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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 March 2020 to 12 June 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD 414 Guideline without deviations.
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
other: Draft Report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
p-mentha-1(7),2-diene
EC Number:
209-081-9
EC Name:
p-mentha-1(7),2-diene
Cas Number:
555-10-2
Molecular formula:
C10H16
IUPAC Name:
3-isopropyl-6-methylenecyclohexene
Constituent 2
Chemical structure
Reference substance name:
(R)-p-mentha-1,8-diene
EC Number:
227-813-5
EC Name:
(R)-p-mentha-1,8-diene
Cas Number:
5989-27-5
Molecular formula:
C10H16
IUPAC Name:
(4R)-isopropenyl-1-methylcyclohexene
Constituent 3
Chemical structure
Reference substance name:
(S)-p-mentha-1,8-diene
EC Number:
227-815-6
EC Name:
(S)-p-mentha-1,8-diene
Cas Number:
5989-54-8
Molecular formula:
C10H16
IUPAC Name:
(4S)-isopropenyl-1-methylcyclohexene
impurity 1
Chemical structure
Reference substance name:
p-cymene
EC Number:
202-796-7
EC Name:
p-cymene
Cas Number:
99-87-6
Molecular formula:
C10H14
IUPAC Name:
1-isopropyl-4-methylbenzene
impurity 2
Chemical structure
Reference substance name:
(-)-pin-2(10)-ene
EC Number:
242-060-2
EC Name:
(-)-pin-2(10)-ene
Cas Number:
18172-67-3
Molecular formula:
C10H16
IUPAC Name:
(1S,5S)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane
impurity 3
Chemical structure
Reference substance name:
p-mentha-1,3-diene
EC Number:
202-795-1
EC Name:
p-mentha-1,3-diene
Cas Number:
99-86-5
Molecular formula:
C10H16
IUPAC Name:
1-isopropyl-4-methylcyclohexa-1,3-diene
impurity 4
Chemical structure
Reference substance name:
(1R,6S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene
Cas Number:
20296-50-8
Molecular formula:
C10H16
IUPAC Name:
(1R,6S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene
impurity 5
Chemical structure
Reference substance name:
(1S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene
EC Number:
207-856-6
EC Name:
(1S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene
Cas Number:
498-15-7
Molecular formula:
C10H16
IUPAC Name:
(1S,6R)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene
impurity 6
Chemical structure
Reference substance name:
7-methyl-3-methyleneocta-1,6-diene
EC Number:
204-622-5
EC Name:
7-methyl-3-methyleneocta-1,6-diene
Cas Number:
123-35-3
Molecular formula:
C10H16
IUPAC Name:
7-methyl-3-methyleneocta-1,6-diene
impurity 7
Chemical structure
Reference substance name:
(5S)-isopropyl-2-methylcyclohexa-1,3-diene
Cas Number:
2243-33-6
Molecular formula:
C10H16
IUPAC Name:
(5S)-isopropyl-2-methylcyclohexa-1,3-diene
impurity 8
Chemical structure
Reference substance name:
(R)-5-isopropyl-2-methylcyclohexa-1,3-diene
EC Number:
224-167-6
EC Name:
(R)-5-isopropyl-2-methylcyclohexa-1,3-diene
Cas Number:
4221-98-1
Molecular formula:
C10H16
IUPAC Name:
(5R)-isopropyl-2-methylcyclohexa-1,3-diene
impurity 9
Chemical structure
Reference substance name:
p-mentha-1,4-diene
EC Number:
202-794-6
EC Name:
p-mentha-1,4-diene
Cas Number:
99-85-4
Molecular formula:
C10H16
IUPAC Name:
1-isopropyl-4-methylcyclohexa-1,4-diene
impurity 10
Chemical structure
Reference substance name:
(1R,5R)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane
Cas Number:
19902-08-0
Molecular formula:
C10H16
IUPAC Name:
(1R,5R)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane
Test material form:
liquid
Details on test material:
Batch No.: F290519
Purity: 74.8% (sum of the three main constituents)
Name of test material (as cited in study report): Reaction mass of beta-phellandrene and d-limonene and l-limonene
Physical state: colourless liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 28 May 2020

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on test animals or test system and environmental conditions:
RATIONALE FOR ANIMAL MODEL:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague Dawley Crl: CD (SD) rat strain was used because of the historical control data available at this laboratory.

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 70 to 83 days old.
- Weight at study initiation: 229 to 307 g
- Housing: Acclimatization - up to four animals; During pairing - one (stock) male and one female; Gestation - one female
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: Six days before commencement of pairing.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: Artificial lighting, 12 h light : 12 h dark
Environmental Enrichment
- Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing) and replaced when necessary.
- Plastic shelter: Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

IN-LIFE DATES: From 15 April 2020 to 15 May 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
RATIONALE FOR ROUTE OF ADMINISTRATION:
The oral gavage route of administration was chosen to simulate the conditions of potential human exposure.

