Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A new comparative study in male rate was performed to investigate the potential toxic effect of terpineol multiconstituent when administered by diet versus by gavage over a period of 28 days. No effect on male fertility was observed in animals from the diet group (12000 ppm). Moreover, in a recent study examining the toxicokinetics of alpha-terpineol in male rats, it was demonstrated that terpineol multiconstituent metabolism is rapidely saturated and revealed that the AUC values at the highest dose level (750 mg/kg) were ~2.3-fold higher than the values predicted from a linear relationship, indicating a systemic overexposure of male rats after gavage adminitration.

Link to relevant study records

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Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 28 to September 03, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD guideline 422 without deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: 303 to 375 g for males and 198 to 253 g for females
- Housing: Up to 5 during pre-mating for all animals and after mating for males and during toxicity phase for unmatted females, individually with litter for females during gestation and lactation.
- Diet (e.g. ad libitum): Standard rodent diet (SDS VRF1 Certified) ad libitum, except overnight before routine blood sampling for Main phase males, Toxicity phase females and Recovery phase animals.
- Water (e.g. ad libitum): Potable water taken from the public supply, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 40 to 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 April 2010 To: 29 June 2010
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Approximately 50% of the final volume of corn oil was added to the required amount of test material. The formulation was mixed using a magnetic stirrer until all of the test material had dissolved and more corn oil was added to make up the required volume. The formulation was then mixed using a magnetic stirrer until homogeneous.
Initially all formulations were prepared freshly on the day of use and used within two hours of completion of preparation. However, following confirmation of the results from a homogeneity and stability, formulations were prepared weekly, subdivided into daily aliquots and used within 8 days of preparation.

VEHICLE
- Concentration in vehicle: 12, 50 and 150 mg/mL
- Amount of vehicle (if gavage): constant dosage-volume of 5 mL/kg bw/day
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Vaginal plug and sperm in vaginal smear referred to as day 0 of gestation
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in the first and last weeks of the dosing period were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed) from all groups. Two assays from each group were analysed. The mean concentrations of Terpineol multiconstituent in test formulations analysed for the study were within applied limits, +10%/-15% of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
Main phase males and Toxicity phase females were dosed daily for a minimum of five consecutive weeks. An additional five males and five females were dosed with the vehicle or at 1000 mg/kg/day for five weeks and then given two weeks of recovery before termination. Main phase females were dosed daily for two weeks before pairing, throughout mating, gestation and until Day 6 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose. Offspring were not dosed.
Frequency of treatment:
Once a day, 7 days a week
Details on study schedule:
None
Remarks:
Doses / Concentrations:
60, 250 and 750 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
- Reproductive subgroup (main phase): 10 males and 10 females/dose (except for control males and at top dose: 5 males/dose)
- Toxicity subgroup: 5 females/dose and same males as for reproductive subgroup
- Recovery subgroup: 5 males and 5 females /dose (control and top dose); Recovery phase males also used for pairing with Main reproductive phase females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a two week preliminary study (Huntingdon Life Sciences Study No. OAD0003) which tested dose levels of 150, 600 and 1000 mg/kg bw/day. In that study animals dosed at 600 and 1000 mg/kg bw/day showed post dose observations of salivation and chin rubbing and some females at 1000 mg/kg bw/day also showed isolated incidences of reduced activity, reduced body tone and unsteady gait. An initial reduction in bodyweight was recorded in males at 600 and 1000 mg/kg bw/day. At 1000 mg/kg bw/day increased water consumption was recorded and at necropsy liver weights was increased whilst the testis and epididymal weight were reduced (67 and 76% of control, respectively). The dose levels selected for the study included a high dose of 750 mg/kg bw/day which was expected to generate some toxic reaction but any effect on testes was expected to be minimal and to not impair the mating performance of these animals. The low and intermediate dose levels were selected to establish a no observed adverse effect level to give suitable safety margins and establish dose response relationships.
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Animals and cages were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS:
Detailed observations were recorded in relation to dose administration. For the Main phase males and Toxicity phase females dosing observations were recorded daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and on one occasion during Week 5. For Main phase females these were recorded daily during the first week of dosing, twice weekly during Week 2 of dosing, on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation. Observations were recorded at the following times in relation to dose administration:
Pre-dose
On return of the animal to its home cage
On completion of dosing of each group
Between one and two hours after completion of dosing of all groups
As late as possible in the working day

Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal (physical condition and behaviour during handling with particular attention to possible signs of neurotoxicity). For the Reproductive subgroup females during the post-mating period, these observations were conducted on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation. A weekly physical examination including arena observations was performed during the recovery period.

BODY WEIGHT:
The weight of the Main phase males and Toxicity phase females was recorded on the day that dosing commenced (Week 0), weekly throughout the dosing and recovery periods and before necropsy. Main phase females were weighed on the day that dosing commenced (Week 0), weekly until mating was detected, on Days 0, 7, 14 and 20 after mating and on Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded on a weekly basis from the start of study for Main phase males and Toxicity phase females and Main phase females until the animals were paired for mating. Food consumption was recorded weekly (g/animal/week) during the recovery period. From these records the mean weekly consumption per animal (g/animal/week) was calculated for each cage. Food consumption was not recorded for Main phase males and females during pairing.
For each Main phase female after mating, the weight of food supplied, that remaining and an estimate of any spilled was recorded for the periods Days 0-6, 7-13 and 14-19 after mating and Days 1-3 and 4-6 of lactation.

WATER CONSUMPTION:
Fluid intake was assessed by daily visual observation.

OTHER:
- SENSORY REACTIVITY:
Sensory reactivity and grip strength assessments were performed (before dosing) on the second 5 main phase (group 5 and 6)/recovery group (control and group 7) males and on toxicity phase (Groups 5 and 6)/recovery phase (Control and Group 7) females during Week 5 of study. The following measurements, reflexes and responses were recorded: approach response, touch response, auditory startle reflex, tail pinch response and grip strength.

- MOTOR ACTIVITY:
During Week 5 of study (before dosing), the motor activity of the second five main phase (Groups 5 and 6)/recovery phase (Control and Group 7) males and on toxicity phase (Groups 5 and 6)/recovery phase (Control and Group 7) females was measured using a Rodent Activity Monitoring System (Version 2.0.3). Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total).

- HAEMATOLOGY:
During Week 5 of treatment (before dosing on each occasion) and after 2 weeks of recovery, blood samples were obtained from the first five main phase males and on the toxicity phase females after overnight withdrawal of food. Animals were held under light general anaesthesia induced by isoflurane and blood samples were withdrawn from the sublingual vein. The following were measured using a Bayer Advia 120 haematology analyser: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total leucocyte count (WBC), Differential leucocyte count (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)) and Platelet count (Plt). The most common morphological changes, anisocytosis, micro/macrocytosis and hypo/hyperchromasia were recorded. Prothrombin time (PT) (using an ACL 3000 Plus analyser and IL PT-Fibrinogen reagent) and Activated partial thromboplastin time (APTT) (using an ACL 3000 Plus Analyser and IL APTT reagent) were also measured.

- BLOOD CHEMISTRY:
At the same time and using the same animals as for peripheral haematology, further blood samples (nominally 0.7 mL) were collected and the plasma was examined using a Roche P Modular Analyser for: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), Bile Acids (BIAC), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot) and Albumin (Alb) (by chemical assay).
Oestrous cyclicity (parental animals):
For 15 days before pairing (including the day of pairing), daily vaginal smears (dry) were taken from all Reproductive subgroup females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed.
Sperm parameters (parental animals):
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify test material related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
Litter observations:
PARAMETERS EXAMINED:
All litters were examined at approximately 24 hours after birth and then daily thereafter for clinical signs (evidence of ill health or reaction to treatment), litter size (mortality and consequent changes in litter size from Days 1-7 of age), sex ratio of each litter (recorded on Days 1, 4 and 7 of age) and individual bodyweight (recorded on Days 1, 4 and 7 of age).

GROSS EXAMINATION OF PUPS:
All offspring killed or dying prior to scheduled termination, and of those killed at the end of the study were subjected to detailed necropsy.
Postmortem examinations (parental animals):
SACRIFICE:
Main phase males and Toxicity phase females were killed in Week 6 after completion of the Week 5 investigations. The recovery phase animals were killed after 2 weeks of recovery and after haematology and blood chemistry sampling.
Main phase females (Groups 1, 5 and 6) were killed on Day 7 of lactation. Main phase females that did not litter (Group 7) were killed on Day 25 after mating. Offspring were killed on Day 7 of age.

GROSS NECROPSY:
All animals were subject to a detailed necropsy. For Reproductive subgroup females, the number of uterine implantation sites was also recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS:
The tissues indicated in Table 1 were weighed.
The following tissues from Main phase male, Toxicity phase female, all recovery phase animals, Main phase females that did not litter (Group 7) and those animals killed or dying prematurely were fixed for histopathology: Adrenal glands, Brain, Pituitary, Prostate, Caecum, Colon, Rectum, Sciatic nerves, Duodenum, Seminal vesicles and coagulation gland, Epididymides (L&R), Skeletal muscle, Skin, Mammary glands (inguinal area), Heart, Spinal cord, Ileum, Spleen, Jejunum, Sternum with marrow, Kidneys, Stomach, Liver, Testes (L&R), Lungs, Thymus, Lymph nodes (mandibular and mesenteric), Thyroid with parathyroids, Trachea, Urinary bladder, Oesophagus, Uterus with cervix and oviducts, Peyer’s patch, Ovaries (L&R) and Vagina.
The following tissues from each Main phase female that did litter (Groups 1, 5 and 6) were fixed for histopathology: Ovaries (L&R), Uterus with cervix and oviducts and vagina. Samples of any abnormal tissues were also retained and processed for examination.
Postmortem examinations (offspring):
All offspring killed or dying prior to scheduled termination, and of those killed at the end of the study were subjected to detailed necropsy.
Statistics:
The following sequence of statistical tests was used for grip strength, motor activity, bodyweight, food consumption, organ weight, litter size and survival indices and clinical pathology data:
1) a parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For comparisons involving two groups only t-tests were used, for all other comparisons the F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.
2) a non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For comparisons involving two groups only, Wilcoxon’s rank sum tests (Wilcoxon 1945) were used. For all other comparisons the H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel 1959) was performed instead.
For organ weight data, analysis of covariance was performed. Sex ratio were analysed by Wald chi-square test.
For gestation length and sperm count estimates an exact two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores, was applied to all groups.
Reproductive indices:
Percentage mating : Number animals mating / Animals paired × 100
Conception rate (%) : Number animals achieving pregnancy / Animals mated × 100
Fertility index (%) : Number animals achieving pregnancy / Animals pairing × 100
Offspring viability indices:
Gestation index (%) : Number of live litters born / Number pregnant × 100
Post - implantation survival index (%) : Total number offspring born / Total number uterine implantation sites × 100
Live birth index (%) : Number live offspring on Day 1 after littering / Total number of offspring born × 100
Viability index (%) : Number live offspring on Day 4 after littering / Number live offspring on Day 1 after littering × 100
Lactation index (%) : Number live offspring on Day 7 after littering / Number live offspring on Day 1 after littering × 100
Percentage of males : Number of males in litter/ Total number of offspring in litter x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No death attributed to the test material. Underactive behaviour and unsteady reactions, in males and females receiving 750 mg/kg/day (during week 1). Dose related increases in post dosing salivation and chin rubbing were seen.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males receiving 750 mg/kg/day showed lower overall weight gain (Week 0-5) compared with Control. No body weight effects in unmatted females receiving up to 750 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males receiving 750 mg/kg/day showed lower overall weight gain (Week 0-5) compared with Control. No body weight effects in unmatted females receiving up to 750 mg/kg/day.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
at 750 mg/kg/day:Bodyweight adjusted liver weights were significantly higher than Control in males and females and bodyweight adjusted kidney weights were significantly higher than Control in males; testis weight was markedly low in males.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Males receiving 750 mg/kg/day showed a range of testicular findings including small, blue and flaccid testes. Epididymides of males receiving 750 mg/kg/day were observed generally small but some contained masses or appeared enlarged.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Adaptive centrilobular hepatocyte hypertrophy in the liver of females receiving 750 mg/kg/day. Histopathological findings in the kidneys of males receiving 250 and 750 mg/kg/day. All these effects resolved after recovery period.
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Absence of spermatozoa observed in 4 animals receiving 750 mg/kg/day and one male at this dose level presented reduced number of spermatozoa. Degenerate spermatogenic cells in duct(s) were observed in all males receiving 750 mg/kg/day.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg/day: no females became pregnant. It is considered that the testicular and epididymal effects observed in males receiving 750 mg/kg/day would have been sufficient to prevent fertilisation.
MORTALITY (PARENTAL ANIMALS):
In the 60 mg/kg/day dose group, one male was found dead on Day 31 of study and one female was killed because of parturition difficulties. This female had given birth to three pups and but still had 15 live pups and one early resorption in utero. The difficulty during parturition may be associated with the presence of an abnormally enlarged placenta as maternal necropsy findings and microscopic evaluation of the organs did not identify any other factors. In the absence of any other death in the intermediate and high dose groups these deaths are not attributed to the test material.

CLINICAL SIGNS (PARENT ANIMALS):
On the first two days of dosing most of the females and a few males receiving 750 mg/kg/day were recorded as having an unsteady gait and some animals were underactive, but all findings resolved within the working day. Over activity was also observed as a post dosing sign during Week 1 in females dosed at 60 mg/kg/day and males and females at 250 mg/kg/day. Signs of salivation and/or chin rubbing were recorded, largely at 750 mg/kg/day, but these are common reactions to the dosing process where the material might be distasteful and probably unrelated to systemic toxicity.

BODY WEIGHT AND FOOD CONSUMPTION (PARENT ANIMALS):
There were no statistically significant effects of CAS 8000-41-7 on bodyweight or bodyweight gain.
Overall weight gain (Weeks 1-5) was slightly, but not significantly reduced for males dosed at 750 mg/kg/day (83% of Control). Much of this effect occurred during pairing and there were minimal or no effects on bodyweight in males receiving 250 or 60 mg/kg/day.
Weight gain of females from Week 0-5 were slightly lower than Control at all dose levels, but in the absence of any consistent trends it was considered to be unaffected by the test material.
During gestation there was no clear effect on bodyweight although gains were slightly lower than Control, and during lactation bodyweight gain of females receiving 250 mg/kg/day were lower than Control.

There were no test material related effects on food consumption. The increase in food consumption observed in all animals during the recovery period was due to cessation of dose administration which used corn oil as the vehicle thereby supplying a portion of the required nutrients.

WATER CONSUMPTION (PARENT ANIMALS):
Qualitative assessment of water consumption by checking residual levels in the water bottles indicated that animals receiving 750 mg/kg/day consumed more water than the Controls.

REPRODUCTIVE FUNCTION (ESTROUS CYCLE) AND REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
There was no effect of CAS 8000-41-7 on oestrous cycles or precoital interval. All females dosed at 750 mg/kg/day failed to litter and were found to have no implantation sites and not to have been pregnant when examined at necropsy on Day 25 after mating. It is considered that the testicular and epididymal effects observed in males receiving 750 mg/kg/day would have been sufficient to prevent fertilisation.
There was no effect of CAS 8000-41-7 on mating performance or fertility at dose levels of 250 mg/kg/day or below.
All females that littered had normal length gestation periods (22 – 23 days duration) but a slightly higher proportion of females at 250 mg/kg/day gave birth after 23 days.

ORGAN WEIGHTS (PARENT ANIMALS): Tables 6 and 7.
After 5 weeks of dosing bodyweight adjusted mean liver weights of males and females receiving 750 mg/kg/day were significantly increased, kidney weights of males were also significantly increased. Testis weight was markedly lower in males receiving 750 mg/kg/day (58% of Control) and there was also a suggestion of slightly lower epididymal weights for these males. Two males at 250 mg/kg/day had combined testis weight below the background range (90 percentile range – males: 2.97-4.07 (n=155)) associated to lower epididymal weights, group mean values for this group were similar to Control.
After two weeks without dosing liver and kidney weights were no longer enlarged but testis and epididymal weights showed no evidence of recovery.

GROSS PATHOLOGY (PARENT ANIMALS): Table 8.
Males receiving 750 mg/kg/day showed a range of testicular findings including small, blue and flaccid testes. Epididymides of males receiving 750 mg/kg/day were observed generally small but some contained masses or appeared enlarged. Two males receiving 250 mg/kg/day also had small testes. Similar findings were still apparent in males killed after two weeks of recovery. There were no significant necropsy findings for females on Day 7 of lactation.

HISTOPATHOLOGY (PARENT ANIMALS): Table 9.
Liver:
Minimal centrilobular hepatocyte hypertrophy was seen in the liver of three toxicity phase females dosed with CAS 8000-41-7 at 750 mg/kg/day. However, these histopathology findings showed complete recovery after 2 weeks.
Kidney:
Histopathological changes associated with hyaline droplets were observed in the kidneys of male rats receiving 250 or 750 mg/kg/day but such changes are commonly associated with administration of volatile hydrocarbons and are of no consequence to human risk assessment.
Epididymes:
Histopathological assessment of the epididymides revealed reduced numbers, or a complete absence of spermatozoa, accompanied by the presence of degenerate spermatogenic cells in duct(s) of males receiving 750 mg/kg/day.Similar changes were still evident following the 2 week recovery period. Spermatocele granuloma(ta) were observed in two males receiving 750 mg/kg/day and one receiving 60 mg/kg/day. However the significance of this change in the single male receiving 60 mg/kg/day is uncertain as spermatocele granuloma(ta) can occur spontaneously in rats of this age.
testes:
Moderate to severe seminiferous tubular atrophy/degeneration was seen in the testes of all animals dosed with Terpineol multiconstituent at 750 mg/kg/day, accompanied by minimal to moderate spermatid giant cells and minimal to slight seminiferous tubular vacuolation. The same changes and reduced organ weights were still evident following the 2 week recovery period but at a lower incidence and severity, indicating a degree of recovery from these changes.


OTHER FINDINGS:
SIGNS AND ARENA OBSERVATIONS:
There were no signs observed among treated males and females at routine physical examination or during the arena observations that were considered to be related to treatment.

SENSORY REACTIVITY OBSERVATIONS AND GRIP STRENGTH:
Sensory reactivity observations and grip strength values for Toxicity subgroup animals were similar to those for Controls, and considered unaffected by treatment.

MOTOR ACTIVITY:
Motor activity scores for males and females showed considerable inter-group variation but no clear dose related trends such that an association with test material was considered unlikely.

HAEMATOLOGY: Tables 2 and 3.
There were no marked effects of Terpineol multiconstituent upon haematology parameters.

BLOOD CHEMISTRY: Tables 4 and 5.
During Week 5 of study, urea and creatinine levels were significantly higher in females receiving 750 mg/kg/day and slightly, but not significantly, high in males at the same dose level. By Week 2 of recovery urea and creatinine levels were still higher than Control for males and females previously receiving 750 mg/kg/day, attaining statistical significance for females only.
Glucose plasma levels were significantly higher than Control in females dosed at 250 and 750 mg/kg/day and marginally high in the males at the same doses but were similar to Control after dosing ceased.
Potassium levels were significantly higher than Control in males and females receiving 750 mg/kg/day, visual inspection of the blood samples did not indicate that they were haemolysed, and levels were similar to respective Controls during the recovery phase.
Bile acid plasma levels for females at all dose levels were higher than the concurrent Control attaining significance at 750 mg/kg/day. A dose related trend was apparent, however, individual values were all within the Historical Control range (90 percentile range – females: 8.7-49.7 (n=38)).


Key result
Dose descriptor:
NOAEL
Effect level:
> 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Impairment of male fertility at 750 mg/kg/day prevented the assessment of effects on female reproduction at 750 mg/kg/day. No effect was reported in females receving 250 mg/day. NOAEL for maternal toxicity was set at least at 250 mg/kg/day.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Males receiving 750 mg/kg/day showed evidence of testicular and epididymal toxicity leading to infertility. NOAEL was set at 250 mg/kg/day where fertility of male was unaffected by the test substance and no other effects were reported.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
LITTER SIZE, SEX RATIO AND SURVIVAL INDICES:
The numbers of implantations, total litter sizes and live litter sizes up to Day 7 of lactation were unaffected by dosing with Terpineol multiconstituent in the 60 and 250 mg/kg/day groups.
Sex ratio (assessed by the percentage of males) at 250 mg/kg/day was slightly, but statistically significantly lower than Control. Individual litter data for this parameter are always variable and as only two litters are outside the concurrent Control data range this intergroup difference is not considered to be toxicologically significant. Sex ratio was unaffected by administration of Terpineol multiconstituent at a dose level of 60 mg/kg/day.
Administration with Terpineol multiconstituent had no effect on post implantation survival index, live birth index and viability index for animals receiving up to 250 mg/kg/day.

CLINICAL SIGNS (OFFSPRING): Clinical signs of offspring did not indicate any reaction to maternal exposure to Terpineol multiconstituent.

BODY WEIGHT (OFFSPRING): .Male and female offspring bodyweights were considered to be unaffected by Terpineol multiconstituent.

GROSS PATHOLOGY (OFFSPRING): Necropsy findings of offspring killed or dying prior to scheduled termination, and of those killed at the end of the study, did not indicate any reaction to maternal dosing with Terpineol multiconstituent.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Male and female offspring were unaffected by the CAS 8000-41-7 up to 250 mg/kg/day.
Reproductive effects observed:
not specified

Table 2: Haematology - group mean values during Week 5 of treatment

Dose (mg/kg/day)

Hct

Hb

RBC

MCH

MCHC

MCV

WBC

N

L

E

B

M

LUC

Plt

PT

APTT

L/L

g/dL

x1012/L

pg

g/dL

fL

x109/L

x109/L

x109/L

x109/L

x109/L

x109/L

x109/L

x109/L

sec

sec

 

Males

Control (n=5)

0.454 ± 0.0185

15.6 ± 0.68

8.44 ± 0.280

18.5 ± 0.90

34.5 ± 0.23

53.7 ± 2.54

13.18 ± 2.573

1.56 ± 0.584

11.08 ± 2.316

0.11 ± 0.021

0.05 ± 0.018

0.27 ± 0.063

0.10 ± 0.032

1228 ± 113.6

13.9 ± 0.37

16.6 ± 1.17

60 (n=4)

0.437 ± 0.0052

14.9 ± 0.45

8.30 ± 0.245

18.0 ± 0.84

34.1 ± 0.66

52.7 ± 1.82

16.30 ± 5.547

3.78 ± 1.968*

11.79 ± 3.309

0.16 ± 0.082

0.08 ± 0.032

0.39 ± 0.219

0.12 ± 0.044

1119 ± 109.7

14.1 ± 0.47

18.6 ± 2.42

250 (n=5)

0.443 ± 0.0161

15.1 ± 0.57

8.34 ± 0.334

18.2 ± 0.33

34.2 ± 0.37

53.1 ± 1.38

12.57 ± 0.972

1.33 ± 0.237

10.82 ± 0.760

0.11 ± 0.023

0.06 ± 0.009

0.17 ± 0.029

0.09 ± 0.016

1193 ± 130.8

13.8 ± 0.30

16.3 ± 3.97

750 (n=5)

0.434 ± 0.0174

14.8 ± 0.68

8.35 ± 0.424

17.8 ± 0.21

34.2 ±0.67

51.9 ± 0.98

14.64 ± 3.813

2.10 ± 0.869

11.94 ± 3.192

0.09 ± 0.057

0.08 ± 0.029

0.32 ± 0.055

0.11 ±0.054

1123 ± 199.7

13.8 ± 0.38

16.2 ± 2.75

 

Females

Control (n=5)

0.414 ± 0.0165

14.6 ± 0.56

7.85 ± 0.347

18.6 ± 0.48

35.2 ± 0.36

52.7 ± 0.93

9.36 ± 2.826

0.87 ± 0.638

8.15 ± 2.166

0.10 ± 0.016

0.03 ± 0.016

0.14 ± 0.045

0.08 ± 0.040

1251 ± 75.2

14.4 ± 0.40

16.2 ± 1.99

60 (n=4)

0.400 ± 0.0037

13.9 ± 0.13*

7.73 ± 0.163

18.0 ± 0.54

34.8 ± 0.58

51.7 ± 0.97

8.69 ± 2.306

0.96 ± 0.897

7.39 ± 1.531

0.11 ± 0.075

0.04 ± 0.006

0.15 ± 0.074

0.05 ± 0.021

1169 ± 69.9

14.6 ± 0.92

13.2 ± 3.22

250 (n=5)

0.403 ± 0.0150

14.0 ± 0.33*

7.66 ± 0.273

18.3 ± 0.34

34.8 ± 0.57

52.5 ± 1.08

8.89 ± 3.411

1.06 ± 0.955

7.48 ± 2.324

0.06 ± 0.031

0.02 ± 0.012

0.20 ± 0.158

0.07 ± 0.044

1219 ± 130.2

13.8 ± 0.18

15.2 ± 1.31

750 (n=5)

0.391 ± 0.0121*

13.5 ± 0.42**

7.31 ± 0.382*

18.5 ± 0.80

34.6 ± 0.64

53.6 ± 1.85

7.54 ± 1.684

0.80 ± 0.462

6.47 ± 1.662

0.07 ± 0.035

0.02 ± 0.016

0.11 ± 0.025

0.05 ± 0.018

1013 ± 297.5

14.4 ± 0.45

13.8 ± 1.99

Table 3: Haematology - group mean values for females after 2 weeks of recovery

Dose (mg/kg/day)

Hct

Hb

RBC

MCH

MCHC

MCV

WBC

N

L

E

B

M

LUC

Plt

PT

APTT

L/L

g/dL

x1012/L

pg

g/dL

fL

x109/L

x109/L

x109/L

x109/L

x109/L

x109/L

x109/L

x109/L

sec

sec

 

Males

Control (n=5)

0.447 ± 0.0137

15.0 ± 0.52

8.93 ± 0.265

16.9 ± 0.63

33.7 ± 0.16

50.0 ± 1.75

11.81 ± 1.308

1.44 ± 0.429

9.85 ± 1.261

0.11 ± 0.020

0.06 ± 0.017

0.28 ± 0.032

0.07 ± 0.010

1239 ± 245.7

13.8 ± 0.42

14.3 ± 1.98

750 (n=5)

0.415 ± 0.0166*

14.2 ± 0.56*

8.28 ± 0.145**

17.1 ± 0.57

34.2 ± 0.33*

50.2 ± 1.75

15.00 ± 3.831

1.94 ± 0.801

12.39 ± 3.435

0.14 ± 0.035

0.08 ± 0.030

0.36 ± 0.159

0.08 ± 0.044

996 ± 130.0

14.6 ± 0.57*

16.3 ± 2.82

 

Females

Control (n=4)

0.405 ± 0.0191

14.2 ± 0.50

7.87 ± 0.369

18.1 ± 0.53

35.2 ± 0.56

51.4 ± 1.38

6.69 ± 1.758

0.77 ± 0.520

5.65 ± 1.326

0.09 ± 0.026

0.02 ± 0.006

0.15 ± 0.070

0.03 ± 0.013

1120 ± 199.5

14.5 ± 0.81

13.0 ± 0.69

750 (n=5)

0.404 ± 0.0131

14.1 ± 0.27

7.83 ± 0.303

18.0 ± 0.56

34.9 ± 0.49

51.7 ± 1.77

7.57 ± 2.070

0.64 ± 0.121

6.60 ± 1.963

0.09 ± 0.018

0.03 ± 0.009

0.16 ± 0.067

0.04 ± 0.018

1031 ± 208.8

14.8 ± 0.50

12.3 ± 2.65

Table 4: Blood chemistry - group mean values taken during Week 5 of treatment

Dose (mg/kg/day)

ALP

ALT

AST

gGT

Bili

BIAC

Urea

Creat

Gluc

Chol

Na

K

Cl

Ca

Phos

Total Prot

Alb

A/G

U/L

U/L

U/L

U/L

µmol/L

umol/L

mmol/L

µmol/L

mmol/L

mmol/L

mmol/L

mmol/L

mmol/L

mmol/L

mmol/L

g/L

g/L

Ratio

 

