Reaction mass of 1-methyl-4-(1-methylethylidene)cyclohexyl acetate and p-menth-1-en-8-yl acetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

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:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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: 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: 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: 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 multi 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 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. Offspring were not dosed.

Frequency of treatment:

Once a day, 7 days a week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

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).

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):

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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no statistically significant effects of Terpineol multi on body weight or body weight 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 body weight 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 body weight although gains were slightly lower than Control, and during lactation body weight gain of females receiving 250 mg/kg/day were lower than Control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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 and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

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 during the dosing period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no marked effects of the substance on any of the haematology parameters assessed.
During week 5 of dosing, haematocrit, haemoglobin concentrations haematocrit and red blood cell counts were slightly, but significantly lower than Control in females receiving 750 mg/kg/day. Statistical significance (p<0.05) was also attained for female haemoglobin concentrations in the low and intermediate dose groups but all values were within background range (90 percentile range 13.2-16.0 (n=168)) and no dose-related trend was apparent suggesting minimal biological importance. By Week 2 of recovery, all previously affected parameters were essentially similar to those of the Controls, indicating complete recovery.
During recovery, males that had previously received 750 mg/kg/day had low haematocrit haemoglobin concentrations and red blood cell counts. Although statistically significant, these slight changes are within the background spectrum and not considered to be of biological significance, particularly because they were not affected during the dosing period, (Background 90 percentile ranges, Hct, 0.378-0.475; Hb, 13.3-16.2; RBC, 7.07-8.81; MCHC, 32.9-36.5 (n=268)).
Prothrombin clotting times were statistically longer than Control for recovery males previously receiving 750 mg/kg/day. These slight changes are considered not to be of biological significance as they were not affected during the dosing period and may be associated with the slightly low platelet counts for these animals.
All other inter-group differences from Controls were minor or lacked dose-relationship and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

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)).
Other findings that gained statistical significance were considered minor in nature, lacked dose relationship and were therefore not considered to be of toxicological significance, this includes the increase in alkaline phosphatase levels in males dosed at 250 and 750 mg/kg/day and the decrease in calcium levels during Week 5 of dosing for males receiving 750 mg/kg/day.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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 values for Toxicity subgroup animals were similar to those for Controls, and considered unaffected by treatment.
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.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: Minimal centrilobular hepatocyte hypertrophy was seen in the liver of three toxicity phase females dosed with Terpineol multi at 750 mg/kg/day. However, these histopathology findings showed complete recovery after 2 weeks without dosing.
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 multi 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.
See table 4 in 'Any other information on results incl. tables'.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect of Terpineol multi on oestrous cycles or precoital interval.

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, whilst spermatocele granuloma(ta) were observed in 2 animals receiving 750 mg/kg/day and in one animal receiving 60 mg/kg/day.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg/day, although all animals had positive evidence of mating none of the females had any evidence of pregnancy 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

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: Males receiving 750 mg/kg/day showed evidence of testicular and epididymal toxicity leading to infertility.

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: Liver showed centrilobular hepatocyte hypertrophy at 750 mg/kg/day indicating an alteration of the metabolic function of the liver following administration. This is considered to be an adaptive response.

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: Males receiving 750 mg/kg/day showed evidence of testicular and epididymal toxicity leading to infertility. These effects seen are discussed together with additional testing information.

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Impairment of male fertility at 750 mg/kg/day prevented the assessment of effects on female reproduction at 750 mg/kg/day.

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

male reproductive system

Organ:

seminiferous tubules

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs of offspring did not indicate any reaction to maternal exposure to the substance.

Mortality / viability:

no mortality observed

Description (incidence and severity):

Administration with Terpineol multi had no effect on post implantation survival index, live birth index and viability index for animals receiving up to 250 mg/kg/day.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Male and female offspring bodyweights were not affected by the substance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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 the substance.

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Generation:

F1

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Impairment of male fertility at 750 mg/kg/day prevented the assessment of effects on female reproduction at 750 mg/kg/day.

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

no

Table 1: Organ weights - group mean unadjusted and adjusted values (g) after 5 weeks

Dose group (mg/kg/day)	Kidneys	Liver	Adrenals	Testes
Males	Unadjusted means
Control	3.456±0.280	20.627±1.135	0.058 ± 0.008	3.548 ± 0.233
60	3.449 ± 0.257	21.042 ± 2.085	0.062 ± 0.009	3.637 ± 0.210
250	3.607 ± 0.354	21.509 ± 1.900	0.057 ± 0.006	3.418 ± 0.372
750	3.873 ± 0.286	24.067 ± 1.858	0.063 ± 0.007	2.013 ± 0.623
	Adjusted means
Control	3.411	20.299	0.058	3.517
60	3.390	20.602	0.062	3.596
250	3.644	21.784	0.057	3.445
750	3.950**	24.637**	0.064	2.067**
Females	Unadjusted means
Control	1.911 ± 0.252	10.293 ± 1.153	0.062 ± 0.013
60	1.882 ± 0.142	10.038 ± 1.300	0.066 ± 0.007
250	1.979 ± 0.189	11.547 ± 1.218	0.067 ± 0.007
750	1.957 ± 0.084	13.311 ± 1.283	0.076 ± 0.011
	Adjusted means
Control	1.871	10.027	0.061
60	1.944	10.456	0.068
250	1.902	11.034	0.065
750	2.011	13.672**	0.078*

Table 2: Organ weights - group mean unadjusted and adjusted values (g) after 2 weeks of recovery

Dose group (mg/kg/day)	Kidneys	Liver	Adrenals	Testes	Epididymides
Males	Unadjusted means
Control	1.262 ± 0.022	23.321 ± 2.798	0.056 ± 0.007	3.877 ± 0.288	3.801 ± 0.316
750	0.898 ± 0.075	21.456 ± 1.059	0.061 ± 0.004	1.741 ± 0.152	3.517 ± 0.465
	Adjusted means
Control	3.786	22.908	0.055	3.879	1.269
750	3.532	21.869	0.062	1.739**	0.891**
Females	Unadjustedmeans
Control	2.029 ± 0.074	11.638 ± 1.268	0.061 ± 0.009
750	1.917 ± 0.265	10.348 ± 1.155	0.069 ± 0.012
	Adjustedmeans
Control	1.991	11.506	0.058
750	1.955	10.480	0.072

Table 3: material-related macroscopic findings in male rats

	After treatment				After recovery
Group	Control	60	250	750	Control	750
Tissue and finding	5	10	10	5	5	5
Testes
Blue	0	0	0	3	0	1
Flaccid	0	0	0	1	0	1
Small	0	0	2	5	0	5
Epididymides
Enlarged	0	0	0	1	-	-
Mass(es)	0	1	0	2	-	-
Small	0	0	0	3	0	5

Table 4: summary of material-related microscopic findings in male rats

	After treatment				After recovery
Group	Control	60	250	750	Control	750
Tissue and finding
Testes
Seminiferous Tubular Atrophy/Degeneration
Minimal	1	0	0	0	0	0
Slight	0	0	0	0	0	0
Moderate	0	0	0	1	0	2
Marked	0	0	0	2	0	3
Severe	0	0	0	2	0	0
Total	1	0	0	5	0	5
Seminiferous Tubular Vacuolation
Minimal	1	0	0	4	0	1
Slight	0	0	0	1	0	0
Total	1	0	0	5	0	1
Spermatid Giant Cells
Minimal	0	0	0	2	0	2
Slight	0	0	0	2	0	2
Moderate	0	0	0	1	0	0
Total	0	0	0	5	0	4
Epididymides
Spermatozoa Absent	0	0	0	4	0	3
Marked reduced numbers of spermatozoa	0	0	0	1	0	2
Degenerate spermatogenic cells in duct(s)
Slight	0	0	0	0	0	4
Moderate	0	0	0	5	0	1
Spermatocele Granuloma
Minimal	0	1	0	1	-	-
Moderate	0	0	0	1	-	-
Kidneys
Cortical Tubules with Hyaline Droplets
Minimal	0	0	5	2
Slight	0	0	1	0
Moderate	0	0	0	3
Total	0	0	6	5	0	1
Granular Casts
Minimal	0	0	0	1
Slight	0	0	0	1
Total	0	0	0	2	-	-
Cortical Tubular Basophilia
Minimal	2	4	3	1
Slight	0	0	3	1
Moderate	0	0	0	1
Total	2	4	6	3	4	4

Conclusions:

Under the conditions of the test (OECD 422, GLP), the systemic NOAEL for males and unmated females was 250 mg/kg/day, the NOAEL for maternal and developmental toxicity was at least 250 mg/kg/day.

Executive summary:

In a GLP study conducted according to OECD TG 422 and in compliance with GLP, three groups, each comprising ten male and ten female rats for the Main (reproductive) phase (exception: five males at control and top dose; additional Recovery phase males were also used for pairing with Main reproductive phase females) and five female rats for the Toxicity phase received Terpineol-Multi 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 750 mg/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-Multi upon haematology parameters. Females showed slight anaemia but males were essentially unaffected. At the end of the two week recovery period, no intergroup differences were present in females whereas haematocrit, haemoglobin and red blood cell count were slightly decreased 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, except for urea and creatinine, showed complete recovery after 2 week recovery period. There were no effects of Terpineol-Multi 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 Terpineol-Multi. 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-Multi but testis and epididymal weights showed no evidence of recovery. Adaptive centrilobular hepatocyte hypertrophy in the liver of females dosed with Terpineol-Multi 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. 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 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-Multi 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 systemic NOAEL for males and unmated females was 250 mg/kg/day, the NOAEL for maternal and developmental toxicity was at least 250 mg/kg/day.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From April 28 to September 03, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for read-across to Terpinyl Acetate multi

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system 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: 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: 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 and 50 mg/mL
- Amount of vehicle: constant dosage-volume of 5 mL/kg bw/day

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 multi in test formulations analysed for the study were within applied limits, +10%/-15% of nominal concentrations, confirming accurate formulation.

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
- 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.

Duration of treatment / exposure:

Toxicity phase females were dosed daily for a minimum of five consecutive weeks. 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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

- Reproductive subgroup (main phase): 10 females/dose
- Toxicity subgroup: 5 females/dose

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.

Maternal 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 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 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 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 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 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 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 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).

- SACRIFICE:
Toxicity phase females were killed in Week 6 after completion of the Week 5 investigations. 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 Toxicity phase female, Main phase females that did not litter (Group 7) and those animals killed or dying prematurely were fixed for histopathology: Adrenal glands, Brain, Pituitary, Caecum, Colon, Rectum, Sciatic nerves, Duodenum, Skeletal muscle, Skin, Mammary glands (inguinal area), Heart, Spinal cord, Ileum, Spleen, Jejunum, Sternum with marrow, Kidneys, Stomach, Liver, 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.

Fetal examinations:

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.

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 estimates an exact two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores, was applied to all groups.

Indices:

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):

On the first two days of dosing most of the females 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 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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the 60 mg/kg/day dose group, 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.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant effects on bodyweight or bodyweight gain. 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.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no substance 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 and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

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 during the dosing period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no marked effects of the substance on any of the haematology parameters assessed.
During week 5 of dosing, haematocrit, haemoglobin concentrations haematocrit and red blood cell counts were slightly, but significantly lower than Control in females receiving 750 mg/kg/day. Statistical significance (p<0.05) was also attained for female haemoglobin concentrations in the low and intermediate dose groups but all values were within background range (90 percentile range 13.2-16.0 (n=168)) and no dose-related trend was apparent suggesting minimal biological importance. By Week 2 of recovery, all previously affected parameters were essentially similar to those of the Controls, indicating complete recovery.
All other inter-group differences from Controls were minor or lacked dose-relationship and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

