Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

OECD 422 (Rat): Oral NOAEL for Reproductive Toxicity: 175 mg/Kg bw/day (females); 500 mg/Kg bw/day (males)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-10-04 to 2018-12-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Deviations did not adversely affect the results of the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Emerald Kalama Chemical Limited (Dans Road, WA8 0RF Widnes, Cheshire United Kingdom); Batch/Lot number: A170524D
- Expiration date of the lot/batch: 2019-06-06
- Purity test date: 2018-07-02

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25 °C, ≤70 RH%), under inert gas, protected from humidity (tight closed container)
- Stability under storage conditions: Stable
- Stability under test conditions: stable for 1 day when stored at room temperature, and at least 15 days when stored below -15°C.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid

OTHER SPECIFICS
- other information:
- Name of substance: Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene
- Public name: Ocimene PQ
- EC# 933-779-9 (pre-registration 222-081-3)
- CAS numbers: n/a (pre-registration 3338-55-4)
- Batch/Lot number: A170524D
- Expiry date: 06 June 2019
- Purity: Considered as 100%

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Wistar) rats

Details on species / strain selection:

The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. The Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were also used for the Dose Range Finding study (study code: 17/180-220PE).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address:Sandhofer Weg 7, D-97633, Sulzfeld, Germany), from SPF colony)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Young adult rats, approximately 11-13 weeks old at start of treatment
- Weight at study initiation: Males: 412–502 g, females: 242–317 g at onset of treatment
- Fasting period before study: Not specified
- Housing: Rodents were group-housed in Type II and III polycarbonate cages, up to 2 animals of the same sex and dose group/cage with the exception of the
mating and gestation/delivery period when they were paired or individually housed, respectively
- Diet (e.g. ad libitum): ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" (Batch number: 840 33675 / 639 38520, Expiry date: 31 January 2019 / 30 April 2019) produced by ssniff Spezialdiäten GmbH (Address: Ferdinand Gabriel Weg 16, D-59494 Soest,
Germany) ad libitum
- Water (e.g. ad libitum): Tap water from the municipal supply, as for human consumption, was provided from a 500 mL bottle, ad libitum
- Acclimation period: At least 14 days

DETAILS OF FOOD AND WATER QUALITY:
Water quality control analysis was performed at least once every three months and microbiological assessment was performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36., Hungary). The diet supplier provided analytical certificates for the batches used. Food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0–25.0°C (target range 22 ± 3°C)
- Humidity (%): 26–69% (target range 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2018-10-4 To: 2018-12-18

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

propylene glycol with 1% polysorbate 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was formulated at appropriate concentrations (0, 12, 35, or 100 mg/mL for doses of 0, 60, 175, or 500 mg/Kg bw/day, respectively) in the vehicle (as a visibly stable homogenous formulation) and formulations were prepared daily. Dosing solutions were administered to the test material or vehicle-treated (control) animals at a constant volume of 5 mL/Kg bw (calculated and adjusted based on each animal’s most recent body weight).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on results of a short solubility test performed at the Test Facility, propylene glycol with 1% polysorbate 80 was selected as vehicle for this study in agreement with the Sponsor based on the formulation and analytical trials. The same vehicle was used in the Dose Range Finding (DRF) study.
- Concentration in vehicle: 0, 12, 35, or 100 mg/mL for doses of 0, 60, 175, or 500 mg/Kg bw/day, respectively.
- Amount of vehicle (if gavage): 5 mL/Kg
- Lot/batch no. (if required):

Name: Propylene glycol
Manufacturer: MERCK
Batch number: K49089078 / K50526178
Expiry date: 31 May 2022/31 August 2023
Storage conditions: Room temperature

Name (Synonym): Polysorbate 80 (Tween 80)
Manufacturer: Sigma-Aldrich
Batch number: BCBV5152
Expiry date: 30 June 2022
Storage conditions: Room temperature

- Purity: Not specified

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Females remained with the same male until copulation occurred; the mating was finished mostly within 5 days except for 2 Mid dose females
(#3501: 8 days, #3506: 11 days)
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: Not required
- After successful mating each pregnant female was caged (how): Sperm positive females were housed individually
- Any other deviations from standard protocol: Not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test material formulations for concentration and homogeneity was performed using a validated HPLC-UV method (High Performance Liquid Chromatography with Ultraviolet detection). Duplicate samples were taken from the top, middle and bottom of the test item formulations three times during the study (during the first and last weeks and approximately midway during the treatment), one set to analyse and one set as a backup, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement. Formulation samples (both sets) were kept frozen (under -15 °C) until shipment. Samples were shipped on dry ice as soon as possible after collection for concentration and homogeneity measurement to the Principal Investigator.

Duration of treatment / exposure:

Males: 28 days (14 days pre-mating and 14 days mating/post-mating)
Females: 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (13 days post-partum dosing).

Frequency of treatment:

Once daily (7 days/week)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 - Control

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

Group 2 - Low Dose

Dose / conc.:

175 mg/kg bw/day (nominal)

Remarks:

Group 3 - Mid Dose

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 4 - High Dose

No. of animals per sex per dose:

12/sex/dose (Two females were used as replacement on Day 3, one female on Day 4 and another female on Day 5. Two additional males were also included in the study to provide mating pairs for the replacement females on Day 4 and Day 5)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected by the study director in consultation with the sponsor based on available acute oral toxicity data (LD50 over 2000 mg/Kg bw in rats (Charles River Laboratories Hungary Kft., 2019; Study code: 17/180-001P) and results of the Dose Range Finding (DRF) study performed at the Test Facility (Charles River Laboratories Hungary Kft., 2019: Study code: 17/180-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. In the DRF study, test material related mortality was seen both at 1000 mg/Kg bw/day and at 600 mg/Kg bw/day. All four females dosed with 1000 mg/Kg bw/day were found dead, and in 600 mg/Kg bw dose group, one was female found dead, and the other females showed signs of systematic toxicity.

- Rationale for animal assignment (if not random): All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges before the first exposure (Day 0). There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately.

- Fasting period before blood sampling for clinical biochemistry: overnight

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily (Not on days when detailed clinical observations were made), after treatment at approximately the same time with minor variations, or in the
afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Performed once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment. Animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult orprolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed at least weekly during the preexposure period, then on Day -1 for randomisation purposes, on Day 0 then at least weekly and at termination. Parental females were weighed on Gestation Days (GD) 0, 3, 7, 10, 14, 17 and 20 and on post-partum days (PPD) 0 (within 24 hours after parturition), 4, 7, 10, 13 and 14 (before termination).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Animal food consumption was determined by re-weighing the non-consumed diet weekly (on the days of body weight measurements).
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (fasted (overnight period of food deprivation, after the litter had been culled))
- How many animals: 5 male and 5 female animals/group (Note: Due to the high mortality of the High dose females, only the 3 surviving females were sampled
and evaluated)
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled necropsy
- Animals fasted: Yes (fasted (overnight period of food deprivation, after the litter had been culled))
- How many animals: 5 male and 5 female animals/group (Note: Due to the high mortality of the High dose females, only the 3 surviving females were sampled
and evaluated)
- Parameters checked in table [No.3] were examined.

SERUM HORMONES: Yes
- Time of blood sample collection: PND14 (females) / PND13 (pups)
- Animals fasted: Yes
- How many animals: all surviving dams and at least two pups per litter on PND14 (females) / PND13 (pups). All adult males.

URINALYSIS: Yes
- Time schedule for collection of urine: prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.4] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Last exposure week (males on Day 25; females on PPD9-12)
- Dose groups that were examined: 5 male and 5 female animals/group (Note: Due to the high mortality of the High dose females, only the 3 surviving females were sampled and evaluated)
- Battery of functions tested: sensory activity / grip strength / motor activity

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the treatments started Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating. Additionally, vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.

Sperm parameters (parental animals):

Parameters examined in all P male parental generations:
testis weight, epididymis weight, weight of seminal vesicles with coagulating glands. Testes and epididymides were weighed paired.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 2 pups/litter ([1]/sex/litter); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead]

Each litter was examined as soon as possible after delivery to detect the presence of gross abnormalities. Observations were reported individually for each adult animal.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

At termination, the surviving adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Gross necropsy was performed on all animals, irrespective of the date of death. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened, and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. The pre-terminally euthanized and the found dead animals were examined similarly to the terminal animals. At the time of termination, body weight and the weight of the following organs from all surviving adult animals were determined:

1) With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus

2) With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroid glands

Testes and epididymides were weighed paired. Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) were calculated and reported. The organ weights of the pre-terminally euthanized animal were measured similarly to the terminal animals. However, no organ weight measurement was performed in the animal found dead.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [5] were prepared for microscopic examination and weighed, respectively.

