p-menth-1-en-8-ol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Key study. Read-across approach. Test method according to OECD Guideline 422 with GLP. Based on the read-across approach from the analogue terpineol, the NOAEL of laevo alpha terpineol for parental reproduction is determined to be 300 mg/kg bw/day in males and 100 mg/kg bw/day in females, and the NOAEL for development toxicity is concluded to be 1000 mg/kg bw/day.

Supporting study. Read-across approach. Repeated dose toxicity study with test method similar to OECD Guideline 408. Based on the read-across approach from the analogue alpha terpinyl acetate after an oral exposure of 20 weeks in rats where no effects were seen in gonads, the NOAEL of L-alpha terpineol  was determined to be higher than 314 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September 2012 – 28 March 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Test method according to OECD guideline 422 and GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(mating females were only administered test chemical up to 4 days of lactation instead of 13 days)

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. Atsugi Breeding Center.
- Age at study initiation: 10 weeks
- Weight at study initiation: 350-420 g (378 g average in males); 215-257 g (231 g average in females)
- Housing: bracket-type metal net cage (W 254 × D 350 × H 170 mm). During mating (1 male: 1 female) and from 17th day of pregnancy to 4th day of lactation a plastic cage (W340 × D400 × H185 mm) was used.
- Diet (e.g. ad libitum): NMF solid form (radiation sterilization, Oriental Yeast Co., Ltd.), ad libitum.
- Water (e.g. ad libitum): Tap water (metal cage) and from bottle (during time of lactation in plastic cage), ad libitum.
- Acclimation period: 20 days (including 3 days of quarantine)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24ºC
- Humidity (%): 39-65 %
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Preparation of the test solutions was conducted by taking a required amount of the test substance with a glass syringe for each concentration and accurately weighing it in a beaker. The weighed test substance was transferred to a measuring cylinder. The beaker was washed several times with a small amount of solvent, and the liquid was also added to the measuring cylinder. The mixture was shaken by hand and mixed, and it was visually confirmed that it was completely dissolved. Next, the solvent was added to the measuring cylinder to the required amount of test material.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1 male/1 female
- Length of cohabitation: up to 14 days
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A sample of 10 mL of each formulation prepared for administration in the first and last week of the dosing period was analysed for achieved concentration of the test substance. The mean concentrations of terpineol in test formulations analysed for the study were within applied limits (99.5 to 103.0% of nominal concentrations), confirming accurate formulation.
The determination of the concentrations was performed by HPLC.

Duration of treatment / exposure:

Males: From 14th day before mating up to 30 days after mating (total 44 days).
Females: From 14th day before mating up to 4 days of lactation (total 41-51 days).
Non-mating female group: 44 days

Frequency of treatment:

Once a day, 7 days a week

Details on study schedule:

Study period: 9 October 2012 (administration start day) – 16 December 2012 (animal test end day)
Note: after administration period some animals from the control and high dose groups were kept for further 14 days without treatment to detect delayed occurrence and recovery from toxic effects.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Mating male group: 12 per sex per dose (including 5 in the control and high dose group for 14-day observation after treatment)
Mating female group: 12 per sex per dose
Non-mating female group: 10 (control group, including 5 for 14-day observation after treatment) and 10 (high dose group, including 3 for 14-day observation after treatment)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
A preliminary range finding study was conducted with doses of 250, 500 and 1000 mg/kg bw/ day. One male in the 1000 mg/kg bw/day dose group showed a decrease in body weight and food intake, a decrease in feces volume, a decrease in locomotor activity and a decrease in respiratory rate, and died on the 12 th day of administration. A high value of liver and kidney weight and a high value of urea nitrogen and total protein were observed in autopsy of surviving males of high dose group. In each female of 500 and 1000 mg/kg bw/day group, a significant reduction in walking and muscle tone and a licking gait were observed only on 1 day of administration. Therefore, in this study, 1000 mg/kg bw/day at which obvious signs of toxicity is expected was set as high dose, and 300 and 100 mg/kg bw/day were set as medium and low dose, respectively, divided by the common ratio of about 3.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily during the quarantine period, three times daily for the administration period (before administration, immediately after administration and 1 to 3 hours after administration), twice daily during the recovery period (morning, afternoon)
- Cage side observations: The presence or absence of abnormalities in general conditions such as life and death, appearance of the body, posture, behavior and emissions (urine, feces) were observed. Regular observation accompanied by handling of animals after 2 weeks of administration was also carried out as observation of detailed general condition.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed general condition observation was performed once for all individuals, before administration. Also, males and non-mating females were observed once a week during the administration period and during the recovery period. Females in the mating group were observed once a week during the pre-mating period, during the mating period, during the pregnancy period and during the lactation period.

BODY WEIGHT: Yes
- Time schedule for examinations: For all individuals, body weight was measured on acclimated days 1, 3, 8, 13 and 20 and on the day of administration 1, 8, 15, 22, 29, 36, 42, 44 and on the day of necropsy. For the recovered animals further recovered the body weight was measured on days 1, 8 and 14 and on the day of necropsy. Body weights of female mating group were measured on day of administration 1, 8, 15, 22, on day of gestation 0, 7, 14, and on day 0 and 4 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes.
-Food consumption was determined daily by weighing the residue. Measurements were not made during the mating period.

