Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: 20 week repeated dose toxicity study
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reliability 2 is assigned in view of its use for read across and because not all fertility parameters have been measured in the 20 week repeated dose study of alpha-Terpinyl Acetate
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
other: OECD TG 408
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Osborne-Mendel
Route of administration:
oral: feed
Details on exposure:
See repeated dose section
Details on mating procedure:
There is information on male and female gonads.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
20 weeks
Frequency of treatment:
daily
Details on study schedule:
See repeated dose section.
No. of animals per sex per dose:
See repeated dose section
Details on study design:
See repeated dose section.
Parental animals: Observations and examinations:
See repeated dose section.
Oestrous cyclicity (parental animals):
No
Sperm parameters (parental animals):
No
Litter observations:
No
Postmortem examinations (offspring):
No
Statistics:
No
Reproductive indices:
No
Offspring viability indices:
No
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
Only when macroscopic effects were seen.
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
not specified
Dose descriptor:
NOAEL
Effect level:
>= 314 mg/kg bw/day (actual dose received)
Based on:
other: The result is converted from alpha-Terpinyl Acetate into alpha-Terpineol using their molecular weights.
Sex:
male/female
Basis for effect level:
other: The animals were dosed via the diet with alpha-Terpinyl Acetate: 1000, 2500 and 10000 ppm, resulting in 400 mg/kg bw
Clinical signs:
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Reproductive effects observed:
not specified
Conclusions:
The NOAEL for fertility from this test is >= 400 mg/kg bw for alpha-Terpinyl Acetate, resulting in 314 mg/kg bw for alpha-Terpineol when using a correction for molecular weight (154/196)
Executive summary:

In repeated dose dietary study of 20 weeks, similar to OECD TG 408, with alpha-Terpinyl Acetate no effect on male and female gonads were seen at the highest dose of 400 mg/kg bw, resulting in a NOAEL of >= 400 mg/kg bw, resulting in a NOAEL for alpha-Terpineol of >=314 mg/kg bw when using a correction for molecular weight (154/196).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat
Quality of whole database:
Three studies are available and needed for assessing the fertility of alpha-Terpineol. Together these fulfill the requirement for this endpoint.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

For the assessment of fertility of alpha-Terpineol data from alpha-Terpinyl Acetate and of Terpineol multi have been used. The read across documentation is presented in the repeated dose section.

For alpha-Terpinyl Acetate a 20 week dietary study is available in which no effects on male and female gonads were seen.

In a reproscreen study (OECD TG 421, being extracted from OECD TG 422) with Terpineol multi (a multi-constituent substance with alpha-Terpineol as its main constituent and gamma-Terpineol as the minor constituent, see the toxico-kinetic section) testicular effects were seen at gavage dosing of 750 mg/kg bw. The testicular effects seen in the study with Terpineol multi are considered to be due to peak exposures which cause an overloading of the metabolic pathway. This reasoning is supported by: 1) testing with Terpineol multi has shown that the testicular effects are only seen during gavage but not during dietary application and; 2) in 20 -week feeding study no testicular effects were seen with alpha-Terpinyl Acetate, which is fully metabolised in alpha-Terpineol (see toxico-kinetic section). This latter study covers a longer exposure duration compared to the OECD TG 421 (20 versus 6 weeks). The other main constituent of Terpineol multi: gamma-Terpineol, is expected to have similar effects because the metabolic pathway will be similar to that of alpha-Terpineol, and therefore the results of Terpineol multi can be used both for alpha-Terpineol and gamma-Terpineol.

A summary of the studies are presented here.

Key study: A summary of the absence of effects for alpha-Terpinyl Acetate is presented in the repeated dose toxicity section. This shows that no effects on male and female gonads are seen and shows that the substance does present a concern for fertility.

Supporting study 1A: There is a GLP study conducted according to OECD guideline 421 (extracted from an OECD TG 422) with Terpineol multi. The methodology of this study is also presented at the repeated dose toxicity section. The focus here will be on the fertility parameters.

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 (Thacker 2010a). 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 doserelated increases in postdosing 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. 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 2week 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 2week recovery period but at a lower incidence and severity suggesting a degree of recovery. Based on the findings in this study, the NOAEL for males for fertility is 250 mg/kg/day, the NOAEL for non-pregnant females for fertility is >= 750 mg/kg bw based on the available parameters at this dose (females were not pregnant due to infertility of the males).