The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerves were retained in modified Davidson’s fixative, testes with epididymides in Bouin’s solution, and all other organs in 10% buffered formalin solution. Thyroid glands from one male and one female PND 13 pup selected by the study director from each litter were preserved in 10% buffered formalin solution. In this case, the thyroid weight (pooled) was determined after fixation. Trimming was done very carefully and only after fixation to avoid tissue damage. For microscopic examination, the retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6 μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. For the adult animals, a detailed histological examination was performed as follows:

• on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group (Due to the high mortality of the High dose females, only the 3 surviving females were evaluated)),
• all found dead High Dose female animals,
• all organs where macroscopic findings (abnormalities) were seen,
• retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups and of all males that failed to sire and all females that failed to deliver healthy pups
• the kidney samples of all the remaining males,
• the stomach samples of remaining males and females.

Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Postmortem examinations (offspring):

SACRIFICE
Surviving pups were sacrificed on PND4 and/or PND13. The anaesthetic product (pentobarbital) was diluted for pups’ euthanasia as required.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities, where possible.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not examined

Statistics:

For information on statistics please see the section 'Any other information on materials and methods incl. tables'.

Reproductive indices:

1) Male Mating Index: (Number of males with confirmed mating / Total Number of males cohabited) x 100

2) Female Mating Index : (Number of sperm − positive females / Total Number of females cohabited) x 100

3) Male Fertility Index: (Number of males impregnating a female / Total Number of males cohabited) x 100

4) Female Fertility Index: (Number of pregnant females / Number of sperm - positive females) x 100

5) Gestation Index: (Number of females with live born pups / Number of pregnant females) x 100

Offspring viability indices:

1) Survival Index (%): (Number of live pups (at designated time) / Number of pups born) x 100

2) Pre-implantation mortality (%): (Number of corpora lutea-Number of implantations / Number of corpora lutea) x 100

3) Intrauterine mortality (%): (Number of implantations-Number of liveborns / Number of implantations) x 100
4) Total mortality (%): (Number of implantations-Number of viable pups (PND0/4/13) / Number of implantations) x 100

5) Sex ratio % (females): (Number of female pups (PND0/4/13) / Number of viable pups (PND0/4/13)) x 100

Note: Survival index on PND13 was calculated from number of pups after culling on PND4 instead of number of pups born.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs were observed in any of the male animals during the study.

The following signs were observed in female rats:
In control group females, piloerection was present in 4 out of 10 animals. In the Low dose group, 6 out of 10 animals showed piloerection at some point during the study; from those 6 animals, 2 animals produced liquid faces (2 out of 10 evaluated animals), and among those two, in one animal hunched back was also observed (1 out of 10 evaluated animals). The observed changes were considered to be incidental or related to the dosing procedure, and not related to treatment. Mid dose group females showed piloerection (8 out of 10 evaluated animals), hunched back (1 out of 10 evaluated animals), and noisy respiration (4 out of 10 evaluated animals). These changes were considered to be treatment-related, adverse changes. Furthermore, brown discharge from the vulva was observed in one dam, and in another animal, thin fur on both forelimbs were also observed. These changes were considered to be incidental, and not treatment-related.

In the surviving High dose female animals, hunched back, piloerection, and noisy respiration was present (3 out of 3 animals), as well as red discharge from the nose in 1 out of 3 animals. These changes, due to their frequency and similarity to findings in the found dead animals, were considered to be treatment-related, adverse effects.

No clinical signs were observed in the two non-pregnant Control group females. Piloerection was present for one occasion in 1 out of 2 animals with total intrauterine mortality in the Low dose group. Noisy respiration was present for one occasion in 1 out of 2 animals with total intrauterine mortality in the Mid dose group. These findings were considered to be incidental, and not treatment-related adverse effects.

In the non-littering High dose females (12 found dead animals and 1 animal with total intrauterine mortality), the symptoms of piloerection (11 out of 13 animals), noisy respiration (11 out of 13 animals), laboured respiration (4 out of 13 animals), hunched back (9 out of 13 animals), decreased activity (2 out of 13 animals), ataxia (1 out of 13 animals), recumbency (3 out of 13 animals), red discharge (5 out of 13 animals) and intermittent tremors (1 out of 13 animals) were present, and these findings were considered to be a treatment-related, adverse effects, although direct correlation with the death of the animal was not established for all of the animals found dead.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Treatment-related mortality was observed in the High dose (500 mg/Kg bw/day) females where 12 animals were found dead either at the beginning of the treatment (between Day 2-6, six animals in total: #4505, #4507, #4508, #4510, #4605 and #4607), or near the end of the pregnancies (Day 24, or between Day 35-38, six animals in total: #4502, #4506, #4509, #4512, #4608 and #4610).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
All body weight and body weight gain values were normal in all dose groups, with the exception of the period between Day 14 to Day 21, where, due to the significantly lower body weight gain data in the control group, the body weight gain data in the high dose group was found to be statistically significantly higher (p < 0.01), despite being normal for a weekly body weight gain. This finding was considered to be incidental and not related to treatment.

Females:
Body weight of the high dose group was normal during the pre-mating and mating period but lower body weight was observed during the gestation period (reaching statistical significance (p<0.05) on GD7) and during the lactation period. Body weight gain data for the same periods showed constantly lower body weight gain values for these animals, reaching statistical significance for the entire gestation period (where at least 9 high dose females were compared to the control females). While no statistically significant difference was observed during the lactation period, it should be noted that for these values, only the 3 surviving high dose females were compared to the control group. The decreased body weight and body weight gain in the high dose females was considered to be a treatment-related adverse effect. For the low- and mid-dose female animals, all body weight and body weight gain values were normal.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males:
Significantly lower (-9.1%, p< 0.01) food consumption was observed in the high dose animals at the beginning of the treatment (Day 0-7), which was considered to be a transient, treatment-related effect. After this period, there were no treatment-related differences in the mean daily food consumption in any treated groups when compared to the control animals. The measured values were within the normal range for this strain and age.

Females:
High dose females showed significantly lower food consumption values during the first two weeks of the treatment (-30.3%, p< 0.01), and during the gestation period (-19.8%, p< 0.01). For the 3 surviving animals, lower food consumption data was measured during the lactation period (-12.3%, not reaching statistical significance when calculated for the entire lactation period), and the difference between high dose and control on the second week was more pronounced (-16.8%).These reduced food consumption values were considered to be treatment-related effects. There were no treatment-trelated differences in the mean daily food consumption in the low- and mid-dose (60 and 175 mg/Kg bw/day) females when compared to the control animals.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed in the haematology parameters. Statistically significant differences were considered to be incidental, since there was no relationship with dose and/or all recorded values were near or within the historical control ranges. These differences were not considered to reflect an effect of the test material.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical chemistry findings were observed. All significant differences were considered to be incidental, since there was no relationship with dose and/or all recorded values were near or within the historical control ranges. These differences were not considered to reflect an effect of the test material.

Endocrine findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in the thyroid hormone levels of the male animals. The observed values were in line with the historical control data, no statistically significant or biologically relevant differences were observed between the treated and control groups.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed in the urinalysis parameters.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups. There was no effect of treatment noted during the assessment of grip strength, foot splay or motor activity.

All dose groups of males and females had a normal locomotor activity; the initial activity was high,with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the treated animals and the control group when evaluating the overall distance travelled (0-60 min, cm). In the high dose male animals, locomotor activity showed an increasing trend between the control/low-dose groups, and through the mid-dose group up until the high-dose group, where the difference reached statistical significance (p<0.05) for the midand high-dose groups in the first five minutes (0-5 minutes), and, for the high-dose group, during the 25-30 minutes. For female animals, and for the rest of the male dose groups, the test material did not increase or decrease the normal locomotor activity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Animals found dead:
Histopathological changes were seen in the stomach of two animals found dead. One animal had diffuse minimal submucosal infiltration of inflammatory cells in the non-glandular part of the stomach.