Males

Control (n=5)

124 ± 23.0

45 ± 12.2

61 ± 3.5

0 ± 0.0

2 ± 0.0

30.6 ± 16.59

4.19 ± 1.019

25 ± 3.1

7.29 ± 1.576

1.36 ± 0.155

142 ± 1.4

4.9 ± 0.27

101 ± 1.3

2.63 ± 0.044

2.13 ± 0.152

63 ± 1.9

35 ± 1.6

1.28 ± 0.120

60 (n=4)

145 ± 22.2

51 ± 8.9

68 ± 13.5

1 ± 0.6

2 ± 0.6

25.1 ± 14.13

4.84 ± 1.262

27 ± 4.5

8.93 ± 1.254

1.41 ± 0.137

142 ± 1.7

5.5 ± 1.03

103 ± 1.4

2.60 ± 0.048

2.22 ± 0.186

63 ± 1.9

33 ± 0.5

1.14 ± 0.090

250 (n=5)

164 ± 18.5*

52 ± 4.0

59 ± 5.5

0 ± 0.0

2 ± 0.4

29.8 ± 8.41

4.90 ± 0.596

27 ± 2.2

9.63 ± 1.264

1.91 ± 0.307

142 ± 2.2

5.6 ± 0.48

102 ± 1.1

2.55 ± 0.083

2.21 ± 0.156

61 ± 2.8

34 ± 1.2

1.27 ± 0.046

750 (n=5)

146 ± 24.6*

46 ± 15.9

69 ± 17.0

0 ± 0.4

2 ± 0.4

31.3 ± 13.30

5.35 ± 1.412

29 ± 4.0

8.72 ± 2.129

1.46 ± 0.463

140 ± 1.8

5.9 ± 0.66*

102 ± 0.5

2.51 ± 0.096*

2.19 ± 0.199

62 ± 2.8

35 ± 0.7

1.29 ± 0.088

 

Females

Control (n=5)

71 ± 14.8

40 ± 7.1

66 ± 8.3

0 ± 0.0

2 ± 0.4

14.2 ± 4.01

5.23 ± 0.853

28 ± 2.1

7.12 ± 0.513

1.51 ± 0.414

142 ± 0.9

4.2 ± 0.42

103 ± 0.9

2.56 ± 0.054

1.63 ± 0.106

66 ± 2.2

39 ± 2.3

1.50 ± 0.179

60 (n=4)

71 ± 19.3

42 ± 8.6

68 ± 11.8

0 ± 0.5

2 ± 0.4

18.7 ± 7.81

5.91 ±1.008

34 ±3.3

7.57 ± 1.426

1.46 ± 0.120

141 ± 1.8

4.7 ± 0.39

103 ± 1.8

2.52 ± 0.087

1.69 ± 0.060

64 ± 6.2

37 ± 2.7

1.36 ± 0.180

250 (n=5)

85 ± 15.8

45 ± 12.7

66 ± 22.9

0 ± 0.0

2 ± 0.7

25.1 ± 11.05

5.40 ± 0.407

32 ± 5.3

8.99 ± 0.997*

1.96 ± 0.394

141 ± 1.5

4.0 ± 0.33

102 ± 1.7

2.52 ± 0.066

1.67 ± 0.208

66 ± 3.4

39 ± 2.3

1.44 ± 0.237

750 (n=5)

75 ± 20.1

40 ± 6.7

58 ± 11.9

1 ± 0.5

2 ± 0.0

31.3 ± 14.04*

7.59 ± 2.350*

41 ± 3.1**

10.00 ± 1.372**

1.91 ± 0.464

139 ± 2.6*

5.6 ± 0.74**

102 ± 2.0

2.53 ± 0.064

1.53 ± 0.212

66 ± 2.3

38 ± 1.5

1.39 ± 0.089

Table 5: Blood Chemistry - Recovery Week 2

Dose (mg/kg/day)

ALP

ALT

AST

gGT

Bili

BIAC

Urea

Creat

Gluc

Chol

Na

K

Cl

Ca

Phos

Total Prot

Alb

A/G

U/L

U/L

U/L

U/L

µmol/L

umol/L

mmol/L

µmol/L

mmol/L

mmol/L

mmol/L

mmol/L

mmol/L

mmol/L

mmol/L

g/L

g/L

Ratio

 

Males

Control (n=5)

125 ± 35.1

50 ± 13.2

69 ± 7.4

0 ± 0.4

2 ± 0.0

32.1 ± 11.48

5.49 ± 0.910

30 ± 3.0

7.79 ± 1.269

2.02 ± 0.357

142 ± 1.2

4.9 ± 0.36

102 ± 1.1

2.66 ± 0.049

2.18 ± 0.145

67 ± 4.0

36 ± 1.5

1.17 ± 0.153

750 (n=5)

98 ± 15.3

42 ± 2.8

65 ± 4.7

0 ± 0.4

2 ± 0.0

30.0 ± 13.01

7.29 ± 1.663

35 ± 5.2

8.77 ± 0.638

1.94 ± 0.668

142 ± 0.7

4.9 ± 0.24

101 ± 0.4

2.63 ± 0.074

2.26 ± 0.086

64 ± 2.4

34 ± 1.8

1.17 ± 0.111

 

Females

Control (n=5)

65 ± 17.9

39 ± 6.6

63 ± 5.2

0 ± 0.4

2 ± 0.4

32.3 ± 18.29

7.17 ± 0.447

38 ± 3.2

7.71 ± 0.735

2.06 ± 0.377

141 ± 1.1

4.2 ± 0.16

102 ± 0.9

2.61 ± 0.064

1.53 ± 0.214

71 ± 4.0

41 ± 2.3

1.35 ± 0.140

750 (n=5)

65 ± 21.7

39 ± 12.0

65 ± 7.9

0 ± 0.4

2 ± 0.0

26.3 ± 27.87

8.59 ± 1.269*

50 ± 7.1*

7.97 ± 0.402

2.51 ± 0.240

142 ± 1.5

4.2 ± 0.24

101 ± 1.8

2.57 ± 0.076

1.61 ± 0.118

67 ± 4.2

38 ± 2.3

1.31 ± 0.019

Conclusions:
The No-Observed-Adverse–Effect-Level (NOAEL) for males and unmated females was 250 mg/k/g/day, the NOAEL and the NOAEL for maternal and developmental toxicity was at least 250 mg/kg/day.
Executive summary:

In a GLP study conducted according to OECD guideline 422, three groups, each comprising of ten male and ten female rats for the Main (reproductive) phase (only five males at top dose) and five female rats for the Toxicity phase received CAS 8000-41-7 at doses of 60, 250 or 750 mg/kg bw/day at a dose volume of 5 mL/kg bw/day. Main phase males and Toxicity phase females were dosed daily for a minimum of five consecutive weeks. An additional ten males and ten females were dosed with the vehicle or at 750 mg/kg/day for five weeks and then given two weeks of recovery before termination. Main phase females were dosed daily for two weeks before pairing, throughout mating, gestation and until Day 6 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose.

During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption (visual), haematology, blood chemistry, oestrous cycles, mating performance and fertility and gestation length. Organ weight, macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.

In the 60 mg/kg/day dose group, one male was found dead on Day 31 of study and one female was killed because of parturition difficulties. In the absence of any other death in the intermediate and high dose groups these deaths are not attributed to the test material. 

No significant findings were recorded for clinical signs,detailed physical examination and arena observations. Underactive behaviour and unsteady reactions, in males and females were observed briefly during Week 1 in animals receiving 750mg/kg/day and dose‑related increases in post‑dosing salivation and chin rubbing were seen.  Behavioural testing during Week 5 of dosing, including sensory reactivity findings, grip strength values and motor activity scores showed no differences considered to be associated with exposure to the test material.

There were no clear effects on bodyweight in males or unmated females receiving up to 750 mg/kg/day. Males receiving 750 mg/kg/day showed lower overall weight gain (Week 0-5) compared with Control. Bodyweight during the recovery phase was similar to Controls. Bodyweight and bodyweight gain were unaffected during gestation. During lactation females receiving 250 mg/kg/day showed lower weight gain than Controls.

There were no adverse effects on food consumption in males, unmated females or females during gestation and lactation but visual assessment of water consumption indicated that males and females receiving 750 mg/kg/day were consuming more water than the Controls during the dosing period.

Among the toxicity subgroup animals, There were no clinically significant effects of Terpineol multiconstituent upon haematology parameters. Females showed slight anaemia but males were essentially unaffected. At the end of the two week recovery period, no ntergroup differences were present in females whereas slightly affected in males.

At 750 mg.kg/day, urea and creatinine levels were significantly higher than Controls in females and slightly high in males. Glucose plasma levels were significantly higher than Control in females dosed at 250 and 750 mg/kg/day and marginally high in males. Potassium levels were significantly higher than Control in males and females receiving 750 mg/kg/day.all the above discussed parameters showed complete recovery after 2 week recovery period.

There were no effects of CAS 8000-41-7 on oestrous cycles, precoital interval or mating. Gestation length was within the normal range but there was a small increase in the numbers of animals at 250 mg/kg/day having longer (23 day) gestation periods. At dose levels up to and including 250 mg/kg/day there were no effects of the test material on the number of implantations, post implantation survival index, live birth index, viability index and lactation index. Male and female offspring bodyweights were not adversely affected by CAS 8000-41-7.

At 750 mg/kg/day, relative liver weights were significantly higher than Control in males and females and relative kidney weights were significantly higher than Control in males. Testis weight was markedly low in males receiving 750 mg/kg/day and there was also an indication of low epididymal weights at this dose. Liver and kidney weights returned to normal after two weeks when the animals did not receive Terpineol multiconstituent but testis and epididymal weights showed no evidence of recovery.

Adaptive centrilobular hepatocyte hypertrophy in the liver of females dosed with Terpineol multiconstituent at 750 mg/kg/day was not present after 2 weeks recovery and histopathological findings in the kidneys of males receiving 250 and 750 mg/kg/day also resolved after the end of dosing.

 

At 750 mg/kg/day, reduced numbers or complete absence of spermatozoa, accompanied by the presence of degenerate spermatogenic cells in duct(s) were observed in the epididymides and were still present following the 2‑week recovery period. Spermatocele granuloma(ta) that were seen in two males receiving 750 mg/kg/day and one receiving 60 mg/kg/day were not seen at the end of the recovery period.Thesignificance of this change in the single male receiving 60 mg/kg/day is uncertain as spermatocele granuloma(ta) can occur spontaneously in rats of this age and considering the absence of other degenerative changes in the testes or epididymides of this animal.

Moderate to severe seminiferous tubular atrophy/degeneration was seen in the testes of all animals dosed with Terpineol multiconstituent at 750 mg/kg/day, accompanied by minimal to moderate spermatid giant cells and minimal to slight seminiferous tubular vacuolation. Similar findings were still evident following the 2‑week recovery period but at a lower incidence and severity suggesting a degree of recovery.

Based on the findings in this study, the No-Observed-Adverse–Effect-Level (NOAEL) for males and unmated females was 250 mg/k/g/day, the NOAEL for maternal and developmental toxicity was at least 250 mg/kg/day.

Endpoint:
reproductive toxicity, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 11 to October 05, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Investigatory study succeeding OECD 422 screening test, performed in GLP laboratory.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Principles of method if other than guideline:
Investigatory study, succeeding OECD 422 screening test, was performed to compare the toxicity of Terpineol multiconstituent to the male reproductive system when administered by dietary or oral gavage routes.
GLP compliance:
no
Remarks:
generally followed good laboratory practice principles, however no specific study-related Quality Assurance procedures were performed and the report may not contain all of the elements required by GLP
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: Approximately 65 days
- Weight at study initiation: 310-350 g
- Housing: Dietary administration - individually housed in polycarbonate or polypropylene cages with stainless steel mesh; Oral gavage administration - housed as five, unless reduced by mortality or isolation.
- Diet (e.g. ad libitum): Rat and Mouse No.1 Maintenance Diet, ad libitum
- Water (e.g. ad libitum): Potable water taken from the public supply, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 11, 2010 To: September 07, 2010
Route of administration:
other: oral (both gavage and dietary)
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Oral gavage formulations were prepared in corn oil weekly or may be in advance of the first day of dosing.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly and may be prepared up to three days in advance of the first day of dosing.
- Mixing appropriate amounts with (Type of food): Rat and Mouse No. 1 Maintenance Diet
- Storage temperature of food: Stored in sealed containers and stored frozen (approximately -20 °C) until issue to the animal room

VEHICLE
- Concentration in vehicle: 750 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw/day
Details on mating procedure:
Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in the first week of the dosing period were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed) from all groups. The samples will be retained frozen (nominally -20 °C) as contingency for any possible future analysis.
Duration of treatment / exposure:
Minimum period: Oral gavage – 2 weeks; Dietary - 2 or 3 weeks
Frequency of treatment:
Dietary administration: continuously; Oral gavage administration: once daily
Details on study schedule:
None
Remarks:
Doses / Concentrations:
7500 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
10000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
750 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Five males
Control animals:
no
Details on study design:
- Dose selection rationale: In a standard OECD 422 study (HLS Study No OAD0004), Terpineol multiconstituent has been screened for possible reproductive toxicity. After 5 weeks of dosing at 750 mg/kg/day all males were sterile and subsequent histopathology of the testes and epididymides showed major reductions in the numbers of sperm in the tubules and a high proportion of animals had spermatoceles within the epididymides. No similar effects were detectable at 250 mg/kg/day. It is postulated that the effects may be related to the high peak doses achieved by oral gavage, resulting in atypical metabolism of the test substance. The dietary levels were selected to provide a daily intake similar to the toxic level by oral gavage.

- See table 1 for description of the different groups of animals tested
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Animals and cages were inspected visually at least twice daily for evidence of ill-health.

