Pyrolysis light oil from waste plastics

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-2022

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Test item formulations were prepared every four days by mixing the calculated amount of the test item (taking into account the density of the liquid) with the calculated volume of corn oil (separately for each concentration), aliquoted to the required volumes of days of the administration, and stored in tightly closed flacks in a cool ventilated place (2 - 8 °C).
Concentration in vehicle: 5, 20 and 80 mg/mL

Details on analytical verification of doses or concentrations:

Verification of dose concentrations was carried by validated GC-MS method. Dosing formulations were analysed for homogeneity, stability and concentration. Stability of the test item in the vehicle prepared at concentrations of 5 and 80 mg/mL was confirmed following 4 days of at +4 - 8 °C temperature. Analysis of 5, 20 and 80 mg/mL formulations for homogeneity and concentration was conducted in the test facility at the beginning, in the middle and at the end of in-life phase. After 4 days of storage, the concentration of 5 and 80 mg/mL formulations was within the acceptable range of the target concentration (85–115% with RSD < 10%) and met the acceptance criteria for test item formulation stability in the vehicle (>85% of the pre-storage value with <10% RSD). During analysis of homogeneity, the mean actual concentration for all strata exceeds the acceptable limit (85 -115%) of the target concentration on date 03 Dec 2021 (all formulations) and 16 Jan 2022 (medium and high-level doses); however, re-analysis of back-up samples from medium levels on these dates revealed acceptance of concentration values. The variability between strata was low for all dates (RSD < 10.0%), which can confirm dose homogeneity. The analyzed concentration was within the acceptable range of 85 -115% of the target concentration at the beginning, middle, and end of the in-life phase of the study, that was confirmed by re-analysis of backup samples for date 03 Dec.2021 and 16 Jan 2022.

Duration of treatment / exposure:

Males: Treatment for 28 days, beginning 14 days prior to mating, throughout the mating period until necropsy.
Females: Females which delivered were treated for 49 – 63 days, beginning 14 days prior to mating until lactation day 13. Females which failed to deliver were treated for 55 days.
Animals of the recovery group were not mated and dosed for 28 days (males) or 55 days (females) with following two weeks recovery period.

Frequency of treatment:

Daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12 (main subgroup, each group)
5 (recovery subgroup, for control and high dose groups)

Control animals:

yes, concurrent vehicle

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

400 mg/kg bw/day dose: one dam had signs of hypolactation with offspring emaciation. Hypolactation was confirmed by histopathological evaluation. Another dam had emaciated litter but hypolactation was not confirmed by mammary gland microscopy (was not extensive). Despite the uniqueness of hypolactation in single female, its relation to the treatment is not excluded.
One female treated with 25 mg/kg bw/day had unilateral chromodacryorrhea and a spot on the cornea identified by ophthalmoscopy presumably of traumatic origin and not treatment-related

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

100 mg/kg bw/day: decrease in monocytes and granulocytes count (significant) and decrease in RBC (significant) in males, non-adverse
400 mg/kg bw/day: decrease in monocytes and granulocytes count (significant) and decrease in RBC (non-significant) in males, decrease in monocytes and granulocytes count (non-significant) in females, and decrease in lymphocyte count (significant) in females, non-adverse.
Haematological changes in the 400 mg/kg bw/day administered males and females correlated to the histopathological findings in the spleen, but not to the findings in the bone marrow smear. No significant change in the relative count of bone marrow monocytes and lymphocytes were observed in animals treated with the high dose. Hematological changes on the test item were reversible and not observed in recovery subgroups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

100 mg/kg bw/day: decreased level of blood glucose (significant) in males, non-adverse.
400 mg/kg bw/day: decreased level of blood glucose (significant) and decreased level of calcium (significant) in males, non-adverse.
Males treated with 100 and 400 mg/kg bw/day doses had decreased level of blood glucose (3.5 ± 0.3 and 3.5 ± 0.4 mmol/L) compared to the control value of 4.1 ± 0.5 mmol/L (p < 0.05). Hypoglycemia can be related to the liver functional insufficient and influence in glycogenolysis, correlated to the microscopic findings in the liver, and considered test item-related but not adverse. The decrease in calcium level was observed in the 400 mg/kg bw/day male group (2.47±0.03 mmol/L compared 2.55±0.07 mmol/L, p<0.05). The change in calcium level was dose dependent, can be due to the renal failure, was associated with the microscopic findings in kidneys, but was slight, reversible and considered to be non-adverse. Decrease in cholesterol level revealed in the 100 mg/kg bw/day treated males (p<0.05) can reflect the hepatic functional insufficiency; however, this change was mild, not dose dependent and without obvious relation to the test item administration.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