One animal had mild focal erosion/ulcer of the non-glandular part of the stomach along with submucosal inflammatory cell infiltration. These findings, which correlated well with the findings from survival animals, were considered to be a treatment-related adverse effect. All other microscopic findings are considered to be post-mortem changes.

Terminal Necropsy (Parental Generation):
Treatment-related findings were observed in the kidney in males and in non-glandular stomach in both sexes.

Minimal to moderate unilateral/bilateral tubular basophilia and minimal to mild accumulation of hyaline droplets were observed in males given 60, 175 or 500 mg/Kg bw/day. Minimal to moderate granular casts were observed in males given 175 or 500 mg/Kg bw/day. The changes in the kidney included hyaline droplet accumulation in the cortical tubules, tubular basophilia and granular casts located at the junction with outer and inner stripes of outer medulla. The hyaline droplets are most likely to be Alpha 2u-globulin which is a low molecular weight protein produced by the liver and excreted through kidney in male rats only (Frazier et al. (2012)). The changes observed in the kidney correlated with the increased kidney weights observed in males given 175 or 500 mg/Kg bw/day. Similar changes were not observed in terminal or found dead high-dose (500 mg/Kg bw/day) females.

Minimal to mild focal erosion/ulcer of the non-glandular stomach was observed in 1/14 high-dose males, 1/3 high-dose females and 1/10 mid-dose females. Minimal to mild squamous cell hyperplasia of the non-glandular stomach was observed in a few treated animals with or without association with erosion/ulcer. Two high-dose females found dead also had lesions in the non-glandular stomach. The single incidence of minimal squamous hyperplasia of the non-glandular stomach in 1/12 low-dose males and 1/10 low-dose females only may be considered within the normal background.

The historical control data for this finding in the laboratory (12 studies) did not show any incidence of this finding. However, it has been reported that gastric erosion and ulceration of the glandular or non-glandular stomach is commonly observed in aged rats with more incidence in gavage studies. The edges of the ulcers in the non-glandular stomach often display epithelial hyperplasia (McInnes. E F (2012), Background lesions in laboratory animals, A colour atlas, Saunders, Elsevier, Edinburgh, pp 23). Occasionally, the non-glandular stomach mucosa of untreated aged rodents exhibit hyperplasia without inflammation or erosion and ulcers. As humans lack a non-glandular stomach, the relevance of the changes produced by chemicals and drugs in the rodent non glandular stomach is often disputed (Greaves, P (2012) Histopathology of preclinical toxicity studies: Interpretation and relevance in drug safety evaluation 4th Edition, Elsevier, Amsterdam pp 343 – 345).

All other changes were seen in control and/or treated animals, or were without meaningful differences in severity and incidence, and therefore regarded as incidental or a common background.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Each female selected for the treatment showed acceptable cycles (mean cycle length was 4.07, 4.00, 4.13 and 4.03 days for the Control, Low, Mid and High dose groups, respectively) before starting the treatment period.

In the first two weeks of the treatment period, the length of oestrus cycle was in the 4.00, 4.03, 3.98 and 4.42 days for each test material treated group for the Control, Low, Mid and High dose groups, respectively. The longer cycle length of High dose females was statistically significant (p<0.05), indicating a possible treatment-related effect, although it should be noted that the results of only 4 High dose females were available for evaluation. The number of females with irregular cycles was significantly higher in the High dose group (6 females) than in Control (1 female) however this dose (500 mg/Kgbw/day) was considered to exceed the maximum tolerated dose based on the severe mortality observed in the High dose group.

Prolonged oestrus was recorded in some control and test material treated females, but the frequency was similar between the groups.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no statistically significant differences observed amongst groups in the weights of reproductive organs (testes, epididymides) when compared to the control animals.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

A treatment-related effect was observed on the reproductive performance of the parental animals in the High dose group (500 mg/Kg bw/day). This dose was considered to exceed the maximum tolerated dose based on the severe mortality observed in the High dose group.

There were no differences between the control and test item treated groups with regard to mating ability; the mating indices were 100% in all groups. Test material administration was considered to have no impact on the duration of the mating period (the mean duration of mating was 4.1, 4.1, 4.0, and 3.2 days in Control, Low, Mid, and High dose groups, respectively). Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within 4 days of pairing (cohabitation) for all females except of two Mid dose animals (#3501: 8 days and #3506: 11 days).

The male and female fertility indices were 83% in the Control, Low, and Mid dose groups, and 80% for High dose males and 80% for High dose females. Although the lower fertility value observed in the High dose females was within the historical range of the Test Facility, it was at the low end of the range. Thus, a possible treatment-related effect could not be fully excluded however this dose (500 mg/Kg bw/day) was considered to exceed the maximum tolerated dose based on the severe mortality observed in the High dose group.

The gestation index was 100% in the Control and Low dose groups, 90% in the Mid dose group and 38% in the High dose group (5 out of 8 pregnant females died near the delivery, on GD20-22) due to the severe maternal toxicity at a concentration clearly exceeding the maximum tolerated dose.

There was no effect of treatment noted during the pre-implantation and gestation periods. The mean duration of pregnancy was comparable in the control and treated groups. The number of corpora lutea and number of implantation sites was comparable to the control mean at all dose groups.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 175 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

mortality

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 175 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Local Effects

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Local Effects

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Reproductive Toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 175 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Growth of the high-dose pups was reduced. This was associated with significantly lower food intake and weight gain of the surviving high dose dams during the lactation period. Low food intake by dams is likely to cause lower milk production, which would be linked to lower pup growth and increased mortality, but this was considered as an adverse reproductive effect in dams and pups at 500 mg/Kg bw/day dose level.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

In the treated groups, the number of viable pups on PND0, 4 and 13 as well as pup survival indices on PND0, 4 and 13 were comparable to control values in the Low and Mid dose groups, but lower survival values were observed on PND4 in the High dose group due to the higher post-natal mortality in the PND 0-4 period (15.6% vs the control level of 2.4%) as well as higher total mortality on PND4 (20.2% vs. the control level of 7.2%). Although those differences were not statistically significant, this might have been due to only 3 High dose litters available for evaluation, due to the high mortality. A similar trend was seen at the end of the lactation period, where higher postnatal mortality in the period of PND0-13 (17.4% vs. the control level of 2.4%) and higher total mortality on PND13 (21.9% vs. 7.2% control level) was noted in High dose group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the lactation period pup body weight gain was lower in the High dose group by about 10%. The difference compared to control was statistically significant (p<0.01) on PND0. The lower pup weight gain corresponded with the pup mortality data, and both correspond with the significantly lower food intake and body weight gain of the surviving High dose dams during the lactation period (where low food intake is likely to cause lower milk production, which would be linked to lower pup growth and increased mortality). The low dose and mid dose groups had similar values to the control.

Food consumption and compound intake (if feeding study):

not examined

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

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the anogenital distance between the treated groups (males/females) and the control on PND0.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There was no nipples/areolae presence observed in any of the male pups on PND13.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The thyroid gland weights of the PND13 pups were not statistically different from the control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Pups found dead:
During the lactation period 2 mid-dose pups were found dead that remained intact (not cannibalized or autolysed). This pup was subjected to necropsy with macroscopicexamination. No macroscopic findings were seen. No other pups that died during the study could be examined for treatment-related macroscopic findings.

Terminal Necropsy (PND13):
No macroscopic changes were observed in F1 offspring generation euthanized and examined externally at scheduled termination on PND13.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any of the PND13 pup dose groups, and the observed
values were consistent with the historical control data.

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 175 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

No test material was detected in the control samples. The mean measured test material concentrations of the individual test material containing dose formulations varied between 92.0% and 99.8%. These results were within the acceptable range (90% - 110%) and were considered suitable for the study purposes. All test material formulation samples were found to be homogeneous.

Table 6. Analytical Results
Dose level (mg/kg bw/day)	Nominal concentration (mg/mL)	Measured concentration (mg/mL)	Percentage of nominal concentration (%)
Analytical sampling #1 (Sampling: 18 October 2018)
0	Control	<LOQ	-
60	12	11.78 ± 0.04	98.2
175	35	33.99 ± 0.63	97.1
500	100	92.01 ± 5.90	92.0
Analytical sampling #2 (Sampling:14 November 2018)
0	Control	<LOQ	-
60	12	11.85 ± 0.79	98.7
175	35	34.95 ± 0.91	99.8
500	100	95.67 ± 4.83	95.7
Analytical sampling #3 (Sampling: 05 December 2018)
0	Control	<LOQ	-
60	12	11.52 ± 1.63	96.0
175	35	34.15 ± 0.68	97.6
500	100	98.88 ± 2.76	98.9

Notes: Mean measured concentrations with the 95% confidence intervals are shown.

The LOQ was 3 μg/mL for the analytical samples, which equals to 3.7 mg/mL formula concentration (counting with 1333xdilution during the sample preparation).

N/A: Not applicable

Table 7. Summary of Total Incidence of Clinical Signs (Parental Generation - Evaluated Animals)
From Day 0 to Day 61	Male Rats				Females Rats
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Total Number of Animals:	12	12	12	14	10	10	10	3
% of no abnormalities detected:	100.0	100.0	100.0	100.0	60.0	40.0	10.0	0.0
% of Animals with Symptoms:	0.0	0.0	0.0	0.0	40.0	60.0	90.0	100.0
Normal
Number of Animals Affected	12	12	12	14	10	10	10	3
% of Affected Animals	100	100	100	100	100	100	100	100
Number of Times Recorded	348	348	348	407	526	495	511	52
Discharge coloured
Number of Animals Affected	0	0	0	0	0	0	1	0
% of Affected Animals	0	0	0	0	0	0	10	0
Number of Times Recorded	0	0	0	0	0	0	1	0
Faeces liquid
Number of Animals Affected	0	0	0	0	0	2	0	0
% of Affected Animals	0	0	0	0	0	20	0	0
Number of Times Recorded	0	0	0	0	0	38	0	0
Fur thin
Number of Animals Affected	0	0	0	0	0	0	1	0
% of Affected Animals	0	0	0	0	0	0	10	0
Number of Times Recorded	0	0	0	0	0	0	1	0
Hunched Back
Number of Animals Affected	0	0	0	0	0	1	1	3
% of Affected Animals	0	0	0	0	0	10	10	100
Number of Times Recorded	0	0	0	0	0	11	1	39
Noisy respiration
Number of Animals Affected	0	0	0	0	0	0	4	3
% of Affected Animals	0	0	0	0	0	0	40	100
Number of Times Recorded	0	0	0	0	0	0	5	39
Piloerection
Number of Animals Affected	0	0	0	0	4	6	8	3
% of Affected Animals	0	0	0	0	40	60	80	100
Number of Times Recorded	0	0	0	0	8	31	16	92
Red discharge
Number of Animals Affected	0	0	0	0	0	0	0	1
% of Affected Animals	0	0	0	0	0	0	0	33
Number of Times Recorded	0	0	0	0	0	0	0	1

Table 8. Summary of Total Incidence of Clinical Signs (Parental Generation - Excluded Animals)
From Day 0 to Day 50	Male Rats				Females Rats
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Total Number of Animals:	-	-	-	-	2	2	2	13
% of no abnormalities detected:	-	-	-	-	100.0	50.0	50.0	0.0
% of Animals with Symptoms:	-	-	-	-	0.0	50.0	50.0	100.0
Normal
Number of Animals Affected	-	-	-	-	2	2	2	13
% of Affected Animals	-	-	-	-	100	100	100	100
Number of Times Recorded	-	-	-	-	94	85	87	46
Activity decreased
Number of Animals Affected	-	-	-	-	0	0	0	2
% of Affected Animals	-	-	-	-	0	0	0	15
Number of Times Recorded	-	-	-	-	0	0	0	6
Ataxia
Number of Animals Affected	-	-	-	-	0	0	0	1
% of Affected Animals	-	-	-	-	0	0	0	8
Number of Times Recorded	-	-	-	-	0	0	0	2
Found Dead
Number of Animals Affected	-	-	-	-	0	0	0	12
% of Affected Animals	-	-	-	-	0	0	0	92
Number of Times Recorded	-	-	-	-	0	0	0	12
Hunched Back
Number of Animals Affected	-	-	-	-	0	0	0	9
% of Affected Animals	-	-	-	-	0	0	0	69
Number of Times Recorded	-	-	-	-	0	0	0	111
Laboured respiration
Number of Animals Affected	-	-	-	-	0	0	0	4
% of Affected Animals	-	-	-	-	0	0	0	31
Number of Times Recorded	-	-	-	-	0	0	0	4
Noisy respiration
Number of Animals Affected	-	-	-	-	0	0	1	11
% of Affected Animals	-	-	-	-	0	0	50	85
Number of Times Recorded	-	-	-	-	0	0	1	123
Piloerection
Number of Animals Affected	-	-	-	-	0	1	0	11
% of Affected Animals	-	-	-	-	0	50	0	85
Number of Times Recorded	-	-	-	-	0	1	0	216
Recumbency
Number of Animals Affected	-	-	-	-	0	0	0	3
% of Affected Animals	-	-	-	-	0	0	0	23
Number of Times Recorded	-	-	-	-	0	0	0	3
Red discharge
Number of Animals Affected	-	-	-	-	0	0	0	5
% of Affected Animals	-	-	-	-	0	0	0	38
Number of Times Recorded	-	-	-	-	0	0	0	11
Tremors Intermittent
Number of Animals Affected	-	-	-	-	0	0	0	1
% of Affected Animals	-	-	-	-	0	0	0	8
Number of Times Recorded	-	-	-	-	0	0	0	2

Table 9. Summary of Bodyweight Data - Male Rats (Parental Generation)
Day(s) Relative to Start Date		Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
0	Mean	476.4 I^1	476.2	472.1	473.9
	SD	31.2	28.9	28.8	37.8
	Max	524	526	524	552
	Min	416	430	424	412
	N	12	12	12	14
	% diff	-	-0.1	-0.9	-0.5
7	Mean	496.4 I^1	499.6	498.0	487.7
	SD	34.7	33.3	32.3	38.3
	Max	545	553	542	560
	Min	438	449	436	424
	N	12	12	12	14
	% diff	-	0.6	0.3	-1.8
14	Mean	517.9 I^1	528.3	524.3	509.1
	SD	33.2	34.5	36.6	42.5
	Max	562	575	578	575
	Min	452	478	463	437
	N	12	12	12	14
	% diff	-	2.0	1.2	-1.7
21	Mean	519.7 I^1	532.8	531.8	521.6
	SD	35.6	32.8	35.8	43.2
	Max	563	578	583	593
	Min	451	492	474	447
	N	12	12	12	14
	% diff	-	2.5	2.3	0.4
27	Mean	535.3 I^1	543.9	545.3	529.8
	SD	35.7	34.9	37.1	40.6
	Max	582	587	598	602
	Min	467	489	481	454
	N	12	12	12	13
	% diff	-	1.6	1.9	-1.0

Statistical Test: Citoxlab DT Transformation: Automatic

[I - Automatic Transformation: Identity (No Transformation)]

