Oligomerisation products of beta-pinene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Oligomerisation products of beta-pinene

EC Number:

701-246-8

Molecular formula:

Variable (dimer = C20-H34)

IUPAC Name:

Oligomerisation products of beta-pinene

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 10 to 14 weeks old
- Weight at study initiation: 177 to 260g
- Fasting period before study: No
- Housing: On arrival and following randomization females were housed individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding equipped with water bottles.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly as a solution, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. A factor of 0.935 was used to correct for the specific gravity of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil is a standard vehicle for toxicity studies and was deemed to be suitable for the test substance based on trial formulation and analytical work.
- Concentration in vehicle: 0, 25, 75 and 250 mg/mL.
- Amount of vehicle (if gavage): 4 mL/Kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was based on the analytical method validated for the test item in vehicle.

Accuracy and homogeneity were determined for formulations prepared for use in Week 1. The chemical analyses were conducted as specified below. Samples and remaining formulation were stored in normal glassware causing the samples stored at room temperature to be exposed to normal laboratory light conditions.

Duplicate samples (approximately 510 or 670 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 50 mL. For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations. The volumetric flasks were filled up to the mark with tetrahydrofuran. When necessary, the solutions were further diluted with 1% corn oil in tetrahydrofuran to obtain concentrations within the calibration range.

Results:
Accuracy: In the Group 1 formulation, no test item was detected. The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in
agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

Homogeniety:
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Details on mating procedure:

- Impregnation procedure: Purchased timed pregnant from reputable supplier.

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 6 to Day 20 post-coitum, inclusive.

Frequency of treatment:

Daily

Duration of test:

Day 6 to Day 20 post-coitum

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 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.:

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

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

22 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of the dose range finder, and in an attempt to produce graded responses to the test item. At 1000 mg/kg/day, no clinical signs were observed, and body weight (gain) and (relative) food consumption were comparable between groups and within the range of available historical control data.

- Rationale for animal assignment (if not random): Randomly assigned

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed at least once daily, beginning on Day 2 post-coitum and lasting up to the day prior to necropsy.
- Clinical observations: The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 1, 2, 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. Cage debris was examined to detect any premature birth.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18 21 post-coitum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes (qualitatively)
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 post-coitum
- Organs examined: All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). The uterus and the thyroid were weighed and organ to bodyweight values calculated.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- The number and distribution of live and dead fetuses: Yes
- The number and distribution of embryo-fetal deaths: Yes

Fetal examinations:

- External examinations: Litters of females surviving to scheduled necropsy, were subjected to detailed external, visceral and skeletal examinations, as described in the following sections. External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

- Soft tissue examinations: The sex of all fetuses was confirmed by internal examination and approximately one-half of the fetuses (live and dead) in each litter (all groups) were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. The heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. All carcasses, including the carcasses without heads, were eviscerated, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons.

- Skeletal examinations: All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S. Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads). All specimens were archived in glycerin with bronopol as preservative. A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

- Fetal sex: The anogenital distance (AGD) was measured for all viable fetuses. The AGD was normalized to the cube root of the fetal body weight.

Statistics:

Statistical analysis:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.

Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and postimplantation loss.

Indices:

Maternal variables:
Bodyweight gains: Calculated against the body weight on Day 6 post-coitum.
Corrected bodyweight gains: Body weight on Day 21 post-coitum minus the body weight on Day 6 post-coitum and the weight of gravid uterus.
Relative food consumption: Calculated against the body weight for scheduled intervals.
Organ weight relative to body weight: Calculated against the body weight on Day 21 post-coitum.

Reproductive and developmental indices:
Pre-implantation loss (%): (number of corpora lutea - number of implantation sites)/(number of corpora lutea) x 100

Postimplantation loss (%): (number of implantation sites - number of live fetuses)/(number of implantation sites) x 100

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:

Viable fetuses affected/litter (%): (number of viable fetuses affected/litter)/(number of viable fetuses/litter) x 100

Historical control data:

Where required HCD has been included in the relevant results sections.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Piloerection was observed for 1/22 females treated at 100 mg/kg/day, for 9/22 females at 300 mg/kg/day, and for 8/22 females at 1000 mg/kg/day. The only other clinical sign observed during the treatment period was alopecia. It occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, body weight gain corrected for gravid uterus was statistically significantly higher (23% higher compared to concurrent controls).

At 1000 mg/kg/day, a trend towards a slightly higher body weight gain was observed on Day 21 post-coitum (1.11x of control, not statistically significant), after correction for gravid uterus this change was more pronounced, with mean body weight reaching 1.23x of control (statistical significant). All mean values for (corrected) body weight and corrected body weight gain remained within the range of available historical control data and are therefore not considered adverse.

HCD for pregnant Wistar Han rats (period 2015-2019):
Bodyweight Day 21 post coitum (g): mean = 325; P5 - P95 = 278 - 373 (n = 1315)
Corrected body weight gain (g): mean = 29; P5 - P95 = 14.9 - 44.2 (n = 1311)
Corrected body weight gain (%): mean = 13; P5 - P95 = 6.9 - 20.5 (n=1311)

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, food consumption before or after correction for body weight was statistically significantly higher on Days 15-21 post-coitum (up to 15% higher compared to controls). This was likely in part due to slightly lower control means in comparison with the historical control data. As all mean values remained within the historical control range , no toxicological relevance was attributed to the observed increases in food consumption.