400 mg/kg bw/day: decreased level of urobilinogen (significant) in males, increased level of ketones (significant) in males, and increase in diuresis (non-significant) in males, non-adverse.
In the 400 mg/kg bw/day dose group, the statistically significant increase in ketones was observed compared to the control vehicle group at the end of the 28-day treatment period. In the 100 mg/kg bw/day dose group, the levels of ketones was also increased (not significantly). Moreover, high dose treated males had decreased level of urobilinogen in urine (p < 0.05). Statistically significant increased ketones and decreased urobilinogen in urine of 400 mg/kg bw/day treated males reflects the liver functional insufficiency, correlated to the microscopic findings in the liver and considered to be test item related. These changes were reversible, not observed in the high dose recovery subgroup and assumed to be non-adverse. Slight increase in the total urine volume and diuresis was notable (by 20%) in the 400 mg/kg bw/day male group. The increased diuresis correlated to the microscopic changes in kidneys, but was statistically non-significant and considered non-adverse.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

400 mg/kg bw/day: increased absolute and relative weight of liver in males (significant) and relative weight of liver in females (non-significant), increased absolute and relative weight of kidney in males (significant) and relative weight of kidney in females (significant), increase in absolute and relative weight of thyroid glands (significant) in females, decrease in relative weight of uterus with cervix (significant).
The increase in kidneys weight in the 400 mg/kg bw/day male group was significant for absolute value (by 7.8 %, p < 0.05) and for relative to body weight value (p < 0.01), and for relative to brain weight value (p < 0.05). In females of a high-dose group, the increase in the kidneys weight was statistically significant for relative to body value (p < 0.05). The increased kidney weight in the 400 mg/kg bw/day group correlated to the clinical biochemistry and being associated with pathomorphological changes in the kidneys can be adverse.
The weight of the liver (absolute and relative to body weight and brain weight) was statistically increased in the 400 mg/kg bw/day male group (by 11.7 % for absolute value, p < 0.05). In lactating females which have functional liver hypertrophy, the increase in the liver weight on the test item was non-significant. The increased liver weight correlated with clinical pathology findings and caused by hepatocellular hypertrophy clearly seen in males. Hepatocellular hypertrophy with increased liver weight is adaptive changes due to metabolic enzyme induction and, hence, non-adverse.
The increase in absolute and relative weight of thyroids in females on 400 mg/kg bw/day dose was significant (by 23.3% for absolute value compared to the control group) and correlated to the epithelial hypertrophy in follicles, which occur with liver enzyme induction and is considered being species-specific and non-adverse. No significant changes in the thyroxin level were revealed in parental animals correlated to the increase in thyroid weight and follicles hypertrophy.
Females treated with 400 mg/kg bw/day dose had decreased weight of the uterus with cervix. Change in uterine weight was notable for absolute values and was significant for relative value with clear dose-dependence. Uterine weights taken from cycling females have a high variance; however, lactating females were not cycling with the identified dioestrus phase. The increase in uterine weight is recognized as test item related and should be considered as the apical endpoints of endocrine influence of the test item according to the OECD Guidance Document 150 (2018).
Females treated with 400 mg/kg bw/day had decreased spleen weight with clear dose-dependence for absolute and relative weight. This change was not statistically significant, but correlated to the hematological effects and atrophy of white pulp in the spleen on the high dose, which is considered to be adverse.
The decrease in relative heart weight was observed in males treated with 400 mg/kg bw/day dose. This change was of notable dose-dependence, correlated to the slight (non-significant) increase in total creatine kinase and significant decrease in calcium level, so can be test item related. However, the decrease in the heart weight was not statistically significant, not associated with histopathological changes in the heart, was reversible and considered non-adverse. In the 400 mg/kg bw/day recovery males, an increase in relative weights of LABC complex (p < 0.05), seminal vesicles (p < 0.05), and prostate (not significant) was observed. There were no alterations in the weight and histology of the male reproductive organs during the treatment period. Therefore, the slight increase in the relative weight of indicated reproductive organs in recovery period is regarded as a change with an unclear relation to the test item.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