PREPARATION OF DOSING SOLUTIONS:
Method of preparation: The required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous. A series of formulations at the required concentrations were prepared in ascending order.

Frequency of preparation: Weekly.
Storage of formulation: Three days at ambient temperature (15 to 25°C) and 14 days refrigerated (2 to 8°C).

VEHICLE
- Concentration in vehicle: 0, 10, 35 or 70 mg/mL
- Dose volume: 1 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Achieved concentration: Samples of each formulation were analyzed for the achieved
concentration of the test item.

Stability and homogeneity: The homogeneity and stability of formulations during storage were determined as part of Covance study BP52WF. In that study, formulations in the concentration range 10 to 200 mg/mL were confirmed to be stable for three days at ambient temperature (15 to 25°C) and 14 days refrigerated (2 to 8°C).
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 with identified stock males
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
- Day 0 of gestation: When positive evidence of mating was detected.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
Females were treated from Day 6 to Day 19 (inclusive) after mating
Frequency of treatment:
Once daily at approximately the same time each day.
Duration of test:
From mating until Day 20 (necropsy).
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
35 mg/kg bw/day (actual dose received)
Dose / conc.:
70 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 mated females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected following the completion of the preliminary embryo-fetal study (Covance No. SD52TY). In this study, groups of three gestating females were exposed to 0, 45, 90 and 180 mg/kg bw/day from gestating days 6 to 20. A significant decrease in bodyweight gain (-30%) in all three animals at 90 mg/kg bw/day was observed which was considered as adverse maternal toxicity.
At 45 mg/kg bw/day, one of the two tested females had a similar bodyweight gain decrease as the 90 mg/kg bw/day group and the other female had bodyweight gain similar to the control group. Therefore 70 mg/kg bw/day, which corresponds to 2/3 way between 45 and 90 mg/kg bw/day, was therefore expected to elicit significant maternal toxicity (about -20% bodyweight gain). 35 mg/kg bw/day and 10 mg/kg bw/day were then chosen to allow the determination of a dose response.

- Rationale for animal assignment: On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.
Method: To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups. Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.

Examinations

Maternal examinations:
MORTALITY: Yes
- A viability check was performed near the start and end of each working day. Animals were
killed for reasons of animal welfare where necessary. A complete necropsy was performed in all cases.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the
occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were recorded daily at the following times in relation to dose administration: A pre-dose observation; One to two hours after completion of dosing all groups; As late as possible in the working day.
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

POST-MORTEM EXAMINATIONS: Yes
- Animals surviving until the end of the scheduled study period were killed on Day 20 after mating by Carbon dioxide asphyxiation.
- Necropsy: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. The organs weighed, tissue samples fixed and sections examined microscopically are detailed in Table 1.
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.
Tissues were routinely preserved in 10% Neutral Buffered Formalin.
Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Ovaries and uterine content:
For females surviving to term, the ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight (including cervix and ovaries): Yes
- Number of corpora lutea: Yes
- Number of implantation sites: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of Fetuses (live and dead): Yes
Blood sampling:
THYROID HORMONE ANALYSIS: Yes
- Blood samples were collected at termination of the study in all surviving adults in sublingual vein after an isoflurane anaesthesia. Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation (2000 g for ten minutes at 4°C).
- Number of aliquots: Two per animal. Aliquot 1: 0.2 mL serum for T3/T4; Aliquot 2: residual serum for TSH.
Fetal examinations:
Method of kill for fetuses: Chilling on a cool plate (approximately 0 °C)
Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex and ano-genital distance of each fetus was recorded.
Examination of nominally 50% of fetuses in each litter: Sexed internally and eviscerated.
Fixation: Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.
Processing: Bouin’s fixed fetuses were subject to free-hand serial sectioning.
IMS fixed fetuses were processed and stained with Alizarin Red.

Fetal Pathology Examination
Bouin’s fixed fetuses: Serial sections were examined for visceral abnormalities.
Alizarin Red stained fetuses: Assessed for skeletal development and abnormalities.
Statistics:
See " Any other information on materials and methods incl. tables"
Indices:
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss
was considered to reflect losses due to non-fertilization of ova and failure to implant. It was
calculated from the formula:
Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100

Where the number of implantations exceeded the number of corpora lutea observed,
pre-implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to
have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%)= [(Number of implantations – Number of live fetuses) / Number of implantations] x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs at 10, 35, or 70 mg/kg bw/day.
There were no signs associated with dose administration at 10, 35, or 70 mg/kg bw/day.
Mortality:
no mortality observed
Description (incidence):
There were no premature deaths which were considered to be related to treatment
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 70 mg/kg bw/day, statistically significantly low mean body weight gain was recorded from Day 6 to Day 20 of gestation (79%) when compared with Controls, leading to statistically significantly low mean bodyweights from Day 14 and a 7% lower final mean bodyweight than Control. Bodyweight gain was unaffected at 10 and 35 mg/kg bw/day.