Table 10. Summary of Bodyweight Data - Female Rats (Parental Generation)
		Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
From Day 0 to Mating Period (Day(s) Relative to Start Date)
Day 0	Mean	277.7 I^1	281.4	281.6	282.1
	SD	19.7	21.5	17.6	19.6
	Max	308	311	310	317
	Min	245	246	248	242
	N	12	12	12	16
	% diff	-	1.4	1.4	1.6
Day 7	Mean	286.1 I^1	288.4	289.5	266.8
	SD	22.8	24.8	25.8	19.3
	Max	324	331	330	296
	Min	254	249	247	234
	N	12	12	12	10
	% diff	-	0.8	1.2	-6.7
Day 14	Mean	292.1 I^1	293.3	297.3	293.2
	SD	23.5	24.9	24.6	16.8
	Max	331	332	337	311
	Min	260	252	246	261
	N	12	12	12	10
	% diff	-	0.4	1.8	0.4
Gestation Period (Day(s) Relative to Mating (Litter: A)
Day 0	Mean	298.2 I^1	297.0	298.8	288.2
	SD	24.8	25.1	26.6	16.9
	Max	346	341	346	310
	Min	263	252	246	255
	N	12	12	12	10
	% diff	-	-0.4	0.2	-3.3
Day 7	Mean	330.8 I, a^2	335.3	335.3	303.7 d^4
	SD	25.3	28.1	23.3	20.8
	Max	377	381	375	332
	Min	300	296	294	272
	N	12	12	12	10
	% diff	-	1.4	1.3	-8.2
Day 14	Mean	363.2 I, a^2	370.2	363.2	329.7
	SD	34.0	36.6	25.1	28.2
	Max	426	420	403	364
	Min	303	291	320	285
	N	12	12	12	9
	% diff	-	1.9	0.0	-9.2
Day 20	Mean	435.9 R^3	438.8	425.0	382.4
	SD	60.9	64.7	48.4	48.9
	Max	522	526	473	440
	Min	302	296	328	307
	N	12	12	12	9
	% diff	-	0.7	-2.5	-12.3
Lactation Period (Day(s) Relative to Littering (Litter: A))
Day 0	Mean	355.8 I^1	364.4	346.2	319.7
	SD	29.8	25.3	26.7	22.6
	Max	408	402	390	341
	Min	311	328	299	296
	N	10	10	9	3
	% diff	-	2.4	-2.7	-10.2
Day 4	Mean	368.3 I^1	378.4	353.6	342.3
	SD	29.7	31.8	26.7	37.9
	Max	422	416	390	369
	Min	320	326	304	299
	N	10	10	9	3
	% diff	-	2.7	-4.0	-7.1
Day 13	Mean	406.6 I^1	397.9	388.4	369.3
	SD	28.9	42.2	29.4	29.0
	Max	460	445	411	398
	Min	357	326	315	340
	N	10	10	9	3
	% diff	-	-2.1	-4.5	-9.2

Statistical Test: Citoxlab DT Transformation: Automatic

1 [I - Automatic Transformation: Identity (No Transformation)]

2 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

3 [R - Automatic Transformation: Rank]

4 [d - Test: Dunnett 2 Sided p < 0.05]

Table 11. Summary of Bodyweight Gain Data - Male Rats (Parental Generation)
Day(s) Relative to Start Date		Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
0 to 7	Mean	20.0^1	23.4	25.9	13.8
	SD	8.7	6.3	8.0	6.9
	Max	37	35	40	27
	Min	4	15	12	-2
	N	12	12	12	14
	% diff	-	17.1	29.6	-31.1
7 to 14	Mean	21.5 I^2	28.8	26.3	21.4
	SD	12.0	6.3	7.6	10.5
	Max	36	37	38	39
	Min	1	15	14	2
	N	12	12	12	14
	% diff	-	33.7	22.5	-0.7
14 to 21	Mean	1.8^1	4.4	7.5	12.6 dd^3
	SD	6.3	5.5	8.4	7.6
	Max	11	15	24	28
	Min	-11	-6	-3	-5
	N	12	12	12	14
	% diff	-	152.4	328.6	618.4
21 to 27	Mean	15.6 I^2	11.2	13.4	13.7
	SD	7.6	7.8	6.0	7.6
	Max	24	22	23	28
	Min	-4	-7	5	5
	N	12	12	12	13
	% diff	-	-28.3	-13.9	-12.1
0 to 27	Mean	58.8 I^2	67.8	73.2	61.9
	SD	14.0	12.8	14.9	19.8
	Max	71	90	98	96
	Min	20	46	47	29
	N	12	12	12	13
	% diff	-	15.2	24.4	5.3

Statistical Test: Citoxlab DT Transformation: Automatic

1 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.01]

2 [I - Automatic Transformation: Identity (No Transformation)]

3 [dd - Test: Dunnett 2 Sided p < 0.01]

 

Table 12. Summary of Bodyweight Gain Data - Female Rats: From Day 0 to Mating Period (Parental Generation)
Day(s) Relative to Start Date		Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Day 0 to 7	Mean	8.4^1	7.0	7.9	-15.0 dd^3
	SD	7.8	7.5	13.1	14.2
	Max	20	20	34	7
	Min	-4	-5	-10	-40
	N	12	12	12	10
	% diff	-	-16.8	-5.9	-278.2
Day 7 to 14	Mean	6.0^1	4.9	7.8	26.4 dd^3
	SD	7.0	11.7	9.4	11.8
	Max	15	25	21	46
	Min	-8	-15	-9	6
	N	12	12	12	10
	% diff	-	-18.1	30.6	340.0
Day 0 to 14	Mean	14.4 I^2	11.9	15.8	11.4
	SD	8.6	10.1	11.5	13.3
	Max	29	25	32	30
	Min	-2	-9	-4	-8
	N	12	12	12	10
	% diff	-	-17.3	9.2	-20.9

Statistical Test: Citoxlab DT Transformation: Automatic

1 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.01]

2 [I - Automatic Transformation: Identity (No Transformation)]

3 [dd - Test: Dunnett 2 Sided p < 0.01]

 

Table 13. Summary of Bodyweight Gain Data - Female Rats: Gestation Period (Parental Generation)
Day(s) Relative to Mating (Litter: A)		Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Day 0 to 7	Mean	32.7^1	38.3	36.5	15.5
	SD	8.5	13.1	13.5	15.1
	Max	43	50	54	31
	Min	13	6	1	-20
	N	12	12	12	10
	% diff	-	17.3	11.7	-52.6
Day 7 to 14	Mean	32.3 R, k^2	34.8	27.9	25.3
	SD	16.4	13.6	13.0	7.2
	Max	57	47	43	34
	Min	-4	-5	-5	13
	N	12	12	12	9
	% diff	-	7.7	-13.7	-21.6
Day 14 to 20	Mean	72.8 R^3	68.7	61.8	52.8
	SD	33.0	37.0	37.2	25.4
	Max	96	110	93	76
	Min	-1	-12	2	1
	N	12	12	12	9
	% diff	-	-5.6	-15.0	-27.5
Day 0 to 20	Mean	137.8 R, k^2	141.8	126.3	94.7 u^4
	SD	50.2	59.1	53.1	44.7
	Max	177	207	172	139
	Min	30	6	17	-3
	N	12	12	12	9
	% diff	-	3.0	-8.3	-31.3

Statistical Test: Citoxlab DT Transformation: Automatic

1 [R,kk - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.01]

2 [R,k - Automatic Transformation: Rank, (All Groups) Test: Kruskal-Wallis p < 0.05]

3 [R - Automatic Transformation: Rank]

4 [u - Test: Dunn 2 Sided p < 0.05]

 

Table 14. Summary of Bodyweight Gain Data - Female Rats: Lactation Period (Parental Generation)
(Day(s) Relative to Littering (Litter: A))		Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Day 0 to 4	Mean	12.5 I^1	14.0	7.3	22.7
	SD	8.0	17.1	5.9	17.6
	Max	22	42	16	37
	Min	-6	-13	-3	3
	N	10	10	9	3
	% diff	-	12.0	-41.3	81.3
Day 4 to 13	Mean	38.3 R^2	19.5	34.9	27.0
	SD	9.2	25.0	16.0	15.1
	Max	51	61	58	41
	Min	24	-36	11	11
	N	10	10	9	3
	% diff	-	-49.1	-8.9	-29.5
Day 0 to 13	Mean	50.8 R^2	33.5	42.2	49.7
	SD	12.9	30.7	16.1	6.7
	Max	73	70	62	57
	Min	32	-41	16	44
	N	10	10	9	3
	% diff	-	-34.1	-16.9	-2.2

Statistical Test: Citoxlab DT Transformation: Automatic

1 [I - Automatic Transformation: Identity (No Transformation)]

2 [R - Automatic Transformation: Rank]

Table 15. Food Mean Consumption (g/animal/day): Male Rats (Parental Generation)
		Day(s) Relative to Start Date
		0 to 7	7 to 14	14 to 21	21 to 27
Group 1 0 mg/Kg bw/day	Mean	27.73 I, a^1	27.21 R^2	25.17 R^2	25.64 I^3
Group 2 60 mg/Kg bw/day	Mean	27.26	26.98	25.76	26.32
Group 3 175 mg/Kg bw/day	Mean	27.02	27.04	27.40	28.07
Group 4 500 mg/Kg bw/day	Mean	25.20 dd^4	26.26	26.69	27.64

Statistical Test: Citoxlab DT Transformation: Automatic

1 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

2 [R - Automatic Transformation: Rank]

3 [I - Automatic Transformation: Identity (No Transformation)]