400 mg/kg bw/day:
- Liver, hepatocellular hypertrophy in 5 of 6 male, adaptive change, non-adverse;
- Thyroid gland, epithelial hypertrophy in follicles (2 of 6 female), test item-related but species specific, non-adverse;
- Kidney. tubule swelling (4 of 6 male and 1 of 6 female) with tubule atrophy and dilation (1 of 6 male), adverse;
- Spleen, atrophy of white pulp (1 of 6 male, and 1 of 6 female), adverse;
- Salivary glands, increased granulation in granular duct (4 of 7 female, including one non-gravid female), non-adverse;
- Mammary glands, hypolactation/involution in 1 of 6 female associated with the vacuolation of cells in pituitary pars nervosa (reproductive end-point, considered to be adverse).
Slight hepatocellular hypertrophy was observed in 5 of 6 examined males in 400 mg/kg bw/day dose group (p < 0.05) which correlated to the increased liver weight. In lactating females having physiological hepatocellular hypertrophy, the test item related changes were not revealed; however, the increase in the relative weight of liver was notable. Hepatocellular hypertrophy with increased liver weight is generally regarded as an adaptive effect associated with enzyme induction or EPR proliferation and in the absence of degenerative or necrotic changes, would not be considered adverse and would have little relevance to man in terms of risk assessment.
Epithelial hypertrophy in follicles of the thyroid gland in high dose treated females correlated to the increase in the thyroids weight, but not to changes in the thyroxin level in parental animals. Follicular epithelial hypertrophy can occur with enzyme induction and increased hepatic clearance of thyroxin, considered to be species-specific and non-adverse.
Epithelium swelling of slight and moderate grade as renal tubule degeneration was observed in males (4 of 6 examined) and females (1 of 6 examined) treated with 400 mg/kg bw/day dose. In one male tubule swelling was associated with atrophy and tubule dilation. Histopathological findings in kidney correlated to the clinical pathology changes, significant increase in kidneys weight in males and females on the high dose, and considered adverse.
One male and one female in the 400 mg/kg bw/day dose group had the atrophy of white pulp of the spleen as a decreased size of follicles. Finding was pronounced in the female, correlated to the hematological changes in males and females and notable decreased weight of the spleen in females received high dose, and considered adverse.
The high incidence of alteration in granular secretory ducts in salivary glands was revealed in 400 mg/kg bw/day treated females (4 of 7 examined females, p < 0.05). The submandibular salivary gland is sexually dimorphic and shows increased granularity of the convoluted (granular) ducts in males compared to females. The high incidence of this finding in females in the study can be due to the hormonal disturbance related to the test item.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Parental females, unlike males, had significant acute thymic involution related to the physiological status of pregnancy and lactating mediated by the neuroendocrine system without enhancement of apoptosis. There was no significant change in the incidence and severity of thymus atrophy observed in treated females compared to the control group. Notably, single female from the high dose group with identified hypolactation had the relatively large thymus compared to other lactating females.
The vacuolation of the tubular epithelium of minimal grade was observed in testes of two males from 400 mg/kg bw/day dose group and one control male. There was no accompanied significant germ cell degeneration and necrosis in these male, so this finding is considered non-adverse and not test item related. In prostate of four males treated with 400 mg/kg bw/day dose and one control male, concretions were noted, which are a very common, associated with acinar atrophy and not treatment-related. During qualitative examination of the testes in the 400 mg/kg bw/day group with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure, no test item related spermatogenic disturbance, tubular vacuolation, contraction, dilatation, necrosis, dilated rete, nor Leydig cell atrophy, hypertrophy, hyperplasia, adenoma were revealed.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Sex:

female

Basis for effect level:

clinical signs

Key result

Dose descriptor:

NOAEL

Effect level:

>= 400 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

clinical signs

Key result

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Critical effects observed:

no

Conclusions:

Based on the results of this study, the no observed adverse effect level (NOAEL) for systemic toxicity was ≥ 100 mg/kg bw/day.
The NOAEL for male reproduction was derived to be ≥ 400 mg/kg bw/day, because no adverse effects on male reproduction function and performance were observed up to the highest tested dose of 400 mg/kg bw/day. The NOAEL for female reproduction was derived to be ≥ 100 mg/kg bw/day based on the decreased uterine weight considered as the reproductive endpoint and apical endpoint of endocrine influence.
The NOAEL for developmental toxicity was considered to be 100 mg/kg bw/day based on the following effects on 400 mg/kg bw/day considered adverse: decrease in the percentage of liveborn pups per group and increase in the percentage of stillborn pups per group, increase in pre-natal loss of offspring; increased incidence of emaciated pups starting from the postnatal day 7; increased incidence of thyroid follicular cells hyperplasia in postnatal day 13 pups.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat