Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No.: 104636
Purity: 74.2% (sum of the three main constituents)
Name of test material (as cited in study report): TERPINEOL
Physical state: colourless liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 01 March 2012

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

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)).


Effect levels (P0)

open allclose all
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.

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.