4 [dd - Test: Dunnett 2 Sided p < 0.01]

 

Table 16. Food Mean Consumption (g/animal/day): Female Rats: From Day 0 to Mating Period (Parental Generation)
		Day(s) Relative to Start Date
		0 to 7	7 to 14
Group 1 0 mg/Kg bw/day	Mean	18.27^1	17.79^1
Group 2 60 mg/Kg bw/day	Mean	19.12	18.90
Group 3 175 mg/Kg bw/day	Mean	18.29	20.04 dd^3
Group 4 500 mg/Kg bw/day	Mean	9.63 dd^3	17.63

Statistical Test: Citoxlab DT Transformation: Automatic

1 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.01]

3 [dd - Test: Dunnett 2 Sided p < 0.01]

 

Table 17. Food Mean Consumption (g/animal/day): Female Rats: Gestation Period (Parental Generation)
		Day(s) Relative to Start Date
		0 to 7	7 to 14	14 to 21
Group 1 0 mg/Kg bw/day	Mean	23.96^1	24.94^1	27.33 I, a^2
Group 2 60 mg/Kg bw/day	Mean	25.81	27.77	28.85
Group 3 175 mg/Kg bw/day	Mean	25.14	25.77	27.28
Group 4 500 mg/Kg bw/day	Mean	18.66 dd^3	20.02 dd^3	22.24

Statistical Test: Citoxlab DT Transformation: Automatic

1 [I,aa - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.01]

2 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

3 [dd - Test: Dunnett 2 Sided p < 0.01]

 

Table 18. Food Mean Consumption (g/animal/day): Female Rats: Lactation Period (Parental Generation)
		Day(s) Relative to Start Date
		0 to 7	7 to 13
Group 1 0 mg/Kg bw/day	Mean	42.49 R^1	69.37 R^1
Group 2 60 mg/Kg bw/day	Mean	45.56	74.03
Group 3 175 mg/Kg bw/day	Mean	40.60	67.19
Group 4 500 mg/Kg bw/day	Mean	39.90	57.72

Statistical Test: Citoxlab DT Transformation: Automatic

1 [R - Automatic Transformation: Rank]

Table 19. Select Haematology Parameters (Parental Generation)
Haematology Parameter	Groups/Concentration (mg/kg bw/day)
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Males
APTT (sec)	11.26	11.44	10.32	11.82
	Differences from control	1.6%	-8.3%	5.0%
	Historical control data (mean ± 2SD)	12.33 ± 1.260
PTT (sec)	10.40	10.26	10.00	10.14
	Differences from control	-1.3%	-3.8%	-2.5%
	Historical control data (mean ± 2SD)	9.86 ± 0.324
Females
APTT (sec)	13.08	12.66	11.98	11.23*
	Differences from control	-3.2%	-8.4%	-14.1%
	Historical control data (mean ± 2SD)	12.95 ± 2.017
PTT (sec)	9.26	8.92	8.70++	8.80
	Differences from control	-3.7%	-6.0%	-5.0%
	Historical control data (mean ± 2SD)	8.86 ± 0.489

*= p<0.05, **= p<0.01; Dunnett two-sided test.

+= p<0.05, ++= p<0.01; Dunn two-sided test.

 

Table 20. Select Clinical Chemistry Parameters (Parental Generation)
Clinical Chemistry Parameter	Groups/Concentration (mg/kg bw/day)
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Males
Albumin (g/L)	30.98	30.88	32.14	33.70+
	Differences from control	-0.3%	3.7%	8.8%
	Historical control data (mean ± 2SD)	30.02 ± 2.053
A/G Ratio	1.36	1.42	1.44	1.56**
	Differences from control	4.4%	5.9%	14.7%
	Historical control data (mean ± 2SD)	1.21 ± 0.093
Alkaline Phosphatase (U/L)	82.8	86.6	84.0	88.6
	Differences from control	4.6%	1.4%	7.0%
	Historical control data (mean ± 2SD)	99.7 ± 24.99
Females
Albumin (g/L)	31.20	31.56	34.14	32.57
	Differences from control	1.2%	9.4%	4.4%
	Historical control data (mean ± 2SD)	29.54 ± 2.126
A/G Ratio	1.25	1.30	1.42	1.30
	Differences from control	4.0%	13.6%	4.0%
	Historical control data (mean ± 2SD)	1.16 ± 0.086
Alkaline Phosphatase (U/L)	72.3	121.6**	87.4	95.0
	Differences from control	68.3%	21.0%	31.5%
	Historical control data (mean ± 2SD)	88.4 ± 27.22

*= p<0.05, **= p<0.01; Dunnett two-sided test

+= p<0.05, ++= p<0.01; Dunn two-sided test

 

Table 21. Kidney and Liver Organ Weights (Parental Generation)
Organ weights	Groups/Concentration (mg/kg bw/day)
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Males
Terminal bodyweight (g)	508.8	513.7	516.0	503.1
	Differences from control	1.0%	1.4%	-1.1%
	Historical control data (mean ± 2SD)	478.5 ± 35.75
Liver (g)	15.640	15.984	17.788*	19.623**
	Differences from control	2.2%	13.7%	25.5%
	Historical control data (mean ± 2SD)	13.272 ± 1.4813
Liver / Bodyweight (%)	3.059	3.107	3.448**	3.892**
	Differences from control	1.6%	12.7%	27.2%
	Historical control data (mean ± 2SD)	2.7721 ± 0.21044
Liver / Brain weight (%)	692.22	707.13	783.36	872.10**
	Differences from control	2.2%	13.2%	26.0%
	Historical control data (mean ± 2SD)	602.6183 ± 70.57230
Kidney (g)	3.346	3.523	3.984**	4.096**
	Differences from control	5.3%	19.1%	22.4%
	Historical control data (mean ± 2SD)	3.065 ± 0.2627
Kidney / Bodyweight (%)	0.657	0.686	0.773**	0.816**
	Differences from control	4.4%	17.6%	24.2%
	Historical control data (mean ± 2SD)	0.6417 ± 0.04463
Kidney / Brain weight (%)	148.19	155.69	175.29**	182.07**
	Differences from control	5.1%	18.3%	22.9%
	Historical control data (mean ± 2SD)	139.1606 ± 12.57275
Females
Terminal bodyweight (g)	363.8	363.0	352.1	314.7
	Differences from control	-0.2%	-3.2%	-13.5%
	Historical control data (mean ± 2SD)	355.5 ± 23.52
Liver (g)	13.660	14.156	14.443	15.077
	Differences from control	3.6%	5.7%	10.4%
	Historical control data (mean ± 2SD)	13.698 ± 1.2264
Liver / Bodyweight (%)	3.762	3.903	4.119*	4.788**
	Differences from control	3.7%	9.5%	27.3%
	Historical control data (mean ± 2SD)	3.8577 ± 0.29215
Liver / Brain weight (%)	656.43	681.44	719.72	767.17*
	Differences from control	3.8%	9.6%	16.9%
	Historical control data (mean ± 2SD)	684.7389 ± 62.85047
Kidney (g)	2.234	2.367	2.375	2.387
	Differences from control	6.0%	6.3%	6.8%
	Historical control data (mean ± 2SD)	2.209 ± 0.1924
Kidney / Bodyweight (%)	0.616	0.656	0.676	0.759**
	Differences from control	6.5%	9.6%	23.2%
	Historical control data (mean ± 2SD)	0.6217 ± 0.03948
Kidney / Brain weight (%)	107.46	113.98	118.25	121.74
	Differences from control	6.1%	10.0%	13.3%
	Historical control data (mean ± 2SD)	110.4571 ± 10.25619

*= p<0.05; **= p<0.01; Dunnett two-sided test.

Table 22. Incidence of Treatment-related Microscopic Changes in the Kidney (Parental Generation)
	Males				Females
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Number of animals	12	12	12	14	10	10	10	3
Accumulation, Hyaline droplets, bilateral/unilateral
Minimal	0	1	4	3
Mild	0	0	5	9	0	0	0	0
Total	0	1	9	12	0	0	0	0
Basophilia, bilateral/unilateral
Minimal	0	5	3	2	0	0	0	0
Mild	0	4	6	7	0	0	0	0
Moderate	0	0	3	5	0	0	0	0
Total	0	9	12	14	0	0	0	0
Cast, granular, bilateral/unilateral
Minimal	0	0	1	2	0	0	0	0
Mild	0	0	3	5	0	0	0	0
Moderate	0	0	2	1	0	0	0	0
Total	0	0	6	8	0	0	0	0
Number of tissues examined	12	12	12	14	5	0	0	3

*Note: Data of two non-pregnant females in the Control Goup, two females with total intrauterine mortality in the Low dose group, two females with total intrauterine mortality in the Mid dose group, and 12 found dead females and one female with total intrauterine mortality in the High dose group were excluded from this evaluation.

