency and further, the mean weekly body weights were comaparable to control.

Treatment had no effects on the body weights, body weight gain and food consumption measured during different intervals of the gestation period at all the tested doses when compared to the control group. Isolated occurrence of significantly lower body weight was observed on GD 11 at 750 mg/kg/day. However, the body weight gain is comparable to control during the same period. Hence, it was considered not related to treatment.

During lactation there was a decrease in absolute body weight and bodyweight gain in the 300 and 750 mg/kg/day groups, but overall bodyweight gain was not statistically different. Food consumption of Groups 3 and 4 was higher than anticipated and actual exposure to the test item was above the nominal dose level; this may have contributed to the slight decrease in bodyweight gain over
the lactation period.

See linked study for bodyweight summary tables

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Generally, the mean food consumption was unaffected throughout treatment in males and during premating period in females at all the tested doses. A slight transient reduction in food consumption was observed during week 1. The sporadic incidences of lower/higher food consumption were considered incidental due to lack of consistency or dose progression.

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

Description (incidence and severity):

Increased total leucocyte count in association with increased lymphocytes/monocytes were noted in females at 300 and 750 mg/kg/day and considered as test item related. However, as no corresponding inflammatory changes were observed histologically at the highest dose group, this finding was considered not to be adverse.

See linked study for haematology summary tables

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Home Cage, Handling, Open field and Sensory Observations:
No treatment-related abnormalities were observed in any of the tested dose groups in both sexes.

Landing hind limb foot splay:
No treatment-related abnormalities were observed in any of the doses tested in both sexes.

Grip strength:
No significant changes were observed at all the tested doses in both sexes, except an incidental finding of marginally higher average grip strength values in males of 100 and 300 mg/kg/day.

Motor activity:
Isolated instances of statistically significant findings were observed in males at 750 mg/kg/day (distance travelled; interval 2 only) and females at 750 mg/kg/day (resting time; interval 3 only).

The changes were considered incidental as they were not dose proportional and/or consistent and the associated findings were not observed during home cage, handling, open field or sensory observations. In addition, the findings were not apparent in the remaining two intervals of data collection.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item related microscopic changes were noted in liver and thyroid in males and females.
Liver: Hepatocyte hypertrophy observed at ≥ 300 mg/kg/day dose in both sexes were considered as test item related. The severity and incidences were slightly higher in females than the males. Hepatocellular hypertrophy was of minimal to mild severity, centrilobular in distribution and was considered as non-adverse adaptive response to test item in the absence of necrosis/degeneration in hepatocyte and in the liver enzyme profiles. This change was grossly associated with enlarged liver (single incidence in male at750 mg/kg/day) and increased weight.

Thyroid: Follicular cell hypertrophy was observed in males at ≥ 300 mg/kg/day with minimal to mild severity in males whereas in females at 750 mg/kg/day with minimal severity and considered as test item-related change. However, associated increased thyroid profile hormone was not noted in either sex. Thyroid follicular cell hypertrophy may be secondary to hepatocellular hypertrophy. The affected thyroid follicles were lined by cuboidal to columnar epithelium with central follicles tightly packed and smaller than normal follicle with depletion of secretion.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The calculated mean oestrous cycle length was 4.3, 4.4, 4.4 and 4.3 days in vehicle control, low, mid and high dose groups, respectively. The mean oestrous
cycle length in the treated groups was comparable to the vehicle control group. All the groups had 2 oestrous cycle measured two weeks during the pre-mating
period.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no test item-related histopathological changes noted in reproductive tissues of the parental male/female rats. The qualitative assessment of
spermatogenesis in testes did not reveal any changes in the examined rats.

Reproductive performance:

no effects observed

Description (incidence and severity):

Fertility parameters
There were no treatment-related effects on the mean pre-coital time, number of pregnancies, gestation length (average days to litter), mating and fertility indices of sires and dams at all the tested dose levels. The mating index was 100 % across all dose groups but one control female failed to litter (found not to be non- pregnant).

One female rat in the mid dose was not littered and sacrificed on GD 26. This animal was found to have one dead foetus in the womb.

Litter data
The test item had no treatment-related effects on the mean litter size, mean viable litter size, number of dead pups at first observation, number of live pups
on LD4 and LD13.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects reported

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no abnormalities observed in the pups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weight of male and female pups per litter and litter mean pup body weight were not affected by treatment at all the tested dose levels. Litters were culled to normalise population number on LD 4. On LD 13 mean bodyweight at 300 and 750 mg/kg/day was lower than the controls, although the difference was not statistically significant. A reduction in maternal weight was also noted during this period.

The lower but statistically not significant lower mean pup body weight at 300 and 750 mg/kg/day doses was observed D13. This lower weight was well within the historical range (HD range is 12.62 – 37.70 with mean value is 29.64g). Hence, this was considered not related to treatment.