Supporting study 1B: In the follow up study, the toxicity of Terpineol multi to the male reproductive system was examined when administered by dietary or oral gavage routes (Thacker 2010c). This study was done to characterise the effects seen in the supporting study 1 with Terpineol multi. Three groups of Crl:CD(SD) male rats (five/dose) were dosed daily with Terpineol multi by dietary and/or oral gavage routes at the following doses: group 1: dietary 7500 ppm + supplementary gavage dose 300 mg/kg/day, group 2: dietary 10000 ppm + supplementary gavage dose 150 mg/kg/day, group 3: Terpineol multi at 750 mg/kg/day by gavage only. During the study, data was recorded on mortality, clinical condition, bodyweight and food consumption. Surviving animals were subjected to a detailed sperm analysis. Testes (L&R), epididymis (L&R), prostate and seminal vesicles were weighed at necropsy and tissues of right testes and epididymis were fixed for histopathological examination. During week 1 of the study, Group 1 and 2 animals consumed less diet than expected; From Day 11 Group 1 animals, receiving 7500 ppm, also received two daily doses of 150 mg/kg/b.i.d. four to five hours apart and average intake was boosted to 663 mg/kg/day whilst Group 2 animals, receiving 10000 ppm, also received a single daily dose of 150 mg/kg boosting intake to 678 mg/kg/day. Clinical signs were generally minimal. Dosing signs for Group 3 animals included salivation and chin rubbing. Food consumption for Group 1 and 2 animals receiving the test material in the diet was low throughout the study; this was attributed to the palatability of the test material. Body weight gains of these animals were also low. Food consumption and bodyweight changes of Group 3 animals were considered to be not adversely affected by the test material. Necropsy data indicated that decreases in reproductive organ weights and changes to macroscopic appearance were most marked in the animals receiving Terpineol multi at 750 mg/kg/day. Occasional animals (1/5 and 1/5 in each of the other groups 1 and 2 respectively) also showed changes to testicular and/or epididymal tissue appearances and/or weights. Sperm analysis showed that motile sperm with normal morphology were present in 4/5 males of Group 2 and 1/5 males of Group 1. The outliers in each group were at the extreme of achieved overall exposure for the group suggesting that absolute exposure was important, although the route of exposure and consequently potential to exceed threshold levels was of greater significance. Microscopic examination indicated there were relatively fewer changes in the testes and epididymides in the animals which were given Terpineol multi by the dietary route with oral gavage supplementation (Groups 1 and 2), whereas there were significant changes in those which received it solely by oral gavage (Group 3). The results of dietary administration suggest that exposure via the dietary route of administration reduces the testicular and sperm toxicity of the test material compared to dosing by oral gavage. The results of this study, in part, support the hypothesis that a high peak plasma level is necessary to induce the observed toxic effects.

This result can be explained by the combined high oral absorption and high dosing. Gavage-dosing result in high peak exposure and can thus results in overloading, the oxygen requiring, glucuronic pathway in the liver, especially when combined with high absorption potential of substances such as the Terpineols. The parent substance (and/or the toxic metabolite) can then enter the systemic circulation instead of being readily excreted via the urine. All organs have the potential to glucuronate substances including the testis. The testis is, however, vulnerable for depletion of oxygen e.g. during glucuronation, because the blood flow in the testicles is low compared to other organs (Aitken and Roman, 2008). This type of testicular effects, including effects on spermatids is related to oxygen depletion by a variety of substances (Aitkin and Roman, 2008). This hypothesis is confirmed with high dietary dosing similar to gavage with Terpineol multi in which no testicular effects were seen. It is also supported by the repeated dose toxicity data of alpha-Terpinyl Acetate in which the rats were dosed much longer (20-wks) and also no such testicular effects were seen up to a dose of 400 mg/kg bw. This latter dose can be roughly converted to 314 mg/kg bw alpha-Terpineol (154/196 Mol x 400 mg/kg bw).

Overall it can be concluded for alpha-Terpineol for fertility for males and females that up to the limit dose of alpha-Terpinyl Acetate of 400 mg/kg bw, no effects were seen. Alpha-Terpinyl Acetate will be fully metabolised in the liver and the gut to alpha-Terpineol. This 400 mg/kg bw of the acetate can be converted to 314 mg/kg bw for alpha-Terpineol. This value is supported by the two supporting studies of Terpineol multi, which contains mainly alpha-Terpineol, showing no effects via the dietary route up to the highest dose of 750 mg/kg bw. The overall NOAEL is therefore >= 314 mg/kg bw.