Gravid uterine weights at 10, 35 and 70 mg/kg bw/day were unaffected by treatment, although slightly lower at 70 mg/kg bw/day. However adjusted weight gain was statistically significantly low at 35 and 70 mg/kg bw/day (70 and 36% of Control, respectively).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption was significantly decreased at 70 mg/kg bw/day over Days 10-14, 14-18 and at 35 and 70 mg/kg bw/day over Days 18-20 when compared with Control. The overall mean food consumption over Days 6-20 was statistically significantly lower (84%) than Control at 70 mg/kg bw/day
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:
not examined
Endocrine findings:
no effects observed
Description (incidence and severity):
There was no effect of treatment on mean T3, T4 or TSH concentrations in serum samples obtained from females that received 10, 35 or 70 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test article-related organ weight changes.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Sprague Dawley rats; therefore, they were considered not test article-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Details on results:
See table of results in the section "Attached background material".

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
All females that mated were pregnant.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of implantations and the pre- and post-implantation losses were considered unaffected by treatment at 10, 35 or 70 mg/kg bw/day.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
One Group 3 female (No. 47), receiving 35 mg/kg bw/day had total litter resorption; since there were no test item related effects on embryo-fetal survival the resorption of this small litter was considered due to chance.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The numbers of resorptions (early, late) were similar to Control and were therefore unaffected by treatment at 10, 35 or 70 mg/kg bw/day.
Dead fetuses:
no effects observed
Description (incidence and severity):
The numbers of live young were similar to Control and were therefore unaffected by treatment at
10, 35 or 70 mg/kg bw/day.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
not examined
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
35 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Placental weight was unaffected by treatment at 10, 35 or 70 mg/kg bw/day.
Total litter and overall fetal weights were unaffected by treatment at 10, 35 or 70 mg/kg bw/day.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was unaffected by treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The litter weight was unaffected by treatment.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
When compared with Control, anogenital distance was unaffected by maternal treatment at
10, 35 or 70 mg/kg bw/day.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants show no relationship to treatment.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants show no relationship to treatment.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of major and minor abnormalities show no relationship to treatment
Other effects:
not specified

Effect levels (fetuses)

Key result
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Formulation analysis:


The mean concentrations were within 7% of the nominal concentration, confirming the accuracy of formulation, with the exception of Group 2 for both the First Preparation and Last Preparation (-30.3 and +14%, respectively). The difference from mean and coefficient of variation remained within 3%, confirming precise analysis. Procedural recoveries remained within the range established at validation, with the exception of two procedural recoveries prepared during the Last Preparation (one from the original analysis and one from the contingency analysis). The procedural recoveries met the SOP acceptance criteria of a minimum of 2 out of 3 samples within range, therefore they confirm the continued accuracy of the analytical method.


For the First Preparation and Last Preparation, Group 2 results were outside of the acceptance criteria. Re-dilutions and contingency analysis were performed. The re-dilutions confirmed the original results, therefore the original results and the contingency results have been reported.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was concluded to be 35 mg/kg bw/day due to the magnitude of the low bodyweight gain and food consumption at 70 mg/kg bw/day and for embryo-fetal survival and development was concluded to be 70 mg/kg bw/day.
Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, three groups of 20 female rats received Reaction mass of beta-phellandrene and d-limonene and l-limonene at doses of 10, 35 or 70 mg/kg bw/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.


Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating, blood samples were taken for thyroid hormone analysis and the gravid uterus weight and thyroid weight were recorded. Microscopic pathology investigations were also undertaken. Ano-genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.


Treatment was generally well tolerated with no test item-related unscheduled deaths. Thyroid hormones (T3, T4 and TSH levels) and maternal clinical condition was not affected at any dose level investigated, and there were no test item-related organ weight, macroscopic or microscopic abnormalities detected at scheduled termination on Day 20 after mating. At 70 mg/kg bw/day, low overall mean bodyweight gain over Days 6-20 (79% of Control) was observed which correlated with low food intake from Day 10 of gestation, leading to an overall statistically significantly lower food consumption than Control (84% of Control); adjusted weight gain was also markedly lower than in Controls at this dose level (36% of Control). There was no adverse effect of maternal treatment on the mean numbers of implantations, resorptions, live young, the extent of pre- and post-implantation losses, sex ratio, ano-genital distance or on placental, fetal and litter weights, and there were no major or minor abnormalities or skeletal variants considered related to treatment at any dose level investigated.


 


Based on the results of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was concluded to be 35 mg/kg bw/day due to the magnitude of the low bodyweight gain and food consumption at 70 mg/kg bw/day and for embryo-fetal survival and development was concluded to be 70 mg/kg bw/day.