Pyrolysis light oil from waste plastics

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-2022

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

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

Justification for study design:

This study was designed to evaluate the potential toxic effects of the test item NAPHTHA when administered to rats for a minimum of 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development.

Test material

Test material information

Constituent 1

Reference substance name:

Pyrolysis light oil from waste plastics

EC Number:

940-514-0

Molecular formula:

Not required for a UVCB-substance (mixture of multiple (>100) organic substances)

IUPAC Name:

Pyrolysis light oil from waste plastics

Test material form:

liquid: volatile

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on 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

Analytical verification of doses or concentrations:

yes

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

Description (incidence):

One female from the control vehicle group died during parturition due to dystocia.

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

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Home cage, handling, open field, sensory, neuromuscular, and physiological parameters evaluated during Functional Observation Battery testing on study day 28 (males) or lactation day 13 (females) and after two weeks recovery period were unaffected by the test item.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Three females treated with 400 mg/kg bw/day dose being cohabitated with males had 7-12-day period of dioestrus, presumably because of pseudopregnancy. These females became pregnant but with resulted increase in the conceiving period. In the 100 mg/kg bw/day dose group, two females were recorded with an irregular cycle. However, one of them had a prolonged period of dioestrus being habituated with male; another one had prolonged the dioestrus period after one week of dosing. So, there are no sufficient grounds to consider the described changes in the oestrous cycle as related to the test item administration. The total number of females with the conceiving period more than 6 days was approximately similar in the control vehicle group and high dose group (4 of 12 and 3 of 12 females, respectively). The duration of the estrous cycle, being evaluated during the administration period before the onset of pregnancy, was approximately the same in all groups.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

400 mg/kg bw/day dose: decreased relative weight of uterus with cervix (significant), adverse; hypolactaion (not significant), non-adverse.
Details:
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 decrease in uterine weight is recognized as test item related and should be considered as the apical endpoint of endocrine influence of the test item according to the OECD Guidance Document 150 (2018).
Two females treated with 400 mg/kg bw/day dose had litters with extremely low body weight gain in all pups. The microscopic evaluation of mammary glands revealed the visual signs of hypolactation in one female associated with the vacuolation of cells in pituitary pars nervosa, which can be a feedback stimulation of oxytocin. Another female with emaciated litter had no significant changes in mammary tissue compared to the control background. Mammary gland is a sensitive tissue to the effects of endocrine disrupting influence of exogenous chemicals and may interfere with any aspect of hormone action. Morphological changes in lactating mammary gland are accompanied by elevated serum prolactin, growth hormone and estrogen levels, the former expected due to the positive regulation of pituitary prolactin secretion by estrogen. No pathomorphological changes in mammary tissue of males and females were revealed that would indicate a pronounced effect of the test item as an endocrine disruptor. However, considering the decreased uterine weight in the high dose group and absence of hypolactation finding in the historical control population, the dependence of hypolactation with the test item treatment cannot be excluded, but it considered non-adverse due to the rarity of finding.

Details on results (P0)

One male from the 100 mg/kg bw/day dose group and one control male did not mate and did not sire a litters; one male from the 400 mg/kg bw/day dose group mated, but female from this group was not pregnant without implantation sites.
One female from the control vehicle group had dystocia and failed to deliver pups.
The vacuolation of the tubular epithelium of minimal grade was observed in two males treated with the high dose and one control male. However, there was no significant germ cell degeneration and necrosis in these male, so this finding is non-adverse and considered to be non-test item-related. In prostate of four males treated with 400 mg/kg bw/day dose and one control male, concretions were noted, which found in the lumen of the prostate are a very common findings associated with age related acinar atrophy and not treatment-related.
No test item-related histological findings in male reproductive organs were revealed. 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.
Indices of mating, fertility, and copulation/conception in the 25, 100 and 400 mg/kg bw/day male and female groups did not differ from the values of control vehicle group and historical control.