Short description of key information:
In the 20-week dietary repeated dose toxicity study no effects on gonads were seen which presents an absence of concern for fertility with alpha-Terpinyl Acetate. This results in a NOAEL for alpha-Terpineol of >= 314 mg/kg bw. The reproductive/developmental toxicity screening test with Terpineol multi in rats, resulted in testicular effects in the rat at 750 mg/kg bw. The results were, however, not seen in follow up studies with 14 day dietary administration of Terpineol multi. Therefore the NOAEL for fertility on this study is considered to be >=750 mg/kg bw also for alpha-Terpineol being the main constituent of Terpineol multi. This means that exposure via the dietary route of administration does not show this testicular and sperm toxicity of the test material compared to dosing via gavage. This supports the hypothesis that a high peak plasma level is necessary to induce the observed toxic effects on the male reproductive system. Therefore the testicular effects seen are likely to be due to 'overloading the metabolic pathway and thus do not have to be considered for risk assessment because these are secondary to the dosing method.

Justification for selection of Effect on fertility via oral route:
For fertility the 20-week repeated dose toxicity study with alpha-Terpinyl Acetate has been selected as key study because: 1) the exposure period is longer than in the OECD TG 421 with Terpineol multi; 2) the dosing has been via the diet, which is not expected to overload the metabolic pathway and; 3) the metabolisation of the acetate is considered to be complete after absorption and resulting in alpha-Terpineol.

Effects on developmental toxicity

Description of key information
In the reproductive/developmental toxicity screening information from OECD TG 421 (extracted from an OECD 422, GLP, study), the NOAEL for developmental toxicity was >=250 mg/kg bw being the highest dose tested, because at one higher dose the females were not pregnant due to infertility of the males. The maternal NOAEL was set to >=250 mg/kg bw (including only pregnant females). 
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From April 28 to September 03, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD guideline 422 without deviation but performed with a very similar structural analogue and not fully covering the developmental toxicity endpoint.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: 303 to 375 g for males and 198 to 253 g for females
- Housing: Up to 5 during pre-mating for all animals and after mating for males and during toxicity phase for unmatted females, individually with litter for females during gestation and lactation.
- Diet (e.g. ad libitum): Standard rodent diet (SDS VRF1 Certified) ad libitum, except overnight before routine blood sampling for Main phase males, Toxicity phase females and Recovery phase animals.
- Water (e.g. ad libitum): Potable water taken from the public supply, ad libitum
- Acclimation period: 7 days

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

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

VEHICLE
- Concentration in vehicle: 12 and 50 mg/mL
- Amount of vehicle (if gavage): 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 CAS 8000-41-7 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
Remarks:
Doses / Concentrations:
60 and 250 mg/kg bw
Basis:
actual ingested
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).
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
Details on maternal toxic effects:
Details on maternal toxic effects:
MORTALITY:
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.

CLINICAL SIGNS:
Over activity was observed as a post dosing sign during Week 1 in females dosed at 60 mg/kg/day.

BODY WEIGHT AND FOOD CONSUMPTION:
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:
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:
There were no statistically significant effects observed.

REPRODUCTIVE FUNCTION (ESTROUS 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. All females that littered had normal length gestation periods (22 – 23 days duration) but a slightly higher proportion of females at 250 mg/kg/day gave birth after 23 days.

ORGAN WEIGHTS:
There were no significant organ weight effects observed.

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

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

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

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

HAEMATOLOGY:
There were no marked effects upon haematology parameters.

BLOOD CHEMISTRY:
Glucose plasma levels were significantly higher than Control in females dosed at 250 mg/kg/day. Bile acid plasma levels for females at all dose levels were higher than the concurrent Control. 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)).
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
LITTER SIZE, SEX RATIO AND SURVIVAL INDICES:
The numbers of implantations, total litter sizes and live litter sizes up to Day 7 of lactation were unaffected by dosing in the 60 and 250 mg/kg/day groups. Sex ratio (assessed by the percentage of males) at 250 mg/kg/day was slightly, but statistically significantly lower than Control. Individual litter data for this parameter are always variable and as only two litters are outside the concurrent Control data range this intergroup difference is not considered to be toxicologically significant. Sex ratio was unaffected at a dose level of 60 mg/kg/day. Administration with CAS 8000-41-7 had no effect on post implantation survival index, live birth index and viability index for animals receiving up to 250 mg/kg/day.

CLINICAL SIGNS: Clinical signs of offspring did not indicate any reaction to maternal exposure by CAS 8000-41-7.

BODY WEIGHT: Male and female offspring bodyweights were considered to be unaffected by CAS 8000-41-7.