DETAILED CLINICAL OBSERVATIONS:
Detailed observations were recorded for oral gavage administration. Observations were recorded daily during the first week of treatment and twice weekly during Weeks 2 to 3 (middle and end of each week). Observations were recorded at the following times during the day:
- Pre-dose
- On return of the animal to its home cage
- On completion of dosing of each group
- Between one and two hours after completion of dosing of all groups or, when the duration of dosing is protracted, 1 to 2 hours after completion of each group.
- As late as possible in the working day

BODY WEIGHT:
Bodyweight was recorded on the day that dosing commenced (Week 0), daily (dietary study) or twice weekly (oral gavage study) throughout the dosing and before necropsy

FOOD CONSUMPTION:
Food consumption was recorded daily (dietary study) or twice weekly (oral gavage study). The food supplied to the cage and food spilled was recorded during cage cleaning on measurement weeks. The food remaining in the cages were recorded at the end of measurement periods.
Oestrous cyclicity (parental animals):
Not applicable
Sperm parameters (parental animals):
- Vas deferens (from left side): Sperm sample assessed for motility using a computer assisted sperm analyser (CASA) on all animals of each group. A manual assessment of sperm morphology will be performed on all animals of each group.
- Cauda epididymis (from left side): The cauda epididymis will be weighed and homogenised and the number of sperm will be counted using a computer assisted sperm analyser (CASA) on all animals of each group.
- Testis (from left side): The testis will be homogenised and the number of homogenisation-resistant spermatids will be counted using a computer assisted sperm analyser (CASA) on all animals of each group.
Litter observations:
Not applicable
Postmortem examinations (parental animals):
SACRIFICE:
Animals were sacrificed by carbon dioxide asphyxiation and subsequent exsanguination.

GROSS NECROPSY:
All animals were subjected to a complete macroscopic examination.

HISTOPATHOLOGY / ORGAN WEIGHTS:
The tissues indicated in Table 2 were weighed and / or fixed for histopathological examinations.
Postmortem examinations (offspring):
Not applicable
Statistics:
- For categorical data, the proportion of animals was analysed using Fisher’s Exact test.
- For continuous data, Bartlett’s test was applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, groups were compared using either a t-test or Wilcoxon rank sum test.
Reproductive indices:
Not applicable
Offspring viability indices:
Not applicable
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs were generally minimal and of the type expected for the age of animals and the duration of the study.
Dosing signs for Group 3 animals receiving oral gavage administration of Terpineol multiconstituent at 750 mg/kg/day, included salivation and chin rubbing. No dosing signs were observed for dietary animals at supplementary oral gavage dosing occasions.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group 1 and 2 animals receiving dietary administration of Terpineol multiconstituent at 7500 or 10000 ppm, showed initial bodyweight loss, between Days 1 and 4 of study, and bodyweight gain thereafter was generally lower than that of animals dosed by gavage.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group 1 and 2 males receiving Terpineol in the diet showed food consumption values lower than expected, particularly at the start of the week 1 suggesting that the test material made the diet less palatable. There was some increase in intake through week one. Males from the 10000 ppm administration group took longer to acclimatise to the diet containing test material however by the end of Week 1 food consumption was approaching normal levels.
On the first day of gavage supplementation (Day 11) a minor decrease in food consumption was recorded for Group 1 and 2 animals, and although food consumption subsequently improved, it never attained the expected levels. This may relate to the nutritive value of corn oil included in the gavage dose.
Group 3 males receiving Terpineol multiconstituent at 750 mg/kg/day, between Days 1-3 of study, had slightly lower than expected food consumption value; this may be associated with the poor bodyweight performance of male 15 within this cage. Food consumption values of Group 3 males receiving Terpineol multiconstituent were within the expected range at all other periods, when the potential effect of the corn oil vehicle is taken into consideration.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When evaluating the data the lower terminal bodyweights of the study animals than the historical control data (HCD) animals has been taken into consideration.

Organ weights of Group 1 males receiving dietary levels of 7500 ppm with 300 mg/kg/day oral gavage supplementation from Day 11, were generally unaffected by dosing. The slightly low testes weights were attributable to Male 1: when this male was excluded the group mean value was 3.493 g. Organ weights of Group 2 males receiving dietary levels of 10000 ppm with 150 mg/kg/day oral gavage supplementation from Day 11, were generally unaffected by dosing. Organ weights of Group 3 males receiving oral gavage administration of Terpineol multiconstituent at 750 mg/kg/day (testes, epididymal prostate and seminal vesicle) were all low.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
One male in Group 1, receiving dietary levels of 7500 ppm with 300 mg/kg/day oral gavage supplementation from Day 11, was recorded to have small, flaccid and blue testes and small epididymides. All other animals in the group were macroscopically normal.
One male in Group 2, receiving dietary levels of 10000 ppm with 150 mg/kg/day oral gavage supplementation from Day 11, was recorded to have an exceptionally small right testis and epididymis and the left testis was enlarged: this pattern of change suggested that damage, or perhaps a congenital condition, had occurred before the start of dosing with compensatory hypertrophy in the left testis.
Small, flaccid and blue testes and small epididymides were recorded for all males in Group 3 receiving 750 mg/kg/day Terpineol multiconstituent by oral gavage.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The most affected group was Group 3 and there were relatively few histopathological changes in the testes and epididimides of the animals where the bulk of the test material had been given by the dietary route with supplementary gavage dosing. Degenerative changes were seen in the testes: seminiferous tubular atrophy, with associated vacuolation and in the epididymides (reduced sperm numbers and increased numbers of degenerated sperm in the ducts) in Group 3 only.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Week 1 group mean achieved dose level for animals receiving Terpineol multiconstituent at 7500 ppm was 410 mg/kg/day and 10000 ppm was 495 mg/kg/day. These lower than expected results reflected lower than expected food consumption and high initial bodyweight. Consequently additional test material was given by oral gavage (2 x 150 mg/kg/day to Group 1 or 1 x 150 mg/kg/day to Group 2) from Day 11 of dosing. This resulted in overall intake levels of 663 or 678 mg/kg/day for Groups 1 and 2 respectively during the final 11 days of the study. Overall achieved dose level (Days 1-21) for Group 1 animals was 546 mg/kg/day and Group 2 animals was 602 mg/kg/day (see table 3).
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Sperm motility (percentage motile and % progressively motile) was low for all groups of animals. The greatest effect was seen in Group 3. One of 5 males in Group 1 and 4 of 5 males in Group 2 showed essentially normal motility patterns and sperm morphology was largely normal in these animals. The males with poor sperm motility showed high incidences of abnormal sperm, usually decapitate but with increasing incidence of sperm with an abnormal mid-piece in the groups where the whole dose had been given by gavage.
The numbers of sperm in the epididymis were generally within normal ranges for males in Group 2 slightly low in Group 1 but for the majority of animals receiving 750 mg/kg/day of Terpineol multiconstituent, sperm numbers were below expected values. In contrast the numbers of spermatids in the testes of these animals were generally high, especially for the pure material, and slightly high spermatid numbers were seen in both of the groups where diet provided most of the test compound.
Reproductive performance:
not examined
Dose descriptor:
LOAEL
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Clinical signs:
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Not examined
Remarks on result:
not measured/tested
Reproductive effects observed:
not specified

Table 3: Achieved dose - group mean values (mg/kg/day)

Group

Mean 1-7

Mean 1-10

Mean 11-14

Mean 8-14

Mean 15-21

Mean 1-21

Dietary 7500 ppm +

supplementary gavage dose 300 mg/kg/day

410

416

368 (668)

566

360 (660)

388 (546)

Dietary 10000 ppm +

supplementary gavage dose 150 mg/kg/day

494

519

511 (661)

625

534 (684)

523 (601)

Numbers in parentheses include oral gavage supplementation

Table 4: Sperm analysis - group mean values

Group

Motile sperm (%)

Progressively motile sperm (%)

Cauda epididymis

Testis

 

Weight (g)

Sperm count (million/g)

Total (million)

Weight (g)

Sperm count (million/g)

Total (million)

1 (n=5)

19 ± 43

7 ± 16

0.171 ± 0.042

388 ± 248

74 ± 52

1.51 ± 0.56

143 ± 82

248 ± 142

2 (n=5)

78 ± 44

39 ± 23 *

0.211 ± 0.019

761 ± 136 *

162 ± 39 *

1.73 ± 0.22

172 ± 35

295 ± 61

3 (n=5)

8 ± 17

0 ± 0

0.126 ± 0.017

458 ± 184

57 ± 20

0.97 ± 0.08

174 ± 157

176 ± 165

Table 5: Sperm motion data - group mean values

Group

VAP (um/s)

VSL (um/s)

VCL (um/s)

ALH (um/s)

BCF (Hz)

STR (%)

LIN (%)

Elongation (%)

Area (um sq)

Rapid (%)

Medium (%)

Slow (%)

Static (%)

1 (n=5)

30 ± 67

16 ± 35

80 ± 179

5 ± 12

8 ± 18

11 ± 25

4 ± 10

6 ± 13

146 ± 327

13 ± 29

1 ± 1

6 ± 13

81 ± 43

2 (n=5)

126 ± 70

74 ± 42 *

287 ± 164

20 ± 11

28 ± 16

48 ± 27

22 ± 13 *

21 ± 12

618 ± 365

57 ± 33

1 ± 1

20 ± 13

22 ± 44

3 (n=5)

0 ± 0

0 ± 0

0 ± 0

0 ± 0

0 ± 0

0 ± 0

0 ± 0

0 ± 0

0 ± 0

0 ± 0

0 ± 0

8 ± 17

92 ± 17

Table 6: Sperm morphology - group mean values

Group

Total number of sperm examined

Normal (%)

Abnormal (%)

Decapitate (%)

Head abnormal (%)

Neck abnormal (%)

Midpiece abnormal (%)

Tail abnormal (%)

1 (n=4)

809

21.4 ± 42.8

78.6 ± 42.8

77.6 ± 44.8

0.5 ± 1.0

0.4 ± 0.7

31.4 ± 36.3

0.5 ± 0.7

2 (n=5)

1019

78.9 ± 43.8

21.1 ± 43.8

20.1 ± 44.1

0.6 ± 0.4

0.1 ± 0.2

1.3 ± 2.9

0.4 ± 0.4

3 (n=5)

1047

0.1 ± 0.2

99.9 ± 0.2

98.0 ± 2.5

0.1 ± 0.2

1.0 ± 2.1

53.0 ± 43.3

1.0 ± 0.9

Table 7: Organ weights - group mean unadjusted and adjusted values (g) for animals killed at scheduled termination

Group

Terminal bodyweight

Epididymides

Prostate

Seminal Vesicles

Testes

Historical control data

487 ± 34.8

1.165 ± 0.079

1.051 ± 0.207

1.918 ± 0.255

3.526 ± 0.307

1 (n=5)

415 ± 51

1.004 ± 0.197

0.752 ± 0.168

1.533 ± 0.311

2.99 ± 1.16

2 (n=5)

423 ± 42

1.021 ± 0.142

0.894 ± 0.157

1.650 ± 0.261

3.07 ± 0.55

3 (n=5)

420 ± 27

0.771 ± 0.033

0.658 ± 0.087

1.365 ± 0.213

1.96 ± 0.16

 

Adjusted means

1 (n=5)

 

1.013

0.756

1.534

3.04

2 (n=5)

 

1.012

0.890

1.649

3.01

3 (n=5)

 

0.770

0.657

1.365

1.96

Historical Control Data, HCD – Rats (IGS CD) males 12.4 - 16.3 weeks of age

Table 8: Macropathology - group distribution of findings for animals killed at scheduled termination

Tissue and finding

Group 1 (n=5)

Group 2 (n=5)

Group 3 (n=5)

Left testis

Blue

1

0

5

Enlarged

0

1

0

Flacid

1

0

5

Small

1

0

5

Left epididymis

Small

1

0

5

Right testis

Blue

1

0

5

Flacid

1

0

5

Small

1

1

5

Right epididymis

Small

1

1

5

Table 9: Histopathology - group distribution of findings for animals killed at scheduled termination

Tissue and finding

Group 1 (n=5)

Group 2 (n=5)

Group 3 (n=5)

Right testis

Seminiferous Tubular Atrophy/Degeneration

1

1

5

Seminiferous Tubular Vacuolation

1

1

4

Spermatid Giant Cells

0

0

5

Right epididymis

Degenerate Spermatogenic Cells in Duct(s)

1

1

5

Epithelial Vacuolation

1

1

0

Inflammation

0

1

1

Reduced Numbers of Spermatozoa

0

1

5

Spermatozoa Absent

1

0

0

Conclusions:
The results of dietary administration suggest that exposure via the dietary route of administration reduces the testicular and sperm toxicity of the test material compared to dosing by oral gavage. The results of this study, in part, support the hypothesis that a high peak plasma level is necessary to induce the observed toxic effects on the male reproductive system.
Executive summary:

In an investigatory reproductive toxicity study, three groups of Crl:CD(SD) male rats (five/dose) were administered daily with CAS 8000-41-7 by dietary and/or oral gavage routes at the following doses:

- group 1: dietary 7500 ppm + supplementary gavage dose 300 mg/kg/day

- group 2: dietary 10000 ppm + supplementary gavage dose 150 mg/kg/day

- group 3: CAS 8000-41-7 at 750 mg/kg/day by gavage only

During the study, data was recorded on mortality, clinical condition, bodyweight and food consumption. Surviving animals were subjected to a detailed sperm analysis. Testes (L&R), epididymis (L&R), prostate and seminal vesicles were weighed at necropsy and tissues of right testes and epididymis were fixed for histopathological examination.

During week 1 of the study, Group 1 and 2 animals consumed less diet than expected; From Day 11 Group 1 animals, receiving 7500 ppm, also received two daily doses of 150 mg/kg/b.i.d. four to five hours apart and average intake was boosted to 663 mg/kg/day whilst Group 2 animals, receiving 10000 ppm, also received a single daily dose of 150 mg/kg boosting intake to 678 mg/kg/day.

Clinical signs were generally minimal. Dosing signs for Group 3 animals included salivation and chin rubbing. 

Food consumption for Group 1 and 2 animals receiving the test material in the diet was low throughout the study; this was attributed to the palatability of the test material. Bodyweight gains of these animals were also low. Food consumption and bodyweight changes of Group 3 animals were considered to be not adversely affected by the test material. 