OTHER:
-URINE EXAMINATION:
During the last week of the administration period and during the recovery period a urinalysis was performed in 5 rats in each group. These animals were housed in a cage prepared with urine collector, and under fasting and free water ingestion urine was collected for 4 and 20 hours. For the first 4 hours urine collected pH and urine volume were taken. Osmotic pressure, electrolytes (sodium, potassium and chlorine) and urine volume were measured for the 20 hours urine obtained.

- HAEMATOLOGY:
The day before necropsy after administration period and after 14-day observation period, blood samples were obtained from 5 animals per sex and per group after overnight withdrawal of food, and under anaesthesia induced by isoflurane. The following were measured using a Siemens Advia 120 haematology analyser: Erythrocyte count (RBC), Haemoglobin concentration (HGB), Haematocrit (HCT), Mean cell volume (MCV), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Reticulocyte count (Retic), Platelet count (PLT) and White blood cell count (WBC). Differential leucocyte count (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M) and Large unstained cells (LUC)) were also measured. Prothrombin time (PT), Activated partial thromboplastin time (APTT) and Fibrinogen (FIB) were measured by automatic blood coagulation analyzer ACL Elite Pro.

- BLOOD CHEMISTRY:
At the same time and using the same animals as for haematology, further blood samples were collected and examined using a Toshiba analyzer TBA-120 FR. Serum was analysed for: Alkaline phosphatase (ALP), Total bile acid (TBA), Total cholesterol (T-CHO), Triglyceride (TG), Phospholipids (PL), Total bilirubin (T-BIL), Glucose (GLU), Urea nitrogen (BUN), Creatinine (CRNN), Sodium (Na), Potassium (K), Chlorine (Cl), Canoesium (Ca), Inorganic phosphorus (P), Total protein (TP) and Anolebumin (ALB). Heparinized blood was examined for Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Lactate dehydrogenase (LDH) and Gamma-glutamyl transpeptidase (GTP).

Oestrous cyclicity (parental animals):

Estrous cycle was measured during pre-mating period: count of estrous, mean duration of cycle, number and index of animals with abnormal estrous cycle.

Sperm parameters (parental animals):

Parameters examined in male parental:
testis: weight, sperm count, vacuolation/atrophy of seminiferous tubular, multinucleated giant cells, etc.
epididymis: weight, sperm count, cell debris (lumen), granuloma

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in offspring: The number of live births and stillbirths were counted on the 0th day of delivery. The sex and weight were measured for each individual. The stillborn pups were fixed with Bouin's solution and stored. Living pups were checked for death once daily until 4th day of lactation. On the 4th day after birth, the sex of all surviving pups was judged, the body weight was measured for each individual, and the presence or absence of abnormalities were observed visually. Then, they were immersed in Bouin's solution under deep anesthesia of isoflurane, fixed and stored.

GROSS EXAMINATION OF DEAD PUPS: yes. The dead pups were observed visually for presence of abnormalities and then fixed with Bouin's solution and stored.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- All animals were subject to a detailed necropsy, which included a visual examination of the external tissues and organs of the head, thorax and abdomen. For mating females, the number of uterine implantation sites and number of corpora lutea were also recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
- ORGAN WEIGHTS:
The absolute weight and relative weight of the following organs were measured: Brain, pituitary, thyroid (including parathyroid gland), adrenal gland, spleen, thymus, tongue, heart, liver, Kidney, testis, epididymis, prostate, seminal vesicle (including coagulated gland), ovary and uterus.
-HISTOPATHOLOGY:
For all individuals, the following organs / tissues were fixed for histopathology examination: cerebrum, cerebellum, sciatic nerve, spinal cord (chest), the eyeball, optic nerve, pituitary, thyroid, parathyroid, adrenal gland, spleen, thymus, submandibular lymph node, mesenteric lymph node, heart, aortic aorta, trachea, lung (including bronchus), tongue, larynx, oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, kidney, testis, ovary, epididymis, uterus, prostate, seminal vesicle (coagulating glands mate), skin (inguinal region), mammary gland (inguinal region), sternum, femur (including bone marrow) and femoral skeletal muscle. Samples of any abnormal tissues were also retained and processed for examination. Testis and epididymis were fixed with Bouin's fixative.

Postmortem examinations (offspring):

GROSS NECROPSY
- Fracture head, facial nostrils and upper and lower jaws, distortion or fracture of the lips, size and shape of the eyes and ears, left and right balance, distortion and size of the chest body, distortion and size of the internal organs were observed with attention to the length, shape, number, loss of the tail, length and shape of limbs, number of fingers, defects and abnormal cases different from normal.

Reproductive indices:

Sex cycle abnormality rate (%) = (number of individuals showing sexual cycle abnormality / number of observed female individuals) × 100
Mating rate (%) = (number of animals mating / number of living animals) × 100
Fertilization rate (%) = (number of males who pregnant female / number of male copulated) × 100
Conception rate (%) = (number of pregnant females / number of mated females) × 100

Offspring viability indices:

Birth rate (%) = (number of births of babies born / number of pregnant females) × 100
Delivery rate (%) = (number of births / number of implantation sites) × 100
4-day survival rate (%) = (number of surviving offspring on 4th day / number of births) × 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

CAGE SIDE OBSERVATIONS: There was no abnormality in any animal.
SIGNS AND ARENA OBSERVATIONS: Wiggling was observed on the first day of pregnancy in one female in the mating group of 1000 mg/kg/day administration group.
In addition, two animals were observed gait abnormality and jumping respectively but they were temporary findings and it was judged that there was no relation with the administration of the test substance because it was not related to the dose. Furthermore, facial cleansing behavior was observed in another animal, which is a normal behavior observed when the animal calms down, and is not related to administration of the test substance.