During Week 5 of study, urea and creatinine levels were significantly higher in females receiving 750 mg/kg/day. By Week 2 of recovery urea and creatinine levels were still higher than Control for females previously receiving 750 mg/kg/day, attaining statistical significance.
Glucose plasma levels were significantly higher than Control in females dosed at 250 and 750 mg/kg/day but were similar to Control after dosing ceased.
Potassium levels were significantly higher than Control in 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)).
Other findings that gained statistical significance were considered minor in nature, lacked dose relationship and were therefore not considered to be of toxicological significance.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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 values for Toxicity subgroup animals were similar to those for Controls, and considered unaffected by treatment.
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.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 5 weeks of dosing body weight adjusted mean liver weights of females receiving 750 mg/kg/day were significantly increased. After two weeks without dosing liver weights were no longer enlarged.
There were no adverse effects on females killed on Day 7 of lactation. Adjusted mean ovary weights for females dosed at 250 mg/kg/day and killed on Day 7 of lactation, were significantly greater than Control values this but this is considered to be of no toxicological significance.
See tables 1 and 2 in 'Any other information on results incl. tables'.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no significant necropsy findings for females on Day 7 of lactation.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: Minimal centrilobular hepatocyte hypertrophy was seen in the liver of three toxicity phase females dosed with Terpineol multi at 750 mg/kg/day. However, these histopathology findings showed complete recovery after 2 weeks without dosing.

Histopathological findings: neoplastic:

no effects observed

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

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. These observations suggest that pregnancy failure occurred before implantation and probably indicates fertilisation failure as the cause.

Other effects:

no effects observed

Description (incidence and severity):

REPRODUCTIVE FUNCTION (OESTROUS CYCLE) AND REPRODUCTIVE PERFORMANCE:
There was no effect of on oestrous cycles or precoital interval. There was no effect on mating performance or fertility at dose levels of 250 mg/kg/day or below.

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: Liver showed centrilobular hepatocyte hypertrophy at 750 mg/kg/day indicating an alteration of the metabolic function of the liver following administration. This is considered to be an adaptive response.

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Impairment of male fertility at 750 mg/kg/day prevented the assessment of effects on female reproduction at 750 mg/kg/day.

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Male and female offspring bodyweights were considered to be unaffected by the substance.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Administration with the substance had no effect on post implantation survival index, live birth index and viability index for animals receiving up to 250 mg/kg/day.

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

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 at a dose level of 60 mg/kg/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The numbers of implantations, total litter sizes and live litter sizes up to Day 7 of lactation were unaffected in the 60 and 250 mg/kg/day groups.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Administration with the substance had no effect on post implantation survival index, live birth index and viability index for animals receiving up to 250 mg/kg/day.

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Impairment of male fertility at 750 mg/kg/day prevented the assessment of effects on female reproduction at 750 mg/kg/day.

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 1: Organ weights - group mean unadjusted and adjusted values (g) after 5 weeks

Dose group (mg/kg/day)	Kidneys	Liver	Adrenals
Females	Unadjustedmeans
Control	1.911 ± 0.252	10.293 ± 1.153	0.062 ± 0.013
60	1.882 ± 0.142	10.038 ± 1.300	0.066 ± 0.007
250	1.979 ± 0.189	11.547 ± 1.218	0.067 ± 0.007
750	1.957 ± 0.084	13.311 ± 1.283	0.076 ± 0.011
	Adjustedmeans
Control	1.871	10.027	0.061
60	1.944	10.456	0.068
250	1.902	11.034	0.065
750	2.011	13.672**	0.078*

Table 2: Organ weights - group mean unadjusted and adjusted values (g) after 2 weeks of recovery