Table 23. Incidence of Treatment-related Microscopic Changes in the Stomach (Parental Generation)
	Males				Females
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Number of animals	12	12	12	14	10	10	10	3
Erosion/ulcer, non-glandular region
Minimal	0	0	0	0	0	0	1	0
Mild	0	0	0	1	0	0	0	1
Total	0	0	0	1	0	0	1	1
Hyperplasia, Squamous, non-glandular region
Minimal	0	1	5	5	0	1	3	0
Mild	0	0	0	1	0	0	0	1
Total	0	1	5	6	0	1	3	1
Infiltrate, Inflammatory Cells, non-glandular region
Minimal	0	0	1	1	0	0	1	0
Mild	0	0	0	1	0	0	0	1
Total	0	0	1	2	0	0	1	1
Number of tissues examined	12	12	12	14	10	10	10	3

*Note: Data of two non-pregnant females in the Control Goup, two females with total intrauterine mortality in the Low dose group, two females with total intrauterine mortality in the Mid dose group, and 12 found dead females and one female with total intrauterine mortality in the High dose group were excluded from this evaluation.

Table 24. Summary of Thyroid Hormone Concentrations (Parental Males)
	Day(s) Relative to Start Date		Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Adult T4 Conc. (ng/mL)	203 to 208	Mean	42.89 R^+	42.43	41.95	41.52
		SD	3.76	4.67	4.92	7.35
		Max	50.8	50.1	50.0	54.8
		Min	35.0	35.3	33.7	31.5
		N	12	12	12	14
		% Diff	-	-1.1	-2.2	-3.2

Table 25. Summary of the pre-natal and post-natal periods with survival indices
Parameters	Group / Concentration (mg/kg bw/day)
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Mean number of implantations	13.75	14.42	11.75	13.25
Number of pups born, mean	16.00	15.10	12.60	16.33
Number of live born pups, mean	15.70	15.10	12.50	16.33
Pre-natal mortality, mean	0.80	2.20	1.60	1.33
Pre-natal mortality (%), mean	4.83	12.65	17.95	6.75
Post-natal mortality on PND0-4, mean	0.40	0.30	0.11	2.67
Post-natal mortality on PND0-4 (%), mean	2.35	2.15	0.58	15.58
Total mortality on PND4, mean	1.20	2.50	1.56	4.00
Total mortality on PND4 (%), mean	7.18	14.44	9.41	20.18
Post-natal mortality on PND0-13, mean	0.40	0.30	0.22	3.00
Post-natal mortality on PND0-13 (%), mean	2.35	2.15	1.17	17.43
Total mortality on PND13, mean	1.20	2.50	1.80	4.33
Total mortality on PND13 (%), mean	7.18	14.44	19.00	21.93
Survival index on PND0 (%)	98.14	100.0	98.49	100.0
Sex ratio (% of females) on PND0	53.67	51.70	52.32	53.16
Survival index on PND4 (%)	97.65	97.85	100.0	84.42
Sex ratio (% of females) on PND4	53.83	51.63	52.32	53.73
Survival index on PND13 (%) (from PND4 after culling)	100.0	100.0	99.31	97.78
Sex ratio (% of females) (PND13)	54.46	52.09	51.92	53.57

 

Table 26. Offspring Mortality and Viability
Parameters	Group / Concentration (mg/kg bw/day)
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Number of evaluated litters	10	10	10	3
Number of pups born	160	151	126	49
Number of liveborns	157	151	125	49
Evaluation of PND0
Number of found dead / stillborn pups on PND0	3	0	2	0
Number of cannibalised pups (PND0)	0	0	0	0
Number of autolysed pups (PND0)	3	0	0	0
Number of found dead, intact pups, FT - (PND0)	0	0	1	0
Number of found dead, intact pups, FT + (PND0)	0	0	1	0
Number of viable pups (PND0)	157	151	124	49
Evaluation of PND1-4 +
Number of dead pups on PND1-4	4	3	0	8**
Number of cannibalised pups (PND1-4)	4	1	0	8**
Number of autolysed pups (PND1-4)	0	2	0	0
Number of found dead, intact pups (PND1-4)	0	0	0	0
Number of viable pups (PND4)	153	148	124	41**
Number of viable pups after culling (PND4)	133	128	108	35
Evaluation of PND5-13 ++
Number of dead pups on PND5-13	0	0	1	1
Number of cannibalised pups (PND5-13)	0	0	0	1
Number of autolysed pups (PND5-13)	0	0	1	0
Number of found dead, intact pups (PND5-13)	0	0	0	0
Number of viable pups (PND13)	133	128	107	34
Notes: * = p<0.05, ** = p<0.01; Chi-squared test; + Counted from end of working day PND0. ++ Counted from end of working day PND4. Abbreviations: FT - : Floating test negative, FT + : Floating test positive

 

Table 27. Body weight data (Offspring)
Parameters	Group / Concentration (mg/kg bw/day)
	Group 1 0 mg/Kg bw/day	Group 2 60 mg/Kg bw/day	Group 3 175 mg/Kg bw/day	Group 4 500 mg/Kg bw/day
Number of evaluated litters	10	10	10	3
Number of viable pups (PND0)	157	151	124	49
Mean pup bodyweight per litter (PND0), g	6.414	6.417	6.564	5.679++
Historical data (mean ± SD):	6.416 ± 0.66
Number of viable pups (PND4)	153	148	124	41**
Mean pup bodyweight per litter (PND4), g	10.056	9.987	10.163	8.698
Historical data (mean ± SD):	10.239 ± 1.46
Mean pup bodyweight gain per litter (PND0-4), g	3.625	3.569	3.599	2.977
Historical data (mean ± SD):	3.798 ± 1.12
Number of viable pups (PND13)	133	128	107	34*
Mean pup bodyweight per litter (PND13), g	26.481	26.435	27.434	23.750
Historical data (mean ± SD):	27.822 ± 3.74
Mean pup bodyweight gain per litter (PND4-13), g	16.442	16.484	17.256	14.955
Historical data (mean ± SD):	17.581 ± 2.78
Mean pup bodyweight gain per litter (PND0-13), g	20.051	20.031	20.873	17.986
Historical data (mean ± SD):	21.379 ± 3.44
Notes: *= p<0.05; **= p<0.01; Chi-squared test.++= p<0.01; Dunnett’s test

Conclusions:

The systemic No Observed Adverse Effect Level (NOAEL) for Ocimene PQ [Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene] was determined to be 175 mg/Kg bw/day, based on mortality observed in female rats at the highest dose tested. The NOAEL for local toxicity (stomach effects) was determined to be 60 mg/Kg bw/day and 175 mg/Kg bw/day for female and male rats, respectively. The NOAEL for reproductive toxicity was determined to be 500 mg/Kg bw/day for male rats and 175 mg/Kg bw/day for female rats while the NOAEL for development / survival of the F1 generation was determined to be 175 mg/Kg bw/day.

Executive summary:

In a key combined repeated dose and reproductive/developmental toxicity screening study, the test material (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene; EC# 933-779-9) was administered via daily oral gavage to male and female Wistar rats at doses of 0, 60, 175, or 500 mg/Kg bw/day (in propylene glycol with 1% polysorbate 80 vehicle). Rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males while the females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13.

 

Signs of morbidity and mortality were observed for twice daily while general and detailed observations for clinical signs were undertaken daily and weekly, respectively. Body weight and food consumption were measured weekly and clinical pathology evaluations, including haematology, coagulation, clinical chemistry and urinalysis were undertaken. Neurological assessments including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. Reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13. At termination, necropsy with macroscopic examination was performed. Weights of selected organs recorded, and representative tissues/organs sampled and preserved in appropriate fixatives from the adult animals. Thyroxine (T4) levels in Day 13 pups and adult males were also assessed. For adult animals, a detailed histological examination was performed on a select list of retained organs in the control and high dose groups and for all those male / female mating pairs where no conceptus and/or no live born pups was achieved. Track down examination of stomach samples of all remaining animals and kidneys of all remaining male rats was also performed.