General condition of surviving animals: In the 1000 mg/kg/day dose group, 4 males had nursing behaviour and 1 male a decrease in feces volume, and in females, a decrease in feces volume was observed. In 3 females of the non - mating group, decrease in fecal volume, scaling, decreased motor activity, wiggling gait or decrease in respiration rate were observed.
These symptoms were not seen during the recovery period, thus it was judged as no treatment related.

Gripping strength: There was no significant difference between the control group and each test substance administration group.

Motor activity: There was no significant difference between the control group and each test substance administration group. Meanwhile, in the females of the non-mating group, a significant low value was observed in the measurement values of 0 - 10 and 10 - 20 intervals in the 1000 mg/kg/day dose group compared with the control group, but there was no significant difference in the total measurement value (0-60 interval) and it was equivalent to the measurement value of the control group at the end of the recovery period week. Thus it was judged to be a change within the variation range.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

6 females in the 1000 mg/kg/day dose group were found dead or euthanized. These animals showed worsening status before moribund and death, suppression and reduction in body weight gain, low food consumption, decreased fecal volume, scraping, wiggling, decreased motor activity, decreased respiratory rate, abdominal drainage and decrease in body temperature. In addition, it was mainly characterized by malnutrition, reduction of spleen and thymus, downsizing of kidney (renal tubular dilation, vacuolization of proximal renal tubule, vacuolation of distal tubule / collecting duct, papillary necrosis, single cell necrosis of teat canal, regeneration of the papillary collecting duct, papillary cell infiltration), single cell necrosis of the transitional epithelial cell, hypertrophy / hyperplasia of the transitional epithelial cell, adrenal gland cell vacuolation, eosinophilic droplets and vacuolation of hepatocytes in the liver. Other secondary changes accompanying state deterioration include atrophy of various lymphoid tissues, atrophy of colonic mucosa, land mucosa and uterine atrophy or reduction of zymogen granules of pancreas, mesenterium lymph nodes, submandibular lymph nodes and thymus.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In males, suppression of body weight gain was observed in the 1000 mg/kg/day dose group, and a significant lower value was observed in body weight gain during the administration period. In the group administered with 300 mg/kg/day or less, the body weight transition was similar to that of the control group, and no significant difference was observed.
In the mating group females, body weight was lower than that of the control group through premating, pregnancy and lactation period in the 1000 mg/kg/day dose group, but no significant difference was observed. A significant low value was observed on the 0th day of lactation compared to the control group in the 300 mg/kg/day administration group, but it was a temporary change, and the weight gain increase in the subsequent lactation period was found to be a significant high value. It was judged that it was not related to administration of the test substance. In the 100 mg/kg/day administration group, no significant difference was observed between the control group and the control group.
In the non-mating group females, mean body weight decreased due to the moribund / death cases on day 4 of administration in the 1000 mg/kg/day dose group, and a significant lower value was observed than in the control group. However, it is a transient fluctuation due to the moribund / death cases, not an influence on the body weight by administration of the test substance.
In the 1000 mg/kg/day dose group, both sexes showed weight gain above the control group during the recovery period, but no significant difference was found between the treated group and the control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In males, a significant high value was observed on the 30th and 44th days of administration in the 1000 mg/kg/day dose group, but it was judged as a change within the variation range because it was temporary. In the group administered with 300 mg/kg/day or less, the food consumption was equivalent to that of the control group, and no significant difference was observed.
In the mating group females, there was no significant difference between the control groups and the test substance administration group in the pre-mating, pregnancy and lactation periods.
In the non-mating group females, a significant low value was observed on the 8th day of administration compared with the control group in the 1000 mg/kg/day dose group, but it was judged that it was a slight temporary change and was not related to the administration of the test substance.
In the 1000 mg/kg/day dose group, males showed food intake over the control group during the recovery period, and a significant difference was observed on administration 1 and 14 days. In females there was no significant difference between the control group and the 1000 mg/kg/day dose group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