Dose group (mg/kg/day)	Kidneys	Liver	Adrenals
	Adjusted means
Control	3.786	22.908	0.055
750	3.532	21.869	0.062
Females	Unadjustedmeans
Control	2.029 ± 0.074	11.638 ± 1.268	0.061 ± 0.009
750	1.917 ± 0.265	10.348 ± 1.155	0.069 ± 0.012
	Adjustedmeans
Control	1.991	11.506	0.058
750	1.955	10.480	0.072

Conclusions:

Under the conditions of the test (OECD 422, GLP), the NOAEL for maternal and developmental toxicity was at least 250 mg/kg bw/day.

Executive summary:

In a GLP study conducted according to OECD TG 422 and in compliance with GLP, three groups, each comprising ten female rats for the Main (reproductive) phase and five female rats for the Toxicity phase received Terpineol-Multi at doses of 60, 250 or 750 mg/kg bw/day at a dose volume of 5 mL/kg bw/day. Toxicity phase females were dosed daily for a minimum of five consecutive weeks. An additional 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 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 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, were observed briefly during Week 1 in animals receiving 750 mg/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 unmated females receiving up to 750 mg/kg/day. 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 but visual assessment of water consumption indicated that 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-Multi upon haematology parameters. Females showed slight anaemia. At the end of the two week recovery period, no intergroup differences were present in females. At 750 mg.kg/day, urea and creatinine levels were significantly higher than Controls in females. Glucose plasma levels were significantly higher than Control in females dosed at 250 and 750 mg/kg/day. Potassium levels were significantly higher than Control in females receiving 750 mg/kg/day. All the above discussed parameters, except for urea and creatinine, showed complete recovery after 2 week recovery period. There were no effects of Terpineol-Multi 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 Terpineol-Multi. At 750 mg/kg/day, relative liver weights were significantly higher than Control in females. Liver weights returned to normal after two weeks when the animals did not receive Terpineol-Multi. Adaptive centrilobular hepatocyte hypertrophy in the liver of females dosed with Terpineol-Multi at 750 mg/kg/day was not present after 2 weeks recovery. Based on the findings in this study, the NOAEL for maternal and developmental toxicity was at least 250 mg/kg/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of α,α-4-trimethyl-(1S)-3-cyclohexene-1-methanol and α,α-4-trimethyl-(1R)-3-cyclohexene-1-methanol and 1-methyl-4-(1-methylethylidene)-cyclohexanol

EC Number:

701-188-3

Molecular formula:

C10H18O

IUPAC Name:

Reaction mass of α,α-4-trimethyl-(1S)-3-cyclohexene-1-methanol and α,α-4-trimethyl-(1R)-3-cyclohexene-1-methanol and 1-methyl-4-(1-methylethylidene)-cyclohexanol

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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: 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: 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 oral 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: constant dosage-volume of 5 mL/kg bw/day

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 multi 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 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. Offspring were not dosed.

Frequency of treatment:

Once a day, 7 days a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

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 body weight 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 were 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

Observations and examinations performed and frequency:

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).

Sacrifice and pathology:

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.

GROSS NECROPSY:
All animals were subject to a detailed necropsy.

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.

Statistics:

The following sequence of statistical tests was used for grip strength, motor activity, bodyweight, food consumption, organ weight, 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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no statistically significant effects on body weight or body weight 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 body weight 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 body weight although gains were slightly lower than Control, and during lactation body weight gain of females receiving 250 mg/kg/day were lower than Control.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

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 and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