Effect levels (P0)

1

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

3

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

4

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

5

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

400 mg/kg bw/day: increased incidence of emaciated pups starting from the postnatal day 7 (significant).
In the 400 mg/kg bw/day dose group, emaciated pups were revealed in four litters: 11 of 85 pups on postnatal day 7, and 20 of 85 pups on postnatal day 13 (p < 0.001). For one of two litters with total affected pups, the offspring emaciation was due to dam lactation disorder. Another female with total emaciated litter had no observable histopathological changes in mammary glands. Two females had single pups in their litters with markedly reduced body weight gain.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

400 mg/kg bw/day: decrease in the percentage of liveborn pups per group (significant), increase in the percentage of stillborn pups per group (significant), increase in pre-natal loss of offspring (non-significant compared control vehicle group).
The change in the mean number of liveborn per litter on 400 mg/kg bw/day dose was notable but insignificant (8.6±2.5 compared 9.8±2.5). However, the percentage of liveborn pups in this group was statistically decreased (88.0% compared 95.4% and 94.9% in the control vehicle group and historical control, p<0.05) and the percentage of stillborn pups was increased (12.0% compared to the control 4.6% and historical control 5.1%, p <0.05). The percentage of pups found dead in the high-dose group during postnatal days 0-1 (including stillborns, died and cannibalized liveborns) was increased (13.0% compared 5.6% in the control group, p <0.05).
At 400 mg/kg bw/day, the increase in the pre-natal loss was observed (20.2%) notable if compared to the control vehicle group (14.2%) and significant compared to the historical control value (11.8%). In the 100 mg/kg bw/day dose group, the tendency to the increase in pre-natal loss (17.8%) can be noted. The total number of females with prenatal and postnatal loss of offspring was approximately the same in all groups, including control.
Postnatal viability indices in the test item treated groups were comparable to the control group. In 25 mg/kg bw/day dose group, one pup had decreased body weight gain to postnatal day 4 and was cannibalized on postnatal day 5. The single pup from the 100 mg/kg bw/day dose group had no external clinical alterations, but was missing and considered cannibalized on postnatal day 2. These findings are not considered test item related.

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Description (incidence and severity):

There were no test item related changes in the male to female ratio per litter. In the control vehicle and medium dose group, the percentage of males was lower (46.3% and 41.5%) compared to historical control value (49.4%), that is considered biological variability. The percentage of newborn male pups in the high-dose group (49.1%) corresponds to the historical control value (49.4%). There was no sex-related pup mortality in the postnatal period, and the percentage of males in the test item groups remained approximately the same.
Absolute and normalized anogenital distance of male and female treated pups did not differ significantly from the values in the control vehicle group and was within the historical control range. The test item did not cause areoles retention in male pups. On a postnatal day 13, there were no males with areolae/nipples in all groups; no signs of demasculinization were found in any male in all groups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

In 400 mg/kg bw/day dose group, the mean value of relative weight of thyroid gland was slightly increased in male pups (0.0198±0.0073 g/100 g body weight) compared to the control vehicle group (0.0166±0.0026 g/100 g body weight). However, this change was not significant, and considered non-adverse.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross observations were revealed in single pup from 25 mg/kg bw/day dose group found dead on postnatal day 1. This pup had low body weight, empty stomach, signs of dehydration and large heart ventricles with dark spots. The uniqueness of gross observations in the low-dose group does not allow it to be associated with the test item. There were no test item-related visceral gross observations in all pups during a scheduled necropsy on postnatal day 13.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

400 mg/kg bw/day: increased incidence of follicular cells hyperplasia (significant).
The hyperplasia of thyroid follicular cells was revealed in 4 of 22 pups in the 100 mg/kg bw/day dose group (non-significant) and in 8 of 19 pups in the 400 mg/kg bw/day dose group (p < 0.01). Hyperplasia of follicular cells in offspring correlated to the follicular cells hypertrophy in parental females in the high dose group, decreased concentration of thyroxin in postnatal day 13 pups and considered to be test item related. Single pup from the 400 mg/kg bw/day dose group had mild signs of follicular cells degeneration. This finding was observed parallel with follicular cells degeneration and its relation to the test item is not excluded.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

2

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Applicant's summary and conclusion

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.