During administration and recovery period, a significant high value of water intake was observed in the 1000 mg/kg/day dose group of males and non-mating group females as compared with the control group.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males, a significant lower value of hemoglobin was observed in the 1000 mg/kg/day dose group compared to the control group. This difference was not seen during the recovery period, thus it was judged as no treatment related.
In the mating group females, no change was observed by administration of the test substance. In addition, compared with the control group, the leukocyte count, the basophil count and the large unstained cell count were significantly lower in the 100 mg/kg/day administration group. Significantly high levels of white blood cell count, eosinophil count and monocyte count were observed in the 300 mg/kg/day administration group, but there was no significant difference in the leukocyte percentage and there was no relation with the dose and the females of the non-mating group. It is considered that it is temporary fluctuation accompanying increase / decrease of mild leukocyte counts at the beginning of lactation after childbirth because there is no such change, and it was judged to be within the physiological variation range.
Moreover, a significant shortening of the prothrombin time was observed in the group administered with 300 mg/kg/day, but it was judged within the physiological variation range because it was slight shortening and there was no toxicological significance.
Significantly low levels of hemoglobin and hematocrit and significant high values of mean red blood cell hemoglobin concentration and significant high levels of fibrinogen were observed in non-mating group females as compared with the control group. In addition, although a significant low value of eosinophil ratio was observed in the 1000 mg/kg/day dose group compared with the control group, it was a slight change and there was no significant difference in the eosinophil count, so it was judged as an accidental change.
During recovery period, significantly low platelet count was observed in the 1000 mg/kg/day dose group compared with the control group, and the lymphocyte ratio was significantly lower in the 1000 mg/kg/day dose group in the non-mating group female, but since it was not recognized during administration period and it was a slight change in the background data, it was judged that it was not related to the administration of the test substance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In male, a significant high value was observed in GTP in the 1000 mg/kg/day dose group compared to the control group. This difference was not seen during the recovery period, thus it was judged as no treatment related.
In the mating group females, a significant high value was found in GTP in the group administered with 300 mg/kg/day compared to the control group, but it was a slight change and it was not related to the dose, so it was judged within the physiological variation range.
In the non-mating group females, significant high values of calcium and inorganic phosphorus were found in the background data of the test facility, but this change was equivalent to the control group of the mating group female, thus it was judged that there was no relation with the administration of the test substance.
At the end of recovery period, significant low value for total bilirubin and significant high value for calcium in the male 1000 mg/kg/day dose group, and significant high value of total cholesterol in the 1000 mg/kg/day dose group of non-mating group females were observed. These were minor changes compared with background data and results at the end of the administration period, thus it was judged that there was no relation with the administration of the test substance.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

During administration period: Regarding qualitative items, neither sex was abnormal in any animals. Regarding quantitative items, a significant high value of urine volume accompanied by a significant high value of water intake and a significant low value of urinary osmotic pressure were observed in the 1000 mg/kg/day dose group of males and non-mating group females as compared with the control group.
During the recovery period: Regarding the qualitative items, neither sex was abnormal in any animals. For quantitative items, high or significant urine volume with a significant high value of water intake and significant low value of urinary osmotic pressure were observed in the 1000 mg/kg/day dose group of males and non-mating females compared with the control group. Sodium, potassium and chlorine emissions were high or significantly high.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

During dosing period, neither animal had any abnormality in the auditory reaction, approach reaction, contact reaction, pain sense reaction, pupillary reflex, and airborne forward reflection.
In the landing leg width, there was no effect of administration of the test substance. In the non-mating group females, a significant high value was observed at 6 weeks of administration in the 1000 mg/kg/day administration group compared with the control group, but since it was equivalent to the measurement value of the control group at the end of the recovery period, it was judged as no treatment related.

During recovery period, neither animal had any abnormality in the auditory reaction, approach reaction, contact reaction, pain sense reaction, pupillary reflex, and airborne forward reflection.
There was no significant difference between the control group and the 1000 mg/kg/day administration group in the landing leg open width.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Adrenal gland: The vacuolation (minor or mild) of cortical cells was found in the group administered 300 mg/kg/day or more in the mating group female and in the 1000 mg/kg/day dose group of the non-mating group female.

Testis: Atrophy (moderate) of seminiferous tubules, multinucleated giant cells (minor to moderate) and vacuolization of seminiferous tubules (minor, mild) were found in the1000 mg/kg/day dose group. The testicular disorder caused by the administration of the test substance was considered to be the cause of female infertility.
At the end of the recovery period, atrophy of seminiferous tubules (mild to moderate), multinucleated giant cells (minor) and vacuolization of seminiferous tubules (minor, mild) were found in the1000 mg/kg/day dose group. Mature spermatozoa were not observed, but the length of seminal epithelium was higher than the examination at the end of administration and recovery was noted.

Epididymis: Reduction of sperm (mild to severe) and cell debris content of the lumen (mild, moderate) were observed in the 1000 mg/kg/day dose group, reflecting the influence on the testes by administration of the test substance.
At the end of the recovery period, reduction of sperm (moderate) and cell debris content of the lumen (mild, moderate) were seen in the 1000 mg/kg/day dose group. Although it was thought that it was an image reflecting the change of the testes, it was thought that it will disappear with the recovery of the testes in the future.