See summary table in "Any other information on results"

Food consumption and compound intake (if feeding study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No changes attributable to the test item were detected in the Ano-genital distance Ano-genital ratio and Ano-genital index in either sex.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

The male pups did not exhibit areola/nipple retention on PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

There were no significant changes in absolute and relative thyroid weight of pups on lactation Day 13.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no developmental and non-developmental gross findings noted in male or female pups.

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic changes in the thyroid gland of 13-Day old pups.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects reported

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Pup bodyweights

Group & Sex	G1F	G2F	G3F	G4F
Dose (mg/kg/day)	0	100	300	750
Number of animals	9	10	9	9
Males (g) D0	6.42	6.6	6.77	6.91
Females (g) D0	6.27	6.38	6.55	6.65
Litter mean pup weight (g) d0	6.25	6.47	6.65	6.79
Litter mean pup weight (g) d13	31.11	28.41	26.24	26.47
SD	4.71	4.8	3.11	3.81

Applicant's summary and conclusion

Conclusions:

Distillates (petroleum), hydrotreated middle (also known as Other Gas Oils CAS 64742-46-7) was administered through diet to Wistar rats at the target dose levels of 100, 300 and 750 mg/kg/day for 4 weeks prior to mating, during mating, and post mating (males) or 4 weeks prior to mating, during mating and pregnancy until 13 days after delivery (females).

As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose of 750 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) for systemic, reproduction and developmental toxicity for the test item is determined to be 750 mg/kg/day corresponding achieved dose level of 707 mg/kg/day for males and 799 mg/kg/day for females (corresponding achieved dose level throughout the treatment period) under the test conditions and d oses employed.

Non-adverse hypertrophy in the liver and follicular cell hypertrophy of the thyroid resulted in a No
Observed Effect level (NOEL) of 100 mg/kg/day in both sexes. Equivalent to 95 (84-103) mg/kg/day in
males and 104 (95-127) mg/kg/day in females.

Executive summary:

Distillates (petroleum), hydrotreated middle (also known as Other Gas Oils CAS 64742-46-7) was administered through diet to Wistar rats at the target dose levels of 100, 300 and 750 mg/kg/day for 4 weeks prior to mating, during mating, and post mating (males) or 4 weeks prior to mating, during mating and pregnancy until 13 days after delivery (females) and resulted in following findings:

There were no effects on clinical observations, or the parameters investigated in the Functional Observation Battery.

Treatment had no effects on mean body weight, body weight gains and food consumption throughout the treatment period in males or during pre-mating period in females. In males, there was a slight but not statistically significant reduction in bodyweight gain at 750 mg/kg/day in the first week of treatment and this corresponded to slightly reduced food consumption. Towards the end of treatment there was a transient but statistically significant reduction in bodyweight at 750 mg/kg/day, but at necropsy (Day 44) all groups were comparable to the controls.

There was a reduction in maternal bodyweight at 300 and 750 mg/kg/day during the lactation period. However, actual achieved dosages were higher than the nominal level during this period due to increased food consumption; it is not clear if this may have contributed to the difference in bodyweight. There was no effect at 100 mg/kg/day.

The mean oestrous cycle length and number of cycles during pre-mating period, pre-coital time was unaffected by the treatment at all of the tested doses. The number of pregnancies, gestation length (average days to litter), mating and fertility indices of sires and dams were unaffected at all the tested doses.

Test item had no treatment-related effects on the mean litter size, mean viable litter size, percentage of male pups, the number of dead pups at first observation, number of live pups on LD4 and LD13 up to the highest dose

tested. No treatment-related changes were observed in the survival index of pups, mean body weight of male and female pups per litter, litter mean pup body weight at birth, ano-genital distance and anogenital ratio. There was a decrease in mean pup weight from Day 7 of lactation at 300 and 750 mg/kg/day, this may be related to the reduced maternal bodyweight gain during this period.

The vaginal smear examined for all the animals prior to necropsy did not show any abnormality.

Test item related increased WBC and lymphocytes/monocytes were noted in females at 300 and 750 mg/kg/day. However, no corresponding inflammatory changes were observed histologically at the highest dose group and this finding was considered not to be adverse.

There were no biologically significant changes noted in thyroid stimulating hormone (TSH) and thyroxine (T4) levels in post-natal Days 4 and 13 pups nor in adult male or females.

Administration of test item resulted in higher liver weight in parental males and females at ≥ 300 mg/kg/day dose levels and was associated with hepatocyte hypertrophy, histologically. The reproductive tissue evaluation did not reveal any test item related effects.

Microscopically, hepatocyte hypertrophy at ≥300 mg/kg/day males and females were accompanied by higher liver weight. Follicular cell hypertrophy in thyroid in males at ≥300 mg/kg/day and females at 750 mg/kg/day were noted may be secondary to the liver findings. This finding was considered as adaptive nonadverse effect of test item administration.

There were no test item related gross and microscopic changes in reproductive organs in both males and females.