Necropsy data indicated that decreases in reproductive organ weights and changes to macroscopic appearance were most marked in the animals receiving Terpineol multiconstituent at 750 mg/kg/day (Table 8). Occasional animals (1/5 and 1/5 in each of the other groups 1 and 2 respectively) also showed changes to testicular and/or epididymal tissue appearances and/or weights (Tables 7 and 8).

Sperm analysis showed that motile sperm with normal morphology were present in 4/5 males of Group 2 and 1/5 males of Group 1 (Tables 4 -6). The outliers in each group were at the extreme of achieved overall exposure for the group suggesting that absolute exposure was important, although the route of exposure and consequently potential to exceed threshold levels was of greater significance.

Microscopic examination indicated there were relatively fewer changes in the testes and epididymides in the animals which were given Terpineol multiconstituent by the dietary route with oral gavage supplementation (Groups 1 and 2), whereas there were significant changes in those which received it solely by oral gavage (Group 3).

The results of dietary administration suggest that exposure via the dietary route of administration reduces the testicular and sperm toxicity of the test material compared to dosing by oral gavage. The results of this study, in part, support the hypothesis that a high peak plasma level is necessary to induce the observed toxic effects.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP study conducted according to OECD Guideline 422 without any deviation (Klimisch score = 1).
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 230 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP study conducted according to OECD Guideline 413 without any deviation (Klimisch score = 1).
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The systemic toxic potential, including reproductive effects, of terpineol multiconstituent was assessed in different studies.

1- First it was assessed in rats by oral gavage administration over a period of five weeks at doses of 0, 60, 250 or 750 mg/kg/day.

Overall, general toxicity was essentially seen at the top dose groups but was of moderate intensity:

· Underactive behavior and unsteady reactions, in males and females were observed briefly during week 1 in animals receiving 750 mg/kg/day

· Males receiving 750 mg/kg/day showed slightly lower overall weight gain (weeks 0-5) compared with Control

· Some minor biological changes were also observed as well as some changes in bodyweight adjusted liver and kidney weights

The main feature was the testicular effects observed at the top dose:

·Testis weight was markedly low compared to control in males receiving 750 mg/kg/day and there was also an indication of low epididymal weights at this dose.

·Reduced numbers or complete absence of spermatozoa accompanied by the presence of degenerate spermatogenic cells in ducts were observed in the epididymides of males receiving 750 mg/kg/day at the end of the 5 week dosing period and was still present following the 2 week recovery period.

·Other related abnormalities were also observed in some animals as spermatocele granuloma, moderate to severe seminiferous tubular atrophy/degeneration accompanied by minimal to moderate spermatid giant cells and minimal to slight seminiferous tubular vacuolation.

In summary: a clear testicular toxicity was observed at 750 mg/kg bw/day while no testicular effect was seen at 250 mg/kg bw/day.

2- In order to clarify these effects and test the hypothesis of a metabolism switch at the top dose due to the mode of administration (gavage), a similar study was conducted to compare gavage and dietary route of exposure.

The duration of two weeks was selected for gavage in order to better capture the earliest effects of the substance and have better information on potential mechanism of action. A group (5 male animals) received Terpineol by gavage at doses of 750 mg/kg/day for two weeks.Two further groups (5 male animals/group – Groups 1 and 2) received Terpineol orally, via the diet, at concentrations of 7500 or 10000 ppm.

From Day 11, due to a food intake insufficient to achieve target levels of 500 and 750 mg/kg bw/day, dietary intake was supplemented with Terpineol by gavage for Group 1 animals at 300 mg/kg/day (150 mg/kg/b.i.d) and Group 2 animals at 150 mg/kg/day. In addition exposure of these two groups was extended to three weeks.

In Group 1 (group mean test substance intake: 546 mg/kg), all animals had normal testis and epipidymis weight as well as normal sperm count except animal one who had a testicular weight at about 30% of the other animals of his group and a sperm count at about 1% of the other animals. This animal was the only one among all the groups to have lost weight during the study which may explain its sensitivity to the substance toxicity. However, all animals in this group, with exception of animal 2, had most of their spermatozoids with abnormal shape and no motility. Animal 2 had a normal sperm count with 97% mobiles and 36% with progressive mobility (slightly lower than in group 2) and he was the one who achieved the lowest test substance intake but not much lower than animal one (527 vs. 551 mg/kg as day one to 21 average).

In Group 2 (group mean test substance intake: 602 mg/kg), all animals were normal with the exception of animal 6 who had no motile sperm, close to 100% of abnormal spermatozoids (decapited) but a normal testis /epipidymis /seminal vesicles weight as well as a normal number of spermatozoa in testis and epipidymis. This animal was the one with the highest test substance intake (639 mg/kg for an average of 592 mg/kg for the other four animals).

Results of the second study were indicative of an effect linked with the bolus dose. However they could not be considered as sufficient to prove it due to the insufficient food intake and the obligation to add some part in gavage to achieve a dose level comparable to the top doses of the first study. For this reason an additional two week study was performed in another laboratory with higher dose in the diet.

3- A comparative two week study was conducted where terpineol multiconstituent was adminitered orally either by diet or by gavage to male rats.

Two groups (5 male animals/group) received Terpineol orally by gavage at 500 and 750 mg/kg bw and two others via the diet, at concentrations of 8000 or 12000 ppm for two weeks. There were two control groups, one vehicle control gavage administration and one pure control.:

Gavage groups

G1 Vehicle control

G4 500 mg/kg bw - TERPINEOL

G5 750 mg/kg bw - TERPINEOL

Diet groups.

G18 8000 ppm - Diet Administration - TERPINEOL

G19 12000 ppm - Diet administration -TERPINEOL

G20 Diet Control

Group numbering was not continuous as there were other substances tested in this study.

Immediately after scheduled sacrifice of each male, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.

The following tests were performed:

·Sperm motility

·Sperm morphology, sperm count and homogenisation-resistant spermatids count were to be performed in a second time and will be reported in the final report

All animals were subject to a detailed necropsy. Epididymis, seminal vesicles, testis and prostate were dissected free of adjacent fat and other contiguous tissue and the weights recorded. The bilateral organs were weighed individually. Organ weights were also adjusted for terminal bodyweight, using the weight recorded before necropsy.

The results available to date (November 2010) are the following:

·Sperm motility

o Motile- the percentage of cells which are moving at or above the minimum speed as defined in the set up parameters

o Progressively motile- the percentage of cells moving with both VAP (average path velocity) > progressive minimum VAP and STR (straightness) > S0. (progressive minimum VAP is a delimiter used in determining whether a motile cell is labelled as rapid or medium. Sois the threshold straightness.)

·Body and organ weights.

Results of the study:

- In the gavage groups, feed intake was decreased in both treated groups and at the end of the two weeks treatment period.

- Test substance administration resulted in an initial decreased body weight gain without complete recovery up to the end of the study.

- In the feeding groups, food intake was very low during the first days of the study but increased rapidly and total substance intake in the high dose group remained slightly over 750 mg/kg during all week two.

- This also resulted in a clear decrease in body weight gain and, even for the high dose group in a body weight loss, with only a partial recovery during week 2.

However, effects on sperm mobility clearly confirms the effects previously observed when the substance is administered by gavage while no effects are detected when administration of via diet.

4- Such discrepancies of effects depending on the mode of dose administration were confirmed in a 90-day toxicity study (i.e. a whole period of spermatogenesis).

Terpineol multiconstituent was dissolved in corn oil, mixed in Ssniff powder feed at the dose level of 12000 ppm and fed to male Sprague-Dawley rats (10/dose) dailyad libitumf or 13 weeks. Rats in the control group were fed basal diet only without any test item admixtures. All rats were observed for clinical signs, mortality, and changes in the body weights and food intake. Sperm evaluations were conducted at termination for all the males from each group. Sperm motility, count and morphology were evaluated for all the groups. All rats were subjected to detailed necropsy at termination and organs were weighed. Histopathological examination of the testes and the epididymides were carried out.

No treatment related mortality or signs of toxicity were noted. The body weights were significantly reduced in rats receiving test item at 12000 ppm. This decrease was associated with a decrease in the food intake throughout the treatment period. The Food consumption was significantly reduced in males receiving test item at 12000 ppm dose during the treatment period. The calculated mean daily test item consumption was 0 and 622.65 mg/kg bw/day corresponding to 0 and 12000 ppm, respectively.

A slight significant increase in the percentage of abnormal (4.8 %) sperms was noted at 12000 ppm as compared to the control group. However, the change was considered incidental as it was well within the range of normal biological variation noted among male rats [the range of the in-house historical control data for mean percentage of abnormal sperms: 0.1- 7.4%]. The sperm motility remained unaffected by dietary administration of test item. There were no test item-related changes observed in cauda epididymal weight/sperm count and testicular weight/spermatid count.

There were no test item-related changes in the terminal fasting body weights. Increased liver weights (absolute-13% and relative-20%) were noted in the treatment group. Increased relative weights (paired and unpaired) of testes and epididymides were observed in the treatment group. There were no test item-related histological changes observed in the testis and the epididymis.

No testicular and epididymal toxicity was evidenced in animals receiving terpineol multiconstituent at 12000 ppm, corresponding to 623 mg/kg bw/day, for 90 days .

Discussion

Terpineol multiconstituent when administered at the dose of 750 mg/kg by gavage produced testicular toxicity with absence of spermatozoids and testicular degeneration. None of these effects were observed at 250 mg/kg.

Hypothesis that these effects could only be observed due to the peak of concentration and a possible metabolism switch at this level was tested in studies where the test substance was administered in the diet trying to achieve this same level.

Results demonstrate clearly that, despite a similar exposure level by gavage and by mixed administration (diet + gavage), a much lower level of toxicity was achieved in the diet groups and singularly in the group two where total test substance intake was higher but the proportion by gavage was twice lower.

In a third study, it was possible to feed the rats at a sufficient level to achieve a dosage around 500 and 750 mg/kg during a period long enough to compare pure gavage and pure diet administration. In this case, while effects on sperm motility in the gavage group were clearly reproduced, no effects were observed on this parameter in the diet groups. The absence of toxicity effect on male fertility was further confirmed in a 90 -day oral toxicity study where male rates were feed at the dose level of 12000 ppm (corresponding to 623 mg/kg/day).

In the 90-day toxicity study conducted by inhalation, no toxic effects were observed in male reproductive organs at any tested concentrations (the NOAEC was established at 2.23 mg/L corresponding to an estimated achieved dose of 566 mg/kg/day). There are therefore strong arguments to consider that the effects observed at high dose by gavage are due to a peak effect and are unlikely to occur, even at the highest achievable dose, by either inhalation, dermal or oral route (in the food).

Gavage administration is a normal way to evaluate drug toxicity (which can be administered as one dose/day) or a convenient surrogate (no need to mix with diet) to evaluate toxicity which could occur due to presence of the substance in the food or to extrapolate to different routes of exposure (dermal or inhalation). However, in some cases, it creates pharmacokinetic (and then pharmacodynamic) circumstances which cannot be encountered in real conditions of exposure and can be considered in this case as a non relevant route of exposure (as would be IV or IP mode of administration).

In a recent study conducted by gavage, pharmacokinetic analysis also confirmed the systemic overexposition of the rat after a single dose at 750 mg/kg. The extent of systemic exposure, characterised by AUCall, was shown to increase with increasing dose over the dose range 75 to 750 mg/kg; however, these increases were greater than the proportionate dose increment in both plasma and whole blood. Overall, the AUCall values at the highest dose level (750 mg/kg) were ca 2.3-fold higher than those values predicted from a linear relationship.

Based on the data currently available, there is strong evidence that no specific reproductive effects will occur when animals are exposed through a route relevant for human exposure.

Conclusion

Based on these findings, there is strong evidence that no reproductive effects are likely to occur by the realistic routes of exposure and no classification for reproductive effects is therefore warranted.

Effects on developmental toxicity

Description of key information

A developmental toxicity study was conducted with terpineol multiconstituent according to OECD guideline 414 and in compliance with GLP in the rat and in the rabbit.

NOAEL (No Observed Adverse Effect Level) for maternal toxicity and embryo-foetal development was determined to be 600 mg/kg bw/day and 500 mg/kg bw/day in the rat and the rabbit, respectively.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 08 November 2013 to 14 January 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD Guideline 414 without deviation.
Reason / purpose:
reference to other study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Number of animals ordered: 88 females; spare animals were removed from the study room after treatment commenced.
- Age of the animals at the start of the study (day 0 of gestation): at least 70 days old.
- Weight range of the animals at the start of the study (day 0 of gestation): 235 to 286 g.

HOUSING
- Cages: cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatisation and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.
- Number of animals per cage during acclimatisation up to four animals; during pairing one (stud) male and one female; during gestation: one female.
- Diet: ad libitum. SDS VRF1 certified pelleted diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Ad libitum.
- Duration of acclimatisation: five days before commencement of pairing.

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23ºC.
- Humidity: 40-70%.
- Air changes: approximately 15 to 20 per hour.
- Air supply: filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (artificial lighting): 12 h dark / 12 h light
- rodent facility: full barrier - to minimise entry of external biological and chemical agents and to minimise the transference of such agents between rooms.

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
METHOD OF PREPARATION
Vehicle was gradually added to the required amount of test substance and mixed to produce a pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser. A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test substance.
The use of plastic or rubber equipment/storage containers was avoided.

VEHICLE
- Concentration in vehicle: determined for each formulation.
- Amount of vehicle (if gavage): 5 mL/kg body weight.
- Justification for use and choice of vehicle (if other than water): substance only slightly soluble in water.
- Formulation frequency: weekly and prepared in advance of the first day of use. The homogeneity and stability of formulations in the concentration range 1 to 200 mg/mL were confirmed for 15 days following refrigerated storage (2-8˚C) and for 2 days following ambient storage (nominally 21˚C) as part of another study, Huntingdon Life Sciences (OAD0004).