Kidney: The vacuolization (minor) of the proximal tubule was observed in the 300 mg/kg/day dose group in the mating group female, the vacuolization (minor, mild) in the distal tubule / collecting duct was in the male, the mating group female and the non-mating group female in the 1000 mg/kg/day dose group, the regeneration of the cortical renal tubule (minor, mild) was in the group administered with 300 mg/kg/day or more in males and in the 1000 mg/kg/day dose group of non-mating females, renal tubule dilation (minor, mild), single cell necrosis of papillary duct (minor) and cortical cell infiltration (minor) were observed in the 1000 mg/kg/day dose group of males and non-mating group females, regeneration (minor) of papillary collecting duct was in the 1000 mg/kg/day male group. Papillary necrosis (minor, mild) and papillary cell infiltration (minor) were found in the 1000 mg/kg/day dose group of mating female and non-mating females. In addition, an increase in the frequency of eosinophilic corpuscles in renal tubules was observed in the male group receiving 300 mg/kg/ day or more. Eosinophilic bodies of renal tubular epithelial cells were positive for α2μ globulin immunohistochemistry and were negative for PAS staining and were considered to be derived from α2μ globulin.
At the end of the recovery period, papillary necrosis (minor) was observed in the 1000 mg/kg/day dose group of male and non-mating females, regeneration (mild) of cortical renal tubules was observed 1000 mg/kg/day dose group of males. Although renal papillary necrosis was not seen at the end of male administration, similar changes were observed in mating and non-mating females, and even in male, regeneration of papillary collecting ducts and single-cell necrosis of papilla were seen, thus it was thought that the change at the end of the administration remained, but the degree of finding was minor. In females, animals with strong papillary changes were deceased or euthanized, and it seemed that they recovered for those with minor changes. Regeneration of cortical renal tubules decreased frequency and showed recovery.

Liver: centrilobular, hepatocytic hypertrophy (minor) was found in the 1000 mg/kg/day dose group of male; hepatocyte vacuolation (minor, mild) was found in the 1000 mg/kg/day dose group of mating female and the non-mating female.

Urinary bladder: Atrophy of umbrella cells (minor, mild) were observed in the 1000 mg/kg/day dose group of males, mating females and non-mating females. Vacuolation of umbrella cells (minor to moderate) were observed in the 300 mg/kg/day dose group of males and mating females. Hypertrophy / hyperplasia of epithelial cells (minor, mild) was observed in the male, mating group female and non - mating group females administered with 1000 mg/kg/day.

Pancreas: An increase in the frequency of decreased zymogen granules (minor to moderate) was observed in the 300 mg/kg/day dose group of mating females and in the 1000 mg/kg/day dose group of non-mating females.

In two female animals all littermate died on 2nd day of lactation in the 300 and 1000 mg/kg/day dose group, respectively. In both cases the death of all littermate was considered to be a possibility of deterioration of the condition of the mother animal.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No sexual cycle abnormalities were found, and no significant difference was observed between the control group and each test substance administration group in the sexual cycle abnormality rate, development of oestrus stages and the average cycle duration.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Testis: Atrophy (moderate) of seminiferous tubules, multinucleated giant cells (minor to moderate) and vacuolization of seminiferous tubules (minor, mild) were found in the1000 mg/kg/day dose group. The testicular disorder caused by the administration of the test substance was considered to be the cause of female infertility.
At the end of the recovery period, atrophy of seminiferous tubules (mild to moderate), multinucleated giant cells (minor) and vacuolization of seminiferous tubules (minor, mild) were found in the1000 mg/kg/day dose group. Mature spermatozoa were not observed, but the length of seminal epithelium was higher than the examination at the end of administration and recovery was noted.

Epididymis: Reduction of sperm (mild to severe) and cell debris content of the lumen (mild, moderate) were observed in the 1000 mg/kg/day dose group, reflecting the influence on the testes by administration of the test substance.
At the end of the recovery period, reduction of sperm (moderate) and cell debris content of the lumen (mild, moderate) were seen in the 1000 mg/kg/day dose group. Although it was thought that it was an image reflecting the change of the testes, it was thought that it will disappear with the recovery of the testes in the future.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The copulation ratio was 100% for control group and each test substance administration group. There was no significant difference in the number of days required for copulation between the control group and each test substance administration group.

Insemination index (males) and fertility index (females) were 100% for control group and for administration doses of 300 mg/kg/day or less. However, in the 1000 mg/kg/day dose group Insemination index (males) was 2/11 (18.2%) and fertility index was 2/10 (20%), with significant difference (p<0.01) from control group.

No significant differences were found between the control group and each test substance-administered group in terms of gestation ratio, pregnancy period, number of corpora lutea, implantation number, implantation ratio, delivery ratio, number of stillborn babies and number of live-borns.

All pregnant animals were delivered on pregnancy day 21 to 23, and the placenta and amniotic membrane were processed normally.

In two female animals all littermate died on 2nd day of lactation in the 300 and 1000 mg/kg/day dose group, respectively. Since these conditions are deteriorated in the histopathologic examination, it is suggested that it was influenced by the administration of the test substance.

In addition, decrease in nursing behaviour was observed from day 2 to 4 of lactation period in 1 animal in the 100 mg/kg/day administration group. This effect is not dose-dependent and thus it was judged as not related with administration of test substance.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive performance

Remarks on result:

other: Effects observed at the highest dose tested (1000 mg/kg bw/day)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: Effects observed at the dose tested of 300 mg/kg bw/day

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no significant difference in the birth rate and 4-day survival rate between the control group and each test substance-administered group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no significant difference between the control group and each test substance administration group.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

None of the dead pups examined had macroscopic abnormality in control and any treated group.
There was no macroscopic abnormality in any pup examined on postnatal day 4.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There was no significant difference in the sex ratio of liveborns and live pups on day 4 between the control group and each test substance administration group.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: No effects observed at the highest dose tested (1000 mg/kg bw/day)

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Based on the findings of this study, the NOAEL for parental reproduction was 300 mg/kg bw/day in males and 100 mg/kg bw/day in females, and the NOAEL for development toxicity was 1000 mg/kg bw/day, the highest dose tested.

Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of terpineol (CAS 8000-41-7) by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. Terpineol was diluted with corn oil and added to Sprague-Dawley SPF rats (12 per sex per dose) at doses of 0, 100, 300 and 1000 mg/kg bw/day, from day 14 before mating up to 30 days after mating for males (total 44 days) and up to 4 days of lactation for females (total 41-51 days). A non-mating female group (10) was exposed at doses of 0 and 1000 mg/kg bw/day for 44 days. Also, after administration period some animals from the control and high dose groups (5 males of mating group, 5 females of control and 3 females of high dose of non-mating group) were kept for further 14 days without treatment to detect delayed occurrence and recovery from toxic effects.

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, haematology, blood chemistry, urinalysis, 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 1000 mg/kg/day dose group of the mating group female, 9/12 cases were infertile, 2/12 cases were pregnant, and 1/12 cases died on day 3 pregnancy. Female infertility was considered to be caused by the effects found in histopathology of testis for the same dose group: Atrophy (moderate) of seminiferous tubules, multinucleated giant cells (minor to moderate) and vacuolization of seminiferous tubules (minor, mild). For epididymis, reduction of sperm (mild to severe) and cell debris content of the lumen (mild, moderate) were observed in the 1000 mg/kg/day dose group, reflecting the influence on the testes by administration of the test substance. No oestrous cycle abnormalities were found, and no significant difference was observed between the control group and each test substance administration group in the sexual cycle abnormality rate, development of oestrous stages and the average cycle duration. The copulation ratio was 100% for control group and each test substance administration group. There was no significant difference in the number of days required for copulation between the control group and each test substance administration group. Insemination index (males) and fertility index (females) were 100% for control group and for administration doses of 300 mg/kg/day or less. However, in the 1000 mg/kg/day dose group, insemination index (males) was 2/11 (18.2%) and fertility index was 2/10 (20%), with significant difference (p<0.01) from control group. No significant differences were found between the control group and each test substance-administered group in terms of gestation ratio, pregnancy period, number of corpora lutea, implantation number, implantation ratio, delivery ratio, number of stillborn babies and number of live-borns. In two female animals all littermate died on 2nd day of lactation in the 300 and 1000 mg/kg/day dose group, respectively. Since these conditions were supported by the histopathological examination, it is suggested that it was influenced by the administration of the test substance.

Based on these findings, the NOAEL for parental reproduction was established at 300 mg/kg bw/day for males and 100 mg/kg bw/day for females, and the NOAEL for development toxicity was determined as the highest dose tested, i.e. 1000 mg/kg bw/day.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance terpineol which shares the same functional groups with the substance L-alpha terpineol also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

other: Read-across from an analogue

Sex:

male

Basis for effect level:

reproductive performance

Remarks on result:

other: read-across from an analogue for which NOAEL = 300 mg/kg bw/day (actual dose received)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

other: Read-across from an analogue

Sex:

female

Basis for effect level:

reproductive performance

Remarks on result:

other: read-across from an analogue for which NOAEL = 100 mg/kg bw/day (actual dose received)

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

other: Read-across from an analogue

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: read-across from an analogue for which NOAEL = 1000 mg/kg bw/day (actual dose received)

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Based on the read-across approach from the analogue terpineol, the NOAEL of laevo alpha terpineol for parental reproduction is determined to be 300 mg/kg bw/day in males and 100 mg/kg bw/day in females, and the NOAEL for development toxicity is concluded to be 1000 mg/kg bw/day.

Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of terpineol (CAS 8000-41-7) by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. Terpineol was diluted with corn oil and added to Sprague-Dawley SPF rats (12 per sex per dose) at doses of 0, 100, 300 and 1000 mg/kg bw/day, from day 14 before mating up to 30 days after mating for males (total 44 days) and up to 4 days of lactation for females (total 41-51 days). A non-mating female group (10) was exposed at doses of 0 and 1000 mg/kg bw/day for 44 days. Also, after administration period some animals from the control and high dose groups (5 males of mating group, 5 females of control and 3 females of high dose of non-mating group) were kept for further 14 days without treatment to detect delayed occurrence and recovery from toxic effects.

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, haematology, blood chemistry, urinalysis, 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 1000 mg/kg/day dose group of the mating group female, 9/12 cases were infertile, 2/12 cases were pregnant, and 1/12 cases died on day 3 pregnancy. Female infertility was considered to be caused by the effects found in histopathology of testis for the same dose group: Atrophy (moderate) of seminiferous tubules, multinucleated giant cells (minor to moderate) and vacuolization of seminiferous tubules (minor, mild). For epididymis, reduction of sperm (mild to severe) and cell debris content of the lumen (mild, moderate) were observed in the 1000 mg/kg/day dose group, reflecting the influence on the testes by administration of the test substance. No oestrous cycle abnormalities were found, and no significant difference was observed between the control group and each test substance administration group in the sexual cycle abnormality rate, development of oestrous stages and the average cycle duration. The copulation ratio was 100% for control group and each test substance administration group. There was no significant difference in the number of days required for copulation between the control group and each test substance administration group. Insemination index (males) and fertility index (females) were 100% for control group and for administration doses of 300 mg/kg/day or less. However, in the 1000 mg/kg/day dose group, insemination index (males) was 2/11 (18.2%) and fertility index was 2/10 (20%), with significant difference (p<0.01) from control group. No significant differences were found between the control group and each test substance-administered group in terms of gestation ratio, pregnancy period, number of corpora lutea, implantation number, implantation ratio, delivery ratio, number of stillborn babies and number of live-borns. In two female animals all littermate died on 2nd day of lactation in the 300 and 1000 mg/kg/day dose group, respectively. Since these conditions were supported by the histopathological examination, it is suggested that it was influenced by the administration of the test substance.