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 during the dosing period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no marked effects of the substance on any of the haematology parameters assessed.
During week 5 of dosing, haematocrit, haemoglobin concentrations haematocrit and red blood cell counts were slightly, but significantly lower than Control in females receiving 750 mg/kg/day. Statistical significance (p<0.05) was also attained for female haemoglobin concentrations in the low and intermediate dose groups but all values were within background range (90 percentile range 13.2-16.0 (n=168)) and no dose-related trend was apparent suggesting minimal biological importance. By Week 2 of recovery, all previously affected parameters were essentially similar to those of the Controls, indicating complete recovery.
During recovery, males that had previously received 750 mg/kg/day had low haematocrit haemoglobin concentrations and red blood cell counts. Although statistically significant, these slight changes are within the background spectrum and not considered to be of biological significance, particularly because they were not affected during the dosing period, (Background 90 percentile ranges, Hct, 0.378-0.475; Hb, 13.3-16.2; RBC, 7.07-8.81; MCHC, 32.9-36.5 (n=268)).
Prothrombin clotting times were statistically longer than Control for recovery males previously receiving 750 mg/kg/day. These slight changes are considered not to be of biological significance as they were not affected during the dosing period and may be associated with the slightly low platelet counts for these animals.
All other inter-group differences from Controls were minor or lacked dose-relationship and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

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)).
Other findings that gained statistical significance were considered minor in nature, lacked dose relationship and were therefore not considered to be of toxicological significance, this includes the increase in alkaline phosphatase levels in males dosed at 250 and 750 mg/kg/day and the decrease in calcium levels during Week 5 of dosing for males receiving 750 mg/kg/day.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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 values for Toxicity subgroup animals were similar to those for Controls, and considered unaffected by treatment.
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.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 5 weeks of dosing body weight 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. See tables 1 and 2 in 'Any other information on results incl. tables'.

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. 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. See table 3 in 'Any other information on results incl. tables'.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: Minimal centrilobular hepatocyte hypertrophy was seen in the liver of three toxicity phase females dosed with Terpineol multi at 750 mg/kg/day. However, these histopathology findings showed complete recovery after 2 weeks without dosing.
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 multi 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.
See table 4 in 'Any other information on results incl. tables'.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: Males receiving 750 mg/kg/day showed evidence of testicular and epididymal toxicity leading to infertility.

2

Dose descriptor:

NOAEL

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: Liver showed centrilobular hepatocyte hypertrophy at 750 mg/kg/day indicating an alteration of the metabolic function of the liver following administration. This is considered to be an adaptive response.

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

male reproductive system

Organ:

seminiferous tubules

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

Table 1: Organ weights - group mean unadjusted and adjusted values (g) after 5 weeks

Dose group (mg/kg/day)	Kidneys	Liver	Adrenals	Testes
Males	Unadjusted means
Control	3.456±0.280	20.627±1.135	0.058 ± 0.008	3.548 ± 0.233
60	3.449 ± 0.257	21.042 ± 2.085	0.062 ± 0.009	3.637 ± 0.210
250	3.607 ± 0.354	21.509 ± 1.900	0.057 ± 0.006	3.418 ± 0.372
750	3.873 ± 0.286	24.067 ± 1.858	0.063 ± 0.007	2.013 ± 0.623
	Adjusted means
Control	3.411	20.299	0.058	3.517
60	3.390	20.602	0.062	3.596
250	3.644	21.784	0.057	3.445
750	3.950**	24.637**	0.064	2.067**
Females	Unadjusted means
Control	1.911 ± 0.252	10.293 ± 1.153	0.062 ± 0.013
60	1.882 ± 0.142	10.038 ± 1.300	0.066 ± 0.007
250	1.979 ± 0.189	11.547 ± 1.218	0.067 ± 0.007
750	1.957 ± 0.084	13.311 ± 1.283	0.076 ± 0.011
	Adjusted means
Control	1.871	10.027	0.061
60	1.944	10.456	0.068
250	1.902	11.034	0.065
750	2.011	13.672**	0.078*

Table 2: Organ weights - group mean unadjusted and adjusted values (g) after 2 weeks of recovery