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration in test formulations:
- Verification: on Day 1 of dosing and the last day of dosing, freshly prepared test formulations were sampled (4 × 1 mL accurately weighed, 2 × 3 mL accurately weighed for controls) and submitted for analysis. Duplicate samples were analysed in accordance with the analytical procedure, and the remaining samples were retained for contingency. These were discarded following acceptable results.
- Results: the mean concentrations of Terpineol multiconstituent in test formulations analysed for the study were within 4% of nominal concentrations, confirming accurate formulation.

Analytical procedure:
The analytical method involved extraction and dilution in acetone followed by gas chromatography analysis with flame ionisation detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 5 μg/mL to 25 μg/mL.
Details on mating procedure:
- Male/female 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.

- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: no data

- Allocation: On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.

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:
Day 6 to 19 after mating, inclusive.
Frequency of treatment:
Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.
Duration of test:
21 days (Days 0-20 post coitum)
No. of animals per sex per dose:
20 mated females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels of 60, 200 and 600 mg/kg bw/day were selected in agreement with the Sponsor.
- Volume dose: 5 mL/kg body weight.
- Rationale for animal assignment, 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.
- Identification of animals: Each animal was assigned a number and identified uniquely within the study by a tail tattoo.
- Identification of cages: Each cage label was colour-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).
Maternal examinations:
CAGE SIDE & CLINICAL OBSERVATIONS: Yes
Time schedule:
- Mortality or signs of morbidity: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s). 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 acclimatisation period, observations of the animals and their cages were recorded at least once per day.
- Clinical signs: 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 then daily from Days 6 to 20 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: 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 inclusive after mating.

POST-MORTEM EXAMINATIONS: Yes
- Time schedule: animals were killed on Day 20 after mating

OTHER:
Preservation of tissues: 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
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (including cervix and ovaries), for pregnant females surviving to term
- Number of corpora lutea: Yes, for all females
- Number of implantations: Yes, for all females
- Number of early resorptions: Yes, for all females
- Number of late resorptions: Yes, for all females
- Other: fetuses (live and dead), for all females
- For apparently empty uterine horns: The number of uterine implantation sites were checked after staining with ammonium sulphide
Fetal examinations:
EXAMINATION OF FETUSES:
- Number of all live fetuses: Yes
- Number of dead fetuses (fetuses at term without spontaneous movements and breathing): Yes
- Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.
- Body weight of fetuses: Body weight of each live fetus was recorded.
- Sex of fetuses: Sex of each fetus was determined.
- External examinations: Yes; each fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices.
- Soft tissue examinations: Yes, 50% of the live fetuses in each litter were sexed internally and eviscerated.
- Skeletal examinations: Yes [all the remaining litters].
- Head examinations: Yes, cleft lip, protruding tongue, craniorachischisis, open eye lid(s), short snout,
- Skin examination: shinny, subcutaneous oedema
Statistics:
- The computer systems that were used on this study to acquire and quantify data include: Liberate, in-house system used for reporting in-life, necropsy, fetal pathology and statistics.
- Pre/post implantation loss and sex ratio were analysed by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect
- Each treated group was compared to control using a Wald chi-square test.
- For resorptions, each treated group was compared to control by exact Wilcoxon rank sum test
- The mean values: group mean values and SD were calculated using individual litter mean values.
Indices:
- Pre-implantation loss (%): [(Number of corpora lutea - Number of implantations) / Number of corpora lutea] X 100
- Post-implantation loss (%): [(Number of implantations - Number of live fetuses) / Number of implantations] X 100
- Sex ratios of the foetuses were calculated as the percentage of males per litter.
- All group values and SD (as appropriate) were calculated from the individual litter values.
- For litter size and survival indices and fetal, placental and litter weight and gravid uterine weight data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed.
Historical control data:
The results of 8 groups corresponding to historical control data are presented in Annex 3 of the report.
We can find the number of fetuses/litters examined, the number of incompletely ossified/unossified skeletals (5th/6th sternebrae, other sternebrae, total number of affected fetuses/litters, mean % fetuses per litter with affected sternebrae).
Clinical signs:
no effects observed
Description (incidence and severity):
Signs in relation to dose administration were limited to a low incidence of salivation or chin rubbing among females receiving 600 mg/kg/day.
Mortality:
no mortality observed
Description (incidence):
No mortality and no relevant clinical signs or signs of reaction to treatment were noted in treated females.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
-Following the commencement of treatment on Day 6 of gestation, females in the 600 mg/kg/day group showed statistically significantly low mean body weight gain compared to Controls between Day 6 and 8 of gestation (3g versus 6g among Controls). A similar statistically significant decrease in mean body weight gain was evident among these females between Day 12 and Day 15 of gestation (17g versus 21g among Control), and mean weight gain from Day 15 to Day 20 of gestation was 0.93X Control although statistical significance was not attained for this difference. As a consequence, overall mean weight gain from Day 6 to Day 20 of gestation for females receiving 600 mg/kg/day was statistically significantly lower than Control (0.92X Control). There was no effect of treatment with Terpineol on the weight gain of females receiving 60 or 200 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Following the commencement of treatment on Day 6 of gestation, mean food intake among females in the 600 mg/kg/day group was slightly lower than Control throughout the treatment period, with statistical significance attained for the differences during Days 6-17 of gestation. Mean food intake for females in the 60 or 200 mg/kg/day groups was unaffected by treatment with Terpineol.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At scheduled termination on Day 20 of gestation, the mean gravid uterine weight of females in the 600 mg/kg/day group was slightly lower than Control (0.94X) and when overall mean body weight gain was adjusted for the contribution of the gravid uterus, net body weight gain during gestation was also lower than Control (0.85X); these differences from Control did not, however, attain statistical significance. Mean gravid uterine weight and net mean body weight gain were similar to Control for females receiving 60 or 200 mg/kg/day and no effect of treatment was inferred.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- There were no treatment-related macroscopic abnormalities detected at scheduled termination at any dose level investigated.
- Two females in the 200 mg/kg/day group (No’s. 47 and 49) were noted to have a misshapen liver with a raised area protruding through the diaphragm, resulting in a diaphragmatic hernia; these abnormalities were deemed to be congenital and unrelated to treatment with Terpineol.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
- With the exception of one female in the 200 mg/kg/day group (No. 52) which had a total litter resorption, all females were found to be pregnant with live young at scheduled termination on Day 20 of gestation.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Abnormalities:
no effects observed
Fetal body weight changes:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 600 mg/kg/day marginally low mean male and female fetal weight was apparent, with statistical significance attained for mean female fetal weight when compared to Control (0.95X), resulting in marginally but statistically significantly low overall mean fetal weight (0.96X Control). In addition, mean placental weight in this dose group was slightly low (0.91X Control) with differences attaining statistical significance. Mean placental, litter and fetal weights at 60 or 200 mg/kg/day were unaffected by maternal treatment with Terpineol.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants showed no relationship to maternal treatment with Terpineol.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 600 mg/kg/day there was a slightly higher incidence of incompletely ossified or unossified 5th and/or 6th sternebrae compared to concurrent control and the Historical Control Data range. In the absence of any related abnormalities this finding was considered to reflect a minor delay in development, indicated by the marginally decreased mean fetal body weight in this group, rather than a direct effect of treatment on fetal development.
Visceral malformations:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 7.8.2/2: Clinical signs – group distribution of observations 

Days 0 - 20

Number of animals affected

Group / sex

1F

2F

3F

4F

Category

Observations

Number in group

20

20

20

20

Coat

Hair loss, forelimbs

 

1

2

4

6

Table 7.8.2/3: Signs associated with dosing - group distribution of observations

Sign

Day number

Group/sex and number of animals showing sign

1F

2F

3F

4F

Behaviour

 

 

Chin Rubbing

17

0

0

0

3

 

 

18

0

0

0

7

 

 

19

0

0

0

6

Salivation

7

0

0

0

3

 

 

8

0

0

0

7

 

 

11

0

0

0

1

 

 

12

0

0

0

2

 

 

13

0

0

0

2

 

 

14

0

0

0

2

 

 

15

0

0

0

1

Table 7.8.2/4: Food consumption - group mean values (g/animal/day) during gestation

 

 

 

Group/Sex

 

Day 0-2

Day3-5

Day 6-9

Day 10-13

Day 14-17

Day 18-19

Statistical test

 

Av

Av

Wi

Wi

Wi

Wi

1F

Mean

25

26

23

25

27

26

 

SD

2.0

2.4

2.5

3.1

2.4

2.5

 

N

20

20

20

20

20

20

2F

Mean

25

26

24

25

28

27

 

 

SD

1.9

2.0

1.7

2.1

2.3

3.2

 

 

N

20

20

20

20

20

20

 

3F

Mean    

25

26

22

25

28

28

 

 

SD

2.3

2.3

1.5

2.3

2.3

3.2

 

 

N

19

19

19

19

19

19

 

4F

Mean

24

25

20**

23**

25*

25

 

 

SD

2.1

2.1

1.8

2.0

2.1

2.4

 

 

N

20

20

20

20

20

20

 

Table 7.8.2/5: Organ weights - group mean unadjusted and adjusted values (g)

Group/Sex

 

Terminal Body Weight

Liver weight

 

Unadjusted Means

Statistical test:

 

Wi

 

 

1F

Mean

420

18.17

 

 

SD

20

1.47

 

 

N

20

20

 

2F

Mean

425

18.48

 

 

SD

18

1.45

 

 

N

20

20

 

3F

Mean    

420

18.37

 

 

SD

23

1.44

 

 

N

19

19

 

4F

Mean

404*

19.14

 

 

SD

18

1.56

 

 

N

20

20

 

Adjusted Means

Statistical test:

 

 

Wi

 

1F

Mean

 

18.02

 

2F

Mean

 

18.06

 

3F

Mean

 

18.22

 

4F

Mean

 

19.84**

 

Table 7.8.2/6: Gravid uterine weight, adjusted body weight and adjusted body weight change - group mean values (g)

 

 

Group/Sex

 

Body weight on Day 6

Terminal Body weight on Day 20

Body weight Change Days 6-20

Gravid Uterine Weight

Adjusted Body weight Day 20

Adjusted Body weight Change Days 6-20

Statistical test

 

Av

Wi

Wi

Wi

Wi

Wi

1F

Mean

292

420

128

95

325

33

 

SD

11.1

20.4

13.6

10.4

16.9

8.8

 

N

20

20

20

20

20

20

2F

Mean

294

425

131

95

330

37

 

 

SD

10.1

18.1

13.2

11.6

12.8

8.3

 

 

N

20

20

20

20

20

20

 

3F

Mean    

291

420

129

94

326

35

 

 

SD

12.9

11.9

14.5

14.6

19.4

10.3

 

 

N

19

19

19

19

19

19

 

4F

Mean

286

404*

118*

89

315

28

 

 

SD

11.7

18.1

11.6

11.9

14.6

8.6

 

 

N

20

20

20

20

20

20

 

Conclusions:
The No Observed Adverse Effect Level (NOAEL) of Terpineol multiconstituent was considered to be 600 mg/kg bw/day for maternal toxicity and 600 mg/kg bw/day for developmental toxicity in Sprague-Dawley rats.
Executive summary:

In a GLP-compliant prenatal developmental toxicity study performed according to OECD guideline 414, Terpineol multiconstituent diluted in corn oil was administered by gavage to groups of mated female Sprague-Dawley rats (20 mated females/dose) at the dose levels of 0, 60, 200, 600 mg /kg bw/ day from Days 6 to 19 after mating. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Detailed observations were recorded daily at the following times in relation to dose administration. A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health. The weight of each adult was recorded on Days 0, 3 and and then daily from Days 6 to 20 after mating. 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 inclusive after mating.

On Day 20 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The gravid uterine weight, number of implantations, live and dead fetuses, early and late resorptions and corpora lutea were recorded. Gross evaluation of the placenta was also performed.

Fetuses were sexed, weighed and examined for external, soft tissue and skeletal malformations.

With the exception of one female in the 200 mg/kg bw/day group (No. 52) which had a total litter resorption, all females were found to be pregnant with live young at scheduled termination on Day 20 of gestation. No mortality was observed.

At scheduled termination on Day 20 of gestation, the adjusted mean liver weight of females receiving 600 mg/kg bw/day was significantly higher than Control (1.10X Control). There were no treatment-related macroscopic abnormalities detected. No relevant clinical signs or signs of reaction to treatment were noted in treated females. Females receiving 60 or 200 mg/kg bw/day showed no treatment-related changes in clinical condition, body weight performance, food intake, liver weight or macropathology.

There was no effect of maternal treatment with Terpineol multiconstituent at any dose level investigated on litter data. Sex ratio, as assessed by the percentage of males per litter, was generally comparable in all groups and in line with expectations. Embryo-fetal growth was slightly reduced by maternal treatment at 600 mg/kg bw/day.

It was considered that there was no adverse effect of maternal treatment on embryo-fetal development; the incidence of major and minor abnormalities and skeletal variants showed no relationship to maternal treatment with Terpineol multiconstituent.

In the 600 mg/kg bw/day group there was a slightly higher incidence of incompletely ossified or unossified 5th and/or 6th sternebrae compared to concurrent control and the Historical Control Data range.

It was considered that this minor finding did not constitute an adverse effect on development.