Based on these results, the read-across approach was applied and the NOAEL of laevo alpha terpineol for parental reproduction was established at 300 mg/kg bw/day for males and 100 mg/kg bw/day for females, and the NOAEL for development toxicity was determined as the highest dose tested, i.e. 1000 mg/kg bw/day.

Reference 3

Endpoint:

reproductive toxicity, other

Remarks:

(20 week repeated dose toxicity study)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD TG 408

Deviations:

yes

Remarks:

(no data on details of test animals and environmental conditions)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Osborne-Mendel

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing: The animals were housed individually in wire cages
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

- DIET PREPARATION
- Rate of preparation of diet (frequency): weekly

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

7% loss of test compound from lab animal diet during a 7-day period.

Duration of treatment / exposure:

20 weeks

Frequency of treatment:

Fresh diets were made and distributed weekly.

Dose / conc.:

10 000 ppm

Remarks:

Equivalent to 400 mg/kg bw/ day (based on food consumption of 40 g/kg bw/day included in the
Guidance on information requirements and chemical safety assessment R8, table R 8 -17)

Dose / conc.:

2 500 ppm

Remarks:

Equivalent to 100 mg/kg bw/ day (based on food consumption of 40 g/kg bw/day included in the
Guidance on information requirements and chemical safety assessment R8, table R 8 -17)

Dose / conc.:

1 000 ppm

Remarks:

Equivalent to 40 mg/kg bw/ day (based on food consumption of 40 g/kg bw/day included in the
Guidance on information requirements and chemical safety assessment R8, table R 8 -17)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes, weekly

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes; At the termination of the experiments the rats were sacrificed and exsanguinated. The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed. Tissues from rats dying during the experiment were examined for gross changes and were preserved if autolysis was not advanced. Organs were not weighed but abnormalities and the suspected reason for death were noted.

HISTOPATHOLOGY: Yes; Organs weighed at termination, the remaining abdominal and thoracic viscera, and one hind leg, for bone, bone marrow, and muscle, were preserved in 10% buffered formalin-saline solution for histopathological examination. For routine histopathology, sections were embedded in paraffin wax and stained with haematoxylin and eosin. Detailed microscopic examinations were done on 6 or 8 rats, evenly divided by sex, from the high dose group and the control group. If changes attributable to the test compound were found in the high dose group, additional animals on lower dosage levels were examined as indicated.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No microscopic change in the tissues in the 10000 ppm exposure group.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

> 10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

haematology

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: Equivalent to NOAEL > 400 mg/kg bw.

Key result

Critical effects observed:

no

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Key result

Reproductive effects observed:

no

Conclusions:

No effects were seen on male and female gonads after 20-weeks of administration of the test substance.

Executive summary:

In a 20 -weeks oral exposure study, Osborne-Mendel rats (10/dose/sex) were administered alpha-Terpinyl Acetate via diet intake at concentrations of 0 (control), 10000, 2500 and 1000 ppm. Animals were then observed for mortality, weight, food intake and general condition. Haematological examinations were made at termination. At the termination of the experiments the rats were sacrificed and exsanguinated. The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed. Detailed microscopic examinations was done on the animals from the high dose group and the control group. No effect on growth or haematology, and no macroscopic or microscopic change in the tissues in the 10000 ppm exposure group were observed. No effect on growth or haematology, and no macroscopic change in the tissues in the 2500 and 1000 ppm exposure groups. No microscopic examination was performed on rats exposed to 2500 and 1000 ppm. According to the Guidance on information requirements and chemical safety assessment R8 table R 8 -17, rats eat 40 to 50 g/kg bw/day. 10000 ppm is equivalent to 1% in the diet. Consequently, rats exposed to 10000 ppm in the diet consumed between 400 and 500 mg Terpinyl-Acetate-Alpha per kg bodyweight per day. Therefore, the NOAEL was calculated to be higher than 400 mg/kg bw.

Reference 4

Endpoint:

reproductive toxicity, other

Remarks:

(20 week repeated dose toxicity study)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance alpha terpinyl acetate undergoes rapid hydrolysis to acetic acid and alpha terpineol which shares the same functional groups with the substance L-alpha terpineol and also has comparable values for the relevant molecular properties.
See attached the reporting format.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

> 7 858 ppm

Based on:

other: read-across from an analogue

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

haematology

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: read-across from an analogue for which NOAEL > 10000 ppm (equivalent to NOAEL > 400 mg/kg bw/ day)

Key result

Critical effects observed:

no

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Key result

Reproductive effects observed:

no

Conclusions:

Based on the read-across approach from the analogue alpha terpinyl acetate after an oral exposure of 20 weeks in rats where no effects were seen in gonads, the NOAEL of L-alpha terpineol was determined to be higher than 314 mg/kg bw/day.