GROSS PATHOLOGY: 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 CAS 8000-41-7.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Based on the findings in this study, the NOAEL for maternal and developmental toxicity was >250 mg/kg bw/day.
Executive summary:

In a GLP study conducted according to OECD guideline 422, three groups of ten female rats for the Main (reproductive) phase and five female rats for the Toxicity phase received CAS 8000-41-7 at doses of 0, 60 or 250 mg/kg bw/day in corn oil at a dose volume of 5 mL/kg bw/day. 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. 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 female was killed because of parturition difficulties. No significant findings were recorded for clinical signs, detailed physical examination and arena observations. 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 250 mg/kg/day. Bodyweight and bodyweight gain were also 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 and water consumption. Also no effects were observed on organ weight and gross pathology. There were no clinically significant effects on haematology parameters, but females showed slight anaemia. Glucose plasma levels were significantly higher than Control in females dosed at 250 mg/kg/day. There were no effects observed 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 all dose levels 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, clinical signs, and gross pathology were not adversely affected. Based on the findings in this study, the No-Observed-Adverse–Effect-Level (NOAEL) for maternal and developmental toxicity was at least 250 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
The database contains a GLP compliant OECD TG 421 information, extracted from an OECD TG 422 study. In addition, reasoning is provided for waiving the developmental toxicity study which together is sufficient to cover this endpoint.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

There is a reproduction / developmental toxicity screening information according to OECD TG 421 available, which is extracted from an OECD TG 422, for Terpineol multi (a multi-constituent substance with alpha-Terpineol as its main constituent and gamma-Terpineol as the minor constituent). For Terpineol multi no adverse effects on developmental toxicity were observed up to the highest dose available for evaluation (>= 250 mg/kg bw; at 750 mg/kg bw there were no litters due to male infertility). Additional information has been included in the waiver for further testing on developmental toxicity which shows that classification and labelling or the substance being assigned as PBT is not expected and further testing is therefore not warranted.


Justification for selection of Effect on developmental toxicity: via oral route:
One reproduction / developmental toxicity screening test is available extracted from the information of the OECD guideline 422 and GLP with Terpineol multi (a multi-constituent substance with alpha-Terpineol as its main constituent and gamma-Terpineol as the minor constituent). This OECD TG 421 information is partly adequate for this endpoint. In the waiver for developmental toxicity information is added to cover the developmental toxicity endpoint.

Justification for classification or non-classification

Fertility information is available from a 20 -week repeated dose toxicity study of alpha-Terpinyl Acetate in which no effects on gonads were seen, which indicates that no fertility effects are expected for alpha-Terpineol because this alcohol is its main metabolite.

Testicular effects were seen for Terpineol multi (a multi-constituent substance with alpha-Terpineol as its main constituent and gamma-Terpineol as the minor constituent). The testicular effects of Terpineol multi are considered to be due to peak exposures such as via gavage which overwhelm the metabolic-glucoronic-pathway. In that case the glucuronidation of alpha-Terpineol (being the main constituent of Terpineol multi) cannot be fully completed and it enters the systemic circulation. Testis and other organs also have the capability of glucuronating alpha-Terpineol, which requires oxygen. Especially the testis is sensitive for oxygen depletion (oxidative stress) and therefore effects may first be seen here, when glucuronic acid is depleted (Aitkin and Roman, 2008). These effects are however secondary, because during repeated human health exposure it is not expected that glucuronic acid will be depleted. This has been shown using similar high doses via the diet and absence of these effects in the 20 -week repeated dose dietary study with alpha-Terpinyl Acetate. Therefore Terpineol multi and thus also alpha-Terpineol does not need to be classified for fertility according to EU Directive 67/548 (DSD) and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (No. 1272/2008). In more detail this is expressed in CLP section 3.7.2.2.1 “Classification is made on the basis of the appropriate criteria, outlined above, and an assessment of the total weight of evidence (see 1.1.1). Classification as a reproductive toxicant is intended to be used for substances which have an intrinsic, specific property to produce an adverse effect on reproduction and substances shall not be so classified if such an effect is produced solely as a non-specific secondary consequence of other toxic effects”. This section is considered applicable for the effects seen for Terpineol multi.

Alpha-Terpineol does not have to be classified for developmental toxicity based on the data of Terpineol multi (being its main constituent). No developmental toxic effects were seen even with peak exposures and high doses overwhelming the metabolic-glucuronic pathway. In addition, information is provided in the waiver why developmental toxicity, not accounted for in the reproductive /developmental screening study, is not expected. Therefore for alpha-Terpineol classification and labelling according to EU Directive 67/548 (DSD) and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (No. 1272/2008) for developmental toxicity is not needed.

Aitken, J.J., and Roman, S.D., 2008, Antioxidant systems and oxidative stress in the testes, Oxid. Med. Cell Longev., 1, 15-24.