On the basis of the results obtained in this study, the dosage of 600 mg/kg bw/day was considered to be the NOAEL (No Observed Adverse Effect Level) for maternal toxicity. The NOAEL for developmental toxicity was considered to be 600 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 February 2018 - 11 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DRT / 199599
- Appearance: Colourless liquid
- Expiration date of the lot/batch: 29 August 2019

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 °C) under nitrogen and kept away from light and humidity.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS
- Age at study initiation: approximately 19 to 23 weeks of age
- Weight at study initiation: 2.48 to 3.86 kg
- Housing: One animal per cage. Suspended cages fitted with perforated floor panels are mounted in batteries. Undertray lined with absorbent paper which is changed at least 3 times a week
- Diet (e.g. ad libitum): Teklad 2930, pelleted diet, restricted (150 g per rabbit per day during acclimatisation up to one week prior to the onset of mating and 200 g/animal/day thereafter).
- Water (e.g. ad libitum): Potable water from the public supply, non -restricted
- Acclimation period: minimum 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-21ºC.
- Humidity (%): 45-70%
- Air supply: filtered, not recirculated
- Photoperiod (hrs dark / hrs light):14 hours light : 10 hours dark

IN-LIFE DATES: From: 25 July 2018 To:11 September 2018
Route of administration:
oral: gavage
Vehicle:
other: 1% Methylcellulose
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
Male/female ratio: 1 : 1 using identified stock New Zealand White bucks.
Checks: natural mating observed.
After mating: each female injected intravenously with 25 i.u. luteinizing hormone.
Day 0 of gestation = Day of mating.
A colony of stud stock males of the same strain is maintained specifically for the purpose of mating; these animals are not part of the study and are maintained as stock animals.
Duration of treatment / exposure:
Days 6 to 28 after mating
Frequency of treatment:
Daily
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In the pilot study, doses of 250 and 400 mg/kg bw/day were well tolerated with no changes in general clinical condition and no overt effects on body weight performance or food intake apparent. Administration at 600 mg/kg bw/day was not well tolerated, with all three females prematurely killed for reasons of animal welfare on Day 2 of study due to post-dosing signs including underactive behavior, constricted pupils, abnormal/unsteady gait and flat posture.
In the preliminary embryo-fetal development study, doses of 0, 150, 300 and 500 mg/kg bw/day were employed. There were no test item-related premature deaths, changes in general clinical condition or post-dosing signs observed. Mean food consumption was slightly lower than Control in all treated groups from Day 6 to Day 16 of gestation, although in the absence of a dose response relationship; body weight performance was not affected. There was no evidence of an effect of maternal treatment on litter data (number of implantations, resorptions, live young, sex ratio or levels of pre- and post-implantation loss) or placental, litter and fetal weights.
The high dose level in this main study was set at 500 mg/kg bw/day since there were no maternal or litter/fetal findings to preclude the use of this dose level. The low and intermediate dose levels were set at 125 and 250 mg/kg bw/day to achieve a dose response and/or aid in the determination of a No-Observed-Adverse-Effect-Level (NOAEL).
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Visually inspected at least twice daily for morbidity, mortality and evidence of reaction to treatment or ill-health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Days 0, 6, 12, 18, 24 and 29 after mating

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimatization. Days 0, 3, 6-29 after mating.

FOOD CONSUMPTION : Yes
- Daily from Day 1 after mating to termination

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 29 after mating
- All adult animals were subject to a complete macroscopic examination. Any abnormal tissue was sampled as appropriate and retained in appropriate fixative
Ovaries and uterine content:
For females surviving to term, the following was recorded:
Uterus Gravid uterine weight (including cervix and ovaries).
The following were recorded for all animals (including those prematurely sacrificed, where possible):
For each ovary/uterine horn Number of: Corpora lutea, Implantation sites, Intrauterine deaths (classified as early or late resorptions), Fetuses (live and dead).

The absence or number of uterine implantation sites was confirmed.

Expelled uterine contents were identified and examined, as appropriate.
Fetal examinations:
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, sampled as appropriate and retained in appropriate fixative. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also
recorded.
Fixation: Approximately 50% of eviscerated fetuses were decapitated; heads were initially stored in Bouin’s fluid. Remaining eviscerated fetuses and torsos were fixed in Industrial Methylated Spirit. Serial sections were examined for soft tissue abnormalities.
Bouin’s fixed fetal heads were subject to free-hand serial sectioning. Industrial Methylated Spirit fixed fetuses and torsos were processed and stained with Alizarin Red for skeletal development and abnormalities assessment.
Statistics:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other analyses the/The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, Dunnett's test (Dunnett 1955, 1964) was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre-treatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon 1945) were made. For all other analyses the/The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel 1959) was performed instead.
Indices:
Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100
Post-implantation loss (%)= [(Number of implantations – Number of live fetuses) / Number of implantations] x 100
Historical control data:
Historical Control data were routinely supplied for selected observations where this information was considered to assist interpretation of study data, and normally include both fetal and litter incidences.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Among pregnant females surviving to scheduled termination there were no test item-related changes in general clinical condition or signs observed in relation to dose administration.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were five premature deaths during the course of the study, none of which were considered to be related to Terpineol multiconstituent administration.
Control Female No. 10 was found dead after dose administration on Day 25 of gestation. No changes in clinical condition or signs related to dose administration had been observed throughout the study, and body weight performance and food intake were in line with expectation. No macroscopic abnormalities were apparent; the uterus contained nine live fetuses.
Female No. 38 receiving 125 mg/kg bw/day was killed for reasons of animal welfare on Day 28 of gestation due to severe respiratory impairment. On Day 27 of gestation this female showed wet rales and during handling had gasping respiration; in light of these signs the female was not dosed. Prior to dose administration on Day 28 of gestation, wet rales was still evident and at a greater severity than the previous day, therefore the female was dispatched to necropsy for welfare reasons. Uterine examination revealed eight live fetuses, and macroscopic examination revealed a large amount of air bubbles present between the skin and its subcutaneous membrane on the dorsal body surface. There was no evidence of mal-dosing in this rabbit, as the trachea and esophagus were intact, so the cause of these
macroscopic findings is unclear, however in the absence of similar findings at this or higher dose levels, this premature death was considered unrelated to treatment.
Two females in the 500 mg/kg bw/day group were killed for reasons of animal welfare on Day 17 of gestation due to general poor clinical condition. Female No. 69 showed a marked decrease in food consumption from Day 10 of gestation and in the final four days prior to
despatch to necropsy had consumed no pelleted diet. Between Day 10 and Day 17 of gestation weight loss of 0.39 kg was recorded and on the day of necropsy clinical signs including thin build, decreased fecal pellet output, loose/mucoid feces, reduced urine output and little diet eaten were apparent. From Day 12 of gestation, Female No. 71 had shown a marked decrease in food consumption with negligible diet consumed during Days 13-16 of gestation. Progressive weight loss was apparent from Day 8 of gestation, totaling 0.35 kg and on the day of necropsy clinical signs including thin build, decreased fecal pellet output, small/pale fecal pellets and little hay and diet eaten were apparent. No macroscopic abnormalities were detected in either of these females. Female No. 69 was pregnant with the uterus containing three live embryos and seven resorptions; Female No. 71 was not pregnant.
Given the low frequency of the premature deaths and the absence of macroscopic abnormalities, a relationship between Terpineol multiconstituent administration and these premature deaths was considered unlikely.
Female No. 73 in the 500 mg/kg bw/day group was found with fetal material in the cage under-tray on Day 27 of gestation and was dispatched to necropsy due to abortion. This female had shown progressive weight loss totaling 0.22 kg over the preceding five days along with negligible food consumption, and clinical signs of reduced fecal pellet output with the pellets being small. Macroscopic examination revealed dark lungs, dark gall bladder with depressed areas on the surface, and the stomach was distended with fetal material. The uterus contained 11 abortion scars, and since only one dead fetus and two pieces of placenta were found in the cage under-tray it was concluded that the female cannibalized all of the remaining fetuses/placentae. Spontaneous abortion is not an uncommon event in the laboratory rabbit, including in Controls, and therefore the occurrence of this isolated abortion was considered unrelated to Terpineol multiconstituent.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The overall body weight performance of pregnant females surviving to scheduled termination was unaffected by Terpineol multiconstituent at doses up to and including 500 mg/kg bw/day.
Females given 500 mg/kg bw/day showed slight mean body weight loss of 0.05 kilos during Days 6-9 of gestation (compared to mean body weight stasis in Controls), however the extent of the body weight loss was not adverse, and thereafter mean body weight gain was
essentially similar to or slightly greater than Control. The mean gravid uterine weight on Day 29 of gestation was similar in all groups. When
overall mean body weight gain was adjusted for the contribution of the gravid uterus, net mean body weight loss was recorded at a similar magnitude in all groups of females and no effect of treatment with Terpineol multiconstituent was inferred.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Among females given 500 mg/kg bw/day, group mean food consumption was slightly lower than pre-treatment values and Control following the onset of treatment on Day 6 of gestation until Day 20 of gestation, with some individual daily recording periods attaining statistical significance. Thereafter, mean food consumption was essentially similar to or slightly higher than Control such that overall mean daily food consumption during Days 6-28 of gestation was only marginally lower than Control (85 grams per day compared to 93 grams per day in
Controls). These differences in mean food consumption were considered not to be adverse at the magnitude observed. Mean food consumption at 125 or 250 mg/kg bw/day was unaffected by treatment with Terpineol multiconstituent.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related macroscopic abnormalities detected among the females at scheduled termination on Day 29 after mating.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 19, 16, 18 and 16 litters were available for assessment at 0, 125, 250 and 500 mg/kg bw/day, respectively.
Other effects:
not examined
Key result
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on mean placental, litter or fetal weights at any dose level investigated.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no evidence that maternal treatment with Terpineol multiconstituent at doses up to and including 500 mg/kg bw/day had any adverse effect on litter data, as assessed by the mean numbers of implantations, resorptions, live young and pre- and post-implantation losses, at any of the doses investigated.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio, as assessed by the percentage of males per litter, was in line with expectations and unaffected by maternal treatment.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus in the 250 mg/kg bw/day showed the major abnormality of gastroschisis. Although there were no cases of gastroschisis in the historical control data range (only studies conducted during 2018), in the absence of similar or related abnormalities in other fetuses in the 250 mg/kg bw/day group or in the 500 mg/kg bw/day group, this single incidence was considered fortuitous and unrelated to Terpineol multiconstituent administration.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
At 500 mg/kg bw/day there were four fetuses in one litter (Litter 81) with the minor macroscopic abnormalities of dark fluid in stomach and brown lungs, along with three fetuses in the same litter with unexpanded lungs. All of these fetuses had no significant underlying skeletal abnormalities, and therefore the observed fresh macroscopic abnormalities in a single litter were considered to be of no toxicological significance and unrelated to maternal treatment.
Key result
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Based on the results obtained in this study of embryo-fetal development, it was concluded that the high dose level of 500 mg/kg bw/day represented the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and for embryo-fetal survival, growth and development.
Executive summary:

In a study conducted according to OECD guideline 414 and in GLP conditions, three groups of 22 females received terpineol multiconstituent at doses of 125, 250 or 500 mg/kg bw/day by oral gavage administration, from Day 6 to 28 after mating at a volume dose of 5 mL/kg body weight. A similarly constituted Control group received the vehicle, 1% methylcellulose, over the same period and at the same volume dose as the treated groups.

Animals were killed on Day 29 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 29 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

Administration of terpineol multiconstituent to pregnant New Zealand White rabbits at dose levels up to and including 500 mg/kg bw/day was well tolerated, with no test item-related premature deaths, clinical signs or post-dosing signs observed.

Females given 500 mg/kg bw/day showed slight mean body weight loss during Days 6-9 of gestation, and a slight reduction in mean food consumption from Day 6 to Day 20 of gestation; these differences from Control were not adverse.

Mean gravid uterine weight and adjusted body weight gain was unaffected by terpineol multiconstituent administration at all dose levels investigated, and there were no test item-related maternal abnormalities detected at scheduled termination.

There was no effect of maternal treatment with terpineol multiconstituent on litter data, as assessed by the number of implantations, resorptions, live young, sex ratio and pre- and post-implantation losses. Placental, litter and fetal weights were similar in all groups.

The incidence of major and minor fetal abnormalities and skeletal variants showed no relationship to maternal treatment with terpineol multiconstituent.

Based on the results obtained in this study of embryo-fetal development, it was concluded that the high dose level of 500 mg/kg bw/day represented the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and for embryo-fetal survival, growth and development.

Endpoint:
developmental toxicity
Remarks:
Preliminary Study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
6 June to 30 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Reason / purpose:
reference to same study
Qualifier:
no guideline available
Principles of method if other than guideline:
The study was conducted to assess the influence of Terpineol multiconstituent on embryo-fetal survival and development in the New Zealand White rabbit and to establish suitable doses for a main embryo-fetal toxicity study.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DRT / 199599
- Appearance: Colourless liquid
- Expiration date of the lot/batch: 29 August 2019

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 °C) under nitrogen and kept away from light and humidity.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS
- Age at study initiation: Phase 1: Approximately 18 to 22 weeks
Phase 2: Approximately 20 to 24 weeks
- Weight at study initiation: 2.44 to 4.13 kg
- Housing: One animal per cage. Suspended plastic cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least three times a week.
- Diet: Teklad 2930 (pelleted) diet. Restricted (150 g per rabbit per day during acclimatisation up to one week prior to the onset of mating and 200 g/animal/day thereafter).
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: Phase 1: 15 days before commencement of mating
Phase 2: 26 days before commencement of mating.

ENVIRONMENTAL CONDITIONS
- Temperature: 15-21 °C
- Humidity: 45-70 %
- Air changes: Filtered, not recirculated
- Photoperiod: 14 h light : 10 h dark

IN-LIFE DATES: From: 6 June 2018 To: 31 July 2018
Route of administration:
oral: gavage
Vehicle:
other: 1% Methylcellulose
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The stability of a homogenous suspension of the test item in the vehicle was demonstrated as part of another study (Envigo Study No. PH67XD) which demonstrated that prepared formulations in the concentration range of 1 to 200 mg/mL in the vehicle were stable for up to 15 days following refrigerated storage (2 to 8°C), and for one day following ambient storage (15 to 25°C).
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 : 1 using identified stock New Zealand White bucks
- After mating: Each female injected intravenously with 25 i.u. luteinizing hormone.
- Day 0 of gestation: Day of mating.
Duration of treatment / exposure:
Females were treated from Day 6 to Day 28 (inclusive) after mating.
Frequency of treatment:
Once daily, at approximately the same time each day.
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
Phase 1 & 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Phase 1 & 2
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Phase 1 & 2
No. of animals per sex per dose:
Phase 1: 3/sex/dose for 0, 150 and 300 mg/kg bw/day; 2/sex/dose for 500 mg/kg bw/day
Phase 2: 3/sex/dose for 0, 150 and 300 mg/kg bw/day; 4/sex/dose for 500 mg/kg bw/day
Control animals:
yes, concurrent vehicle
Details on study design:
As no adverse effects of treatment were observed at 500 mg/kg bw/day in Phase 1 after five days of dosing, which precluded further investigation of this dose level, the remaining required animals were assigned to each group in Phase 2 in order to form the full complement of six females at each dose level.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals and their cages were visually inspected at least twice daily for evidence of reaction to treatment or ill-health.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were performed daily during the treatment period to establish and confirm a pattern of signs in association with dosing at the following times during the day: Pre-dose observation; 10 to 20 minutes after the end of dosing each group; 1 to 2 hours after completion of dosing; As late as possible in the working day.
Physical examination were performed on Days 0, 6, 12, 18, 24 and 29 after mating

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during acclimatization; the weight of each adult was recorded on Days 0, 3 and 6-29 after mating.