Executive summary:

In a 20 -weeks oral exposure study, Osborne-Mendel rats (10/dose/sex) were administered alpha-Terpinyl Acetate via diet intake at concentrations of 0 (control), 10000, 2500 and 1000 ppm. Animals were then observed for mortality, weight, food intake and general condition. Haematological examinations were made at termination. At the termination of the experiments the rats were sacrificed and exsanguinated. The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed. Detailed microscopic examinations was done on the animals from the high dose group and the control group. No effect on growth or haematology, and no macroscopic or microscopic change in the tissues in the 10000 ppm exposure group were observed. No effect on growth or haematology, and no macroscopic change in the tissues in the 2500 and 1000 ppm exposure groups. No microscopic examination was performed on rats exposed to 2500 and 1000 ppm. According to the Guidance on information requirements and chemical safety assessment R8 table R 8 -17, rats eat 40 to 50 g/kg bw/day. 10000 ppm is equivalent to 1% in the diet. Consequently, rats exposed to 10000 ppm in the diet consumed between 400 and 500 mg Terpinyl-Acetate-Alpha per kg bodyweight per day. Therefore, the NOAEL was calculated to be higher than 400 mg/kg bw. Based on these results, the read-across approach was applied and the NOAEL of laevo alpha terpineol was determined to be higher than 314 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Key study with Klimisch score = 2

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Key study. Read-across approach. A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of terpineol (CAS 8000-41-7) by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. Terpineol was diluted with corn oil and added to Sprague-Dawley SPF rats (12 per sex per dose) at doses of 0, 100, 300 and 1000 mg/kg bw/day, from day 14 before mating up to 30 days after mating for males (total 44 days) and up to 4 days of lactation for females (total 41-51 days). A non-mating female group (10) was exposed at doses of 0 and 1000 mg/kg bw/day for 44 days. Also, after administration period some animals from the control and high dose groups (5 males of mating group, 5 females of control and 3 females of high dose of non-mating group) were kept for further 14 days without treatment to detect delayed occurrence and recovery from toxic effects.

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, haematology, blood chemistry, urinalysis, 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 1000 mg/kg/day dose group of the mating group female, 9/12 cases were infertile, 2/12 cases were pregnant, and 1/12 cases died on day 3 pregnancy. Female infertility was considered to be caused by the effects found in histopathology of testis for the same dose group: Atrophy (moderate) of seminiferous tubules, multinucleated giant cells (minor to moderate) and vacuolization of seminiferous tubules (minor, mild). For epididymis, reduction of sperm (mild to severe) and cell debris content of the lumen (mild, moderate) were observed in the 1000 mg/kg/day dose group, reflecting the influence on the testes by administration of the test substance. No oestrous cycle abnormalities were found, and no significant difference was observed between the control group and each test substance administration group in the sexual cycle abnormality rate, development of oestrous stages and the average cycle duration. The copulation ratio was 100% for control group and each test substance administration group. There was no significant difference in the number of days required for copulation between the control group and each test substance administration group. Insemination index (males) and fertility index (females) were 100% for control group and for administration doses of 300 mg/kg/day or less. However, in the 1000 mg/kg/day dose group, insemination index (males) was 2/11 (18.2%) and fertility index was 2/10 (20%), with significant difference (p<0.01) from control group. No significant differences were found between the control group and each test substance-administered group in terms of gestation ratio, pregnancy period, number of corpora lutea, implantation number, implantation ratio, delivery ratio, number of stillborn babies and number of live-borns. In two female animals all littermate died on 2nd day of lactation in the 300 and 1000 mg/kg/day dose group, respectively. Since these conditions were supported by the histopathological examination, it is suggested that it was influenced by the administration of the test substance.

Based on these results, the read-across approach was applied and the NOAEL of laevo alpha terpineol for parental reproduction was established at 300 mg/kg bw/day for males and 100 mg/kg bw/day for females, and the NOAEL for development toxicity was determined as the highest dose tested, i.e. 1000 mg/kg bw/day.

Supporting study. Read-across approach. In a 20 -weeks oral exposure study, Osborne-Mendel rats (10/dose/sex) were administered alpha-Terpinyl Acetate via diet intake at concentrations of 0 (control), 10000, 2500 and 1000 ppm. Animals were then observed for mortality, weight, food intake and general condition. Haematological examinations were made at termination. At the termination of the experiments the rats were sacrificed and exsanguinated. The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed. Detailed microscopic examinations was done on the animals from the high dose group and the control group. No effect on growth or haematology, and no macroscopic or microscopic change in the tissues in the 10000 ppm exposure group were observed. No effect on growth or haematology, and no macroscopic change in the tissues in the 2500 and 1000 ppm exposure groups. No microscopic examination was performed on rats exposed to 2500 and 1000 ppm. According to the Guidance on information requirements and chemical safety assessment R8 table R 8 -17, rats eat 40 to 50 g/kg bw/day. 10000 ppm is equivalent to 1% in the diet. Consequently, rats exposed to 10000 ppm in the diet consumed