 

Treatment-related mortality was observed in the high-dose females (12 out of 16 animals), either at the beginning of the treatment (between Days 2-6) or near the end of the pregnancies (on GD20-22). No further mortality, clinically adverse effects (except for minor, transient symptoms), or changes in neurological assessment were observed through the study period. Adverse effects on the body weight of females in the high dose (500 mg/Kg bw/day) group were observed during the gestation (reaching statistical significance) and lactation period, with similar changes observed in food consumption.

 

Haematology, clinical chemistry, and urinalysis parameters evaluated remained unaffected by treatment and no treatment-related changes were observed in the thyroid hormone levels of the adult male animals. Gross necropsy did not reveal any remarkable treatment-related adverse effects.

 

Higher liver and kidney weights were observed in both sexes in the mid- and high-dose animals (175 and 500 mg/Kg bw/day). These organ weight changes were correlated with the microscopic kidney findings in the males (most likely to be Alpha 2u globulin which is specific to male rat kidney only). These changes were considered to be non-adverse in the context of human health. Histopathological findings in the stomach included mild focal erosion/ulcer of the nonglandular stomach in 1 out of 14 high-dose males and in 1 out of 3 high-dose females; minimal focal erosion/ulcer in the same region in 1 out of 10 mid-dose females. These observations were considered to be an adverse, treatment-related local irritant effect.

 

Slight perturbation in oestrus cycle (slightly longer cycle length and increased number of females with irregular cycles) was observed in the high-dose females, although the results of only four high-dose females were available for evaluation because the maximum tolerated dose was clearly exceeded due to the toxicity observed. No treatment-related changes were observed in the reproductive parameters during mating, but a treatment-related adverse effect was observed on gestation of the high-dose group female rats (5 out of 8 pregnant high-dose (500 mg/Kg bw/day) females died prior to the delivery).

 

Compared to the control group, lower survival values and higher post-natal mortality or total mortality values during the PND 0-4 or PND 0-13 periods were observed in the high-dose group. Although those differences were not statistically significant, this might have been due to the limited number of high-dose litters available for evaluation (only three litters available due to high mortality). This was considered to be an adverse treatment-related effect.

 

No adverse effect on PND0 body weight was recorded in the litters of the three surviving high-dose females (500 mg/Kg bw/day) or at lower dose levels. Growth and survival of the high-dose group pups was reduced, which was associated with significantly lower food intake and weight gain of the surviving high-dose dams during the lactation period. Low food intake by dams is likely to cause lower milk production, which would be linked to lower pup growth and increased mortality, but this was considered to be an adverse reproductive effect in dams and pups at 500 mg/Kg bw/day dose level. No developmental or endocrine changes (anogenital distance, thyroid gland weights, thyroid hormone level) were observed in the pups at any of the dose levels.

 

The systemic No Observed Adverse Effect Level (NOAEL) for Ocimene PQ [Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene] was determined to be 175 mg/Kg bw/day, based on mortality observed in female rats at the highest dose tested. The NOAEL for local toxicity (stomach effects) was determined to be 60 mg/Kg bw/day and 175 mg/Kg bw/day for female and male rats, respectively. The NOAEL for reproductive toxicity was determined to be 500 mg/Kg bw/day for male rats and 175 mg/Kg bw/day for female rats while the NOAEL for development / survival of the F1 generation was determined to be 175 mg/Kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

175 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

One key substance specific OECD Guideline 422 study available for assessment.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a key OECD Guideline 422 combined repeated dose and reproductive/developmental toxicity screening study (Charles River Laboratories Hungary Kft., 2021), the test material (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene; EC 933-779-9) was administered via daily oral gavage to male and female Wistar rats at doses of 0, 60, 175, or 500 mg/Kg bw/day (in propylene glycol with 1% polysorbate 80 vehicle). Rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males while the females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13.

 

Signs of morbidity and mortality were observed for twice daily while general and detailed observations for clinical signs were undertaken daily and weekly, respectively. Body weight and food consumption were measured weekly and clinical pathology evaluations, including haematology, coagulation, clinical chemistry and urinalysis were undertaken. Neurological assessments including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. Reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13. At termination, necropsy with macroscopic examination was performed. Weights of selected organs recorded, and representative tissues/organs sampled and preserved in appropriate fixatives from the adult animals. Thyroxine (T4) levels in Day 13 pups and adult males were also assessed. For adult animals, a detailed histological examination was performed on a select list of retained organs in the control and high dose groups and for all those male / female mating pairs where no conceptus and/or no live born pups was achieved. Trackdown examination of stomach samples of all remaining animals and kidneys of all remaining male rats was also performed.

 

Treatment-related mortality was observed in the high-dose females (12 out of 16 animals), either at the beginning of the treatment (between Days 2-6) or near the end of the pregnancies (on GD20-22). No further mortality, clinically adverse effects (except for minor, transient symptoms), or changes in neurological assessment were observed through the study period. Adverse effects on the body weight of females in the high dose (500 mg/Kg bw/day) group were observed during the gestation (reaching statistical significance) and lactation period, with similar changes observed in food consumption.

 

Haematology, clinical chemistry, and urinalysis parameters evaluated remained unaffected by treatment and no treatment-related changes were observed in the thyroid hormone levels of the adult male animals. Gross necropsy did not reveal any remarkable treatment-related adverse effects.

 

Higher liver and kidney weights were observed in both sexes in the mid- and high-dose animals (175 and 500 mg/Kg bw/day). These organ weight changes were correlated with the microscopic kidney findings in the males (most likely to be Alpha 2u globulin which is specific to male rat kidney only). These changes were considered to be non-adverse in the context of human health. Histopathological findings in the stomach included mild focal erosion/ulcer of the nonglandular stomach in 1 out of 14 high-dose males and in 1 out of 3 high-dose females; minimal focal erosion/ulcer in the same region in 1 out of 10 mid-dose females. These observations were considered to be an adverse, treatment-related local irritant effect.

 

Slight perturbation in oestrus cycle (slightly longer cycle length and increased number of females with irregular cycles) was observed in the high-dose females, although the results of only four high-dose females were available for evaluation because the maximum tolerated dose was clearly exceeded due to the toxicity observed. No treatment-related changes were observed in the reproductive parameters during mating, but a treatment-related adverse effect was observed on gestation of the high-dose group female rats (5 out of 8 pregnant high-dose (500 mg/Kg bw/day) females died prior to the delivery).

 

Compared to the control group, lower survival values and higher post-natal mortality or total mortality values during the PND 0-4 or PND 0-13 periods were observed in the high-dose group. Although those differences were not statistically significant, this might have been due to the limited number of high-dose litters available for evaluation (only three litters available due to high mortality). This was considered to be an adverse treatment-related effect.

 

No adverse effect on PND0 body weight was recorded in the litters of the three surviving high-dose females (500 mg/Kg bw/day) or at lower dose levels. Growth and survival of the high-dose group pups was reduced, which was associated with significantly lower food intake and weight gain of the surviving high-dose dams during the lactation period. Low food intake by dams is likely to cause lower milk production, which would be linked to lower pup growth and increased mortality, but this was considered to be an adverse reproductive effect in dams and pups at 500 mg/Kg bw/day dose level. No developmental or endocrine changes (anogenital distance, thyroid gland weights, thyroid hormone level) were observed in the pups at any of the dose levels.

 

The systemic No Observed Adverse Effect Level (NOAEL) for Ocimene PQ [Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene] was determined to be 175 mg/Kg bw/day, based on mortality observed in female rats at the highest dose tested. The NOAEL for local toxicity (stomach effects) was determined to be 60 mg/Kg bw/day and 175 mg/Kg bw/day for female and male rats, respectively. The NOAEL for reproductive toxicity was determined to be 500 mg/Kg bw/day for male rats and 175 mg/Kg bw/day for female rats while the NOAEL for development / survival of the F1 generation was determined to be 175 mg/Kg bw/day.

Justification for classification or non-classification

Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene does not meet the criteria to be classified for Reproductive toxicity under EU Regulation (EC) No 1272/2008 (CLP).