FOOD CONSUMPTION: Yes
- All adult animals Daily from Day 1 after mating to termination.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Animals surviving until the end of the scheduled study period were killed on Day 29 after mating, by intravenous injection of sodium pentobarbitone.
Ovaries and uterine content:
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
Fetal examinations:
SACRIFICE: Method of kill for fetuses - Subcutaneous injection of sodium pentobarbitone

FETAL EXAMINATION:All fetuses and placentae were dissected from the uterus and weighed individually. Fetuses were individually identified within the litter, using a coding system based on their position in the uterus. Each placenta and fetus was externally examined and an internal examination of the neck and the contents of the thoracic and abdominal cavities of each fetus was performed and any abnormalities were recorded, sampled as appropriate and retained in appropriate fixative. The sex of each fetus was recorded. Grossly normal fetuses were discarded.
Statistics:
No statistical analyses were performed on this study.
Indices:
Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100
Post-implantation loss (%)= [(Number of implantations – Number of live fetuses) / Number of implantations] x 100
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs or signs observed following dose administration that were clearly attributable to treatment with Terpineol multiconstituent.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female No. 11 receiving 150 mg/kg bw/day was killed for reasons of animal welfare on Day 17 of gestation due to general poor clinical condition. This female had shown gradual and persistent weight loss of 290 grams between Days 12 and 17 of gestation, with a concomitant reduction in food consumption during the same period. Signs of pale faeces, reduced faecal pellet output, small faecal pellets, cold to touch, dull eyes (grey/bloodshot), dark muzzle, decreased activity and subdued behaviour were apparent from Day 15-17 of gestation, and thin build was also recorded on Day 17 of gestation. Uterine examination revealed eight implantation sites with six live embryos and two resorptions. Macroscopic examination revealed clear fluid and gelatinous material in the stomach. In the absence of any similar findings in other animals in this or higher dose groups, this premature death was considered incidental and unrelated to Terpineol multiconstituent administration.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Following the beginning of treatment on Day 6 of gestation, body weight gain for all groups of treated females was essentially similar to Control and considered unaffected by treatment. Gravid uterine weight and net bodyweight gain was essentially similar across all groups of
females.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean food consumption for females receiving Terpineol multiconstituent was slightly lower than Control in all treated groups from Day 6 to Day 16 of gestation, although in the absence of a clear dose response. Thereafter, from Day 17 of gestation mean food consumption was considered unaffected in all treated groups when compared to Controls.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination on Day 29 of gestation did not reveal any abnormalities in the dams.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
With the exception of one female at 150 mg/kg bw/day (No. 7) and one female at 500 mg/kg/day (No. 20), all females were pregnant with live young.
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean placental and fetal weights in all treated groups were lower than Control, although there was no dose response apparent. Control values were, however, influenced by two litters (No’s. 1 and 2) where only one live fetus was present, resulting in an atypically high mean fetal weight for those litters. When these two litters were excluded from the Control group mean values, there was no evidence of an effect of Terpineol multiconstituent on mean placental or fetal weights.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
Based on the results of this preliminary study, it was therefore concluded that the 500 mg/kg bw/day dose level would be suitable for use as the high dose level on the main rabbit embryo-fetal development study.
Executive summary:

A preliminary study was conducted to assess the influence of Terpineol multiconstituent on embryo-fetal survival and development in the New Zealand White rabbit and to establish suitable doses for a main embryo-fetal toxicity study. A cautious approach was taken, with the study conducted in two phases. Initially, in Phase 1 three females were allocated to each of Groups 1 to 3, and two females to Group 4. If no adverse effects of treatment were observed at 500 mg/kg bw/day in Phase 1 after 5 days of dosing which would preclude further investigation at this dose level, additional females were assigned to each group in Phase 2 of the study in order to form the full complement of six females at each dose level. If animals receiving 500 mg/kg bw/day in Phase 1 showed a marked adverse reaction to treatment no further animals were treated at this level.

Administration of terpineol multiconstituent to pregnant New Zealand White rabbits at dose levels up to and including 500 mg/kg bw/day was well tolerated, with no test item-related premature deaths, clinical signs or post-dosing signs observed. Mean food consumption in all groups of treated females was slightly lower than Control during Days 6-16 of gestation although in the absence of a dose response relationship; thereafter, mean food consumption was essentially similar in all groups of females. Body weight performance, gravid uterine weight and adjusted body weight gain were unaffected by Terpineol multiconstituent administration at all dose levels investigated, and there were no test item-related maternal or fetal macroscopic abnormalities detected at scheduled termination.

There was no effect of maternal treatment with terpineol multiconstituent on litter data, as assessed by the number of implantations, resorptions, live young, sex ratio and pre- and post-implantation losses. Placental, litter and fetal weights were similar in all groups.

Previous pilot studies in the rabbit showed that dose levels of terpineol multiconstituent above 500 mg/kg bw/day were not tolerated. Based on the results of this preliminary study, it was therefore concluded that the 500 mg/kg bw/day dose level would be suitable for use as the high dose level on the main rabbit embryo-fetal development study.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rabbit
Quality of whole database:
GLP study conducted according to OECD Guideline 414 without any deviation (Klimisch score = 1).
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a GLP-compliant prenatal developmental toxicity study performed according to OECD guideline 414, terpineol multiconstituent diluted in corn oil was administered by gavage to groups of mated female Sprague-Dawley rats (20 mated females/dose) at the dose levels of 0, 60, 200, 600 mg /kg bw/ day from Days 6 to 19 after mating. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Detailed observations were recorded daily at the following times in relation to dose administration. A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health. The weight of each adult was recorded on Days 0, 3 and and then daily from Days 6 to 20 after mating. 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 inclusive after mating.

On Day 20 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The gravid uterine weight, number of implantations, live and dead fetuses, early and late resorptions and corpora lutea were recorded. Gross evaluation of the placenta was also performed.

Fetuses were sexed, weighed and examined for external, soft tissue and skeletal malformations.

With the exception of one female in the 200 mg/kg bw/day group (No. 52) which had a total litter resorption, all females were found to be pregnant with live young at scheduled termination on Day 20 of gestation. No mortality was observed.

At scheduled termination on Day 20 of gestation, the adjusted mean liver weight of females receiving 600 mg/kg bw/day was significantly higher than Control (1.10X Control). There were no treatment-related macroscopic abnormalities detected. No relevant clinical signs or signs of reaction to treatment were noted in treated females. Females receiving 60 or 200 mg/kg bw/day showed no treatment-related changes in clinical condition, body weight performance, food intake, liver weight or macropathology.

There was no effect of maternal treatment with terpineol multiconstituent at any dose level investigated on litter data. Sex ratio, as assessed by the percentage of males per litter, was generally comparable in all groups and in line with expectations. Embryo-fetal growth was slightly reduced by maternal treatment at 600 mg/kg bw/day.

It was considered that there was no adverse effect of maternal treatment on embryo-fetal development; the incidence of major and minor abnormalities and skeletal variants showed no relationship to maternal treatment with terpineol multiconstituent.

In the 600 mg/kg bw/day group there was a slightly higher incidence of incompletely ossified or unossified 5th and/or 6th sternebrae compared to concurrent control and the Historical Control Data range.

It was considered that this minor finding did not constitute an adverse effect on development.

On the basis of the results obtained in this study, the dosage of 600 mg/kg bw/day was considered to be the NOAEL (No Observed Adverse Effect Level) for maternal toxicity. The NOAEL for developmental toxicity was considered to be 600 mg/kg bw/day.

In a study conducted according to OECD guideline 414 and in GLP conditions, three groups of 22 females received terpineol multiconstituent at doses of 125, 250 or 500 mg/kg bw/day by oral gavage administration, from Day 6 to 28 after mating at a volume dose of 5 mL/kg body weight. A similarly constituted Control group received the vehicle, 1% methylcellulose, over the same period and at the same volume dose as the treated groups.

Animals were killed on Day 29 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 29 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

Administration of terpineol multiconstituent to pregnant New Zealand White rabbits at dose levels up to and including 500 mg/kg bw/day was well tolerated, with no test item-related premature deaths, clinical signs or post-dosing signs observed.

Females given 500 mg/kg bw/day showed slight mean body weight loss during Days 6-9 of gestation, and a slight reduction in mean food consumption from Day 6 to Day 20 of gestation; these differences from Control were not adverse.

Mean gravid uterine weight and adjusted body weight gain was unaffected by terpineol multiconstituent administration at all dose levels investigated, and there were no test item-related maternal abnormalities detected at scheduled termination.

There was no effect of maternal treatment with terpineol multiconstituent on litter data, as assessed by the number of implantations, resorptions, live young, sex ratio and pre- and post-implantation losses. Placental, litter and fetal weights were similar in all groups.

The incidence of major and minor fetal abnormalities and skeletal variants showed no relationship to maternal treatment with Terpineol multiconstituent.

Based on the results obtained in this study of embryo-fetal development, it was concluded that the high dose level of 500 mg/kg bw/day represented the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and for embryo-fetal survival, growth and development.


Toxicity to reproduction: other studies

Additional information

The systemic toxic potential, including reproductive effects, of terpineol multiconstituent was assessed in Sprague Dawley rats by oral gavage administration over a period of five weeks at doses of 0, 60, 250 or 750 mg/kg/day and according to OECD guideline 422. This study indicated that the kidneys, the liver and the testes were the main target organs.

A significant dose-related increase in liver weight was reported in both males and females. Minimal centrilobular hepatocyte hypertrophy was seen in the liver of three toxicity phase females receiving 750 mg/kg/day of Terpineol and accounted for the increases in liver weight at necropsy. Other biochemical findings in this study, such as bile acids and cholesterol levels in females at 750 mg/kg/day may also indicate an alteration of the metabolic function of the liver following administration of Terpineol. However, the changes in liver weight and histopathology findings showed complete recovery after 2 weeks.

Testis weight was markedly low compared to control in males receiving 750 mg/kg/day and there was also an indication of low epididymal weights at this dose.Reduced numbers or complete absence of spermatozoa accompanied by the presence of degenerate spermatogenic cells in ducts were observed in the epididymides of males receiving 750 mg/kg/day at the end of the 5-week dosing period. None of these effects were observed at 250 mg/kg/day.

A new comparative study was performed to investigate the potential toxic effect of terpineol multiconstituent when administered by diet versus by gavage over a period of 28 days. Similar effects on the liver were reported in all treated animals whatever the route of administration (i.e. marked dose-related increase in liver weight). On the contrary, effects on testis weight and sperm motility were observed in the gavage group, but no effects were observed on these parameters in animals exposed to the same dose level by diet. There are therefore strong arguments to consider that the testicular effects observed at 750  mg/kg/day after gavage administration were due to a peak effect of terpineol multiconstituent and are unlikely to occur, even at the highest achievable dose, by either inhalation, dermal or oral route (in the food).

The gavage administration is a normal way to evaluate drug toxicity (which can be administered as one dose/day) or a convenient surrogate (no need to mix with diet) to evaluate toxicity which could occur due to the presence of the substance in food or to extrapolate to different routes of exposure (dermal or inhalation). However, in some cases, it creates pharmacokinetic (and then pharmacodynamic) circumstances which cannot be encountered in real conditions of exposure and can be considered in this case as a non-relevant route of exposure (as would be intravenous or intraperitoneal modes of administration).

In the recent in vivo toxicokinetic study, results obtained after administration of single doses of terpineol multiconstituent revealed the non-linear pharmacokinetics of alpha-terpineol. The AUC values at the highest dose level (750 mg/kg) were ~2.3-fold higher than the values predicted from a linear relationship. These results indicate a systemic overexposure to alpha-terpineol and its metabilites after gavage administration. Further investigations after repeated dosing should confirm this effect and would probably indicate a much higher systemic exposure at 750 mg/kg/day than the one obtained after a single dose. 

Based on the complementary information provided in the dossier, the driving hypothesis is that the saturation of alpha-terpineol metabolism led to disproportionately high systemic concentrations that should be considered as unrelated to human exposure, especially after gavage administrations of high dose of terpineol multiconstituent (750 mg/kg/day). After single dose of terpineol multiconstituent, the AUCs increased more than dose-proportionally, leading to much higher systemic exposure than expected. This should be considered as non-relevant plasmatic concentrations compared to real conditions of exposure.

Moreover, a 90-day inhalation study, requested by ECHA, was conducted at dose levels up to 2.23 mg/L (corresponding to an estimated achieved dose of 566 mg/kg/day) and no toxicity was reported in male reproductive organs at top dose.

Justification for classification or non-classification

Based on findings reported above on the effect on male fertility, and taking into account recent data on toxicokinetics, it is considered that there is strong evidence that no reproductive effects are likely to occur by the possible routes of human exposure:

-         The effects on testes are reported at top dose (750 mg/kg/day) and only after gavage administration.

-         No effects on testes were observed by dietary administration while the effects on the liver were reported after both gavage and diet administrations.

- No effects on male reproductive organs were reported after 90 days exposure by inhalation at top dose (2.23 mg/L, corresponding to an estimated achieved dose of 566 mg/kg/day).

-         Oral gavage with terpineol multiconstituent has shown to result for alpha-terpineol in

o  higher peak in plasma (Cmax)

o  higher area under concentration-time curve in plasma (AUC)

In the context of terpineol multiconstituent dossier, oral gavage at high dose clearly resulted in much higher systemic exposure than expected, leading to biologically non-relevant effects that should not be considered for classification purposes.

Based on the available data, classification for reproductive effects is then not warranted at this stage.