Historical control data for pregnant Wistar Han rats (period 2015-2019):
Food consumption (gram/animal/day):
Days 15-18 post coitum: mean = 23; P5 – P95 = 17.7 – 28 (n=1328)
Days 18-21 post coitum: mean = 23; P5 – P95 = 18.0 – 28 (n=1319)
Relative food consumption (gram/kg body weightl/day):
Days 15-18 post coitum: mean = 79; P5 – P95 = 64.9 – 94.9 (n=1328)
Days 18-21 post coitum: mean = 71; P5 – P95 = 58.1 – 85.4 (n=1313)

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Increased serum levels of thyroid stimulating hormone (TSH) were noted at 300 and 1000 mg/kg/day (1.53x and 1.57x of controls, respectively). Values remained within the available historical control range.

Serum levels of triiodothyronine (T3) and total thyroxine (T4) were statistically significantly lower at 1000 mg/kg/day (0.75x and 0.84x of controls, for T3 and T4, respectively) and remained within the available historical control data for T4 values. The remaining serum levels were comparable to the concurrent control.

At 100 mg/kg/day, one female was not gravid and was therefore excluded from the data tables.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Test item-related higher thyroid gland weights (absolute and relative to body weights) were noted in the 1000 mg/kg/day group females. However, mean values remained well within the available historical control data .

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test item. Findings that were noted among control and/or treated animals were considered to be of no toxicological significance, as they are occasionally seen among rats used in these types of study and/or remained within the range of biological variation for rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the thyroid gland. Follicular cell hypertrophy was present in the thyroid gland at increased incidence in females treated at 300 mg/kg/day, and at increased incidence and severity (up to moderate) at 1000 mg/kg/day.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

Abortions are not a common feature in rats.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The numbers of pregnant females and implantation sites, and pre- and postimplantation loss in the control and test groups were similar and in the range of normal biological variation.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 1000 mg/kg/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There were no test item-related effects on litter size of any group. Fetal weights (male, female and combined) were comparable to the controls for all treated groups.

At 300 mg/kg/day, the mean fetal weight of males was just above the 95-percentile of the historical control data (5.6 gram versus P5-P95 HCD: 5.2-5.5 gram). However, as this occurred at the mid dose only and the magnitude of change was only minimal, this was considered to be unrelated to treatment with the test item.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no test item-related effects on external morphology following treatment up to 1000 mg/kg/day.

One control fetus was observed with omphalocele. This was the only external malformation that occurred, and external variations were not seen in any group.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were no test item-related effects on skeletal morphology following treatment up to 1000 mg/kg/day.

Three fetuses at 100 mg/kg/day and one at 300 mg/kg/day were observed with bent limb bones. As this malformation occurred without dose relationship and is among historical control data, it was considered to be unrelated to treatment with the test item.

At 100 and 300 mg/kg/day, statistically significantly higher incidences of the variation bent ribs were observed compared to concurrent controls (3.6x of controls). Mean litter incidences were 6.4%, 23.3%, 23.3% and 15.4% per litter in the control, 100, 300 and 1000 mg/kg/day groups. Nevertheless, the higher incidences at the low and mid dose level were considered not to be test item-related as there was no dose relationship and incidences were within the historical control data range (1.9% – 27.6% per litter).

The other variations occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data. Therefore, they were considered to be unrelated to treatment with the test item.

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no test item-related effects on visceral morphology following treatment up to 1000 mg/kg/day.

At 300 mg/kg/day, cephalic examination revealed internal hydrocephaly in one fetus. As this occurred singly at the mid dose level, it was considered a chance finding.
The two visceral variations that were noted (small supernumerary liver lobes and convoluted ureter) occurred at low incidences and at frequencies that were within the range of available historical control data. Therefore, they were considered to be unrelated to treatment with the test item.

Other effects:

not examined

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Time-mated female Wistar Han rats were treated with the test substance from Day 6 to Day 20 post-coitum, inclusive, by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg/day. The rats of the control group received the vehicle, corn oil,
alone.
Test formulations prepared were considered homogeneous at the concentrations tested, and analysis of the accuracy revealed acceptable levels.

Piloerection was observed for 1/22 females treated at 100 mg/kg/day, 9/22 females at 300 mg/kg/day and 8/22 females at 1000 mg/kg/day. In the absence of a clear dose-related response and supportive changes indicative of a reduced clinical condition, this was considered not to represent an adverse effect of the test item.

At 1000 mg/kg/day, a trend towards a slightly higher body weight gain was observed on Day 21 post-coitum (not statistically significant), which was more pronounced after correction for gravid uterus (statistically significant). Similarly, food consumption before or after correction for body weight was statistically significantly higher on Days 15-21 post-coitum. As all values remained within the historical control range, the changes observed in body weights and food consumption were considered not of toxicologically relevance.

At 300 and 1000 mg/kg/day, test item-related morphologic alterations in the thyroid gland were present. Follicular cell hypertrophy was recorded at 300 mg/kg/day at an increased incidence, but the severity did not exceed the severity of control females. In addition, there were no macroscopic findings or organ weight changes in the thyroid gland at 300 mg/kg/day. At 1000 mg/kg/day, the follicular cell hypertrophy was recorded at both an increased incidence and increased severity compared to concurrent controls. As a result, also thyroid organ weight (absolute and relative to body weight) was increased. Furthermore, serum levels
of total triiodothyronine (T3) and total thyroxine (T4) were decreased at this high dose level and TSH was increased at 300 and 1000 mg/kg/day. However, mean values of thyroid organ weight, total T4 and TSH remained within historical control data ranges. Therefore, the observed changes in thyroid gland morphology and organ weight were considered to be non-adverse. In the 90-day study performed with this test item liver enzyme induction was reported. The relation to the thyroid findings in the present study could not be evaluated as the relevant investigations are outside the remit of the study design.

No test item-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e. mortality/moribundity, uterine contents, corpora lutea, implantation sites, and pre- and postimplantation loss).

No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, and external, visceral and skeletal malformations and developmental variations).

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for the test substance was established as being at least 1000 mg/kg/day for all endpoints.