Dose group (mg/kg/day)	Kidneys	Liver	Adrenals	Testes	Epididymides
Males	Unadjusted means
Control	1.262 ± 0.022	23.321 ± 2.798	0.056 ± 0.007	3.877 ± 0.288	3.801 ± 0.316
750	0.898 ± 0.075	21.456 ± 1.059	0.061 ± 0.004	1.741 ± 0.152	3.517 ± 0.465
	Adjusted means
Control	3.786	22.908	0.055	3.879	1.269
750	3.532	21.869	0.062	1.739**	0.891**
Females	Unadjusted means
Control	2.029 ± 0.074	11.638 ± 1.268	0.061 ± 0.009
750	1.917 ± 0.265	10.348 ± 1.155	0.069 ± 0.012
	Adjusted means
Control	1.991	11.506	0.058
750	1.955	10.480	0.072

Table 3: material-related macroscopic findings in male rats

	After treatment				After recovery
Group	Control	60	250	750	Control	750
Tissue and finding	5	10	10	5	5	5
Testes
Blue	0	0	0	3	0	1
Flaccid	0	0	0	1	0	1
Small	0	0	2	5	0	5
Epididymides
Enlarged	0	0	0	1	-	-
Mass(es)	0	1	0	2	-	-
Small	0	0	0	3	0	5

Table 4: summary of material-related microscopic findings in male rats

	After treatment				After recovery
Group	Control	60	250	750	Control	750
Tissue and finding
Testes
Seminiferous Tubular Atrophy/Degeneration
Minimal	1	0	0	0	0	0
Slight	0	0	0	0	0	0
Moderate	0	0	0	1	0	2
Marked	0	0	0	2	0	3
Severe	0	0	0	2	0	0
Total	1	0	0	5	0	5
Seminiferous Tubular Vacuolation
Minimal	1	0	0	4	0	1
Slight	0	0	0	1	0	0
Total	1	0	0	5	0	1
Spermatid Giant Cells
Minimal	0	0	0	2	0	2
Slight	0	0	0	2	0	2
Moderate	0	0	0	1	0	0
Total	0	0	0	5	0	4
Epididymides
Spermatozoa Absent	0	0	0	4	0	3
Marked reduced numbers of spermatozoa	0	0	0	1	0	2
Degenerate spermatogenic cells in duct(s)
Slight	0	0	0	0	0	4
Moderate	0	0	0	5	0	1
Spermatocele Granuloma
Minimal	0	1	0	1	-	-
Moderate	0	0	0	1	-	-
Kidneys
Cortical Tubules with Hyaline Droplets
Minimal	0	0	5	2
Slight	0	0	1	0
Moderate	0	0	0	3
Total	0	0	6	5	0	1
Granular Casts
Minimal	0	0	0	1
Slight	0	0	0	1
Total	0	0	0	2	-	-
Cortical Tubular Basophilia
Minimal	2	4	3	1
Slight	0	0	3	1
Moderate	0	0	0	1
Total	2	4	6	3	4	4

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test (OECD 422, GLP), the NOAEL was determined to be 250 mg/kg bw/day for males and unmated females.

Executive summary:

In the combined repeated dose toxicity study with reproduction/developmental toxicity screening test, conducted following GLP guidelines and OECD guideline 422, three groups, each comprising of ten male and ten female rats for the Main (reproductive) phase and five female rats for the Toxicity phase received Terpineol-Multi by gavage 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. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose. 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. During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (visual), haematology, blood chemistry. 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 body weight 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 750 mg/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 body weight 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. Body weight during the recovery phase was similar to Controls. Body weight and body weight 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-Multi upon haematology parameters. Females showed slight anaemia but males were essentially unaffected. At the end of the two week recovery period, no intergroup differences were present in females whereas haematocrit, haemoglobin and red blood cell count were slightly decreased 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, except for urea and creatinine, showed complete recovery after 2 week recovery period. 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-Multi but testis and epididymal weights showed no evidence of recovery. Adaptive centrilobular hepatocyte hypertrophy in the liver of females dosed with Terpineol-Multi 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. 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 and there was no other degenerative change 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-Multi 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/kg bw/day.