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Developmental toxicity / teratogenicity

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Administrative data

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: Reliability 2 is assigned because the information is used for read across. The information as such is a GLP study conducted according to OECD guideline 422.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
other: Audited Draft Study Report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
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

Test material

Constituent 1
Reference substance name:
Terpineol multi: 8000-41-7
IUPAC Name:
Terpineol multi: 8000-41-7
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Terpineol; CAS 8000-41-7
- Source: Les Dérivés Résiniques et Terpéniques, France
- Physical state: Clear, colourless, lightly viscous liquid
- Storage condition of test material: Room temperature in the dark

Test animals

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 (e.g. ad libitum): Standard rodent diet (SDS VRF1 Certified) ad libitum, except overnight before routine blood sampling for Main phase males, Toxicity phase females and Recovery phase animals.
- Water (e.g. ad libitum): Potable water taken from the public supply, ad libitum
- Acclimation period: 7 days

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

IN-LIFE DATES: From: 28 April 2010 To: 29 June 2010

Administration / exposure

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

VEHICLE
- Concentration in vehicle: 12 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
Doses / concentrations
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).

Examinations

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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)).

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 750 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

Results (fetuses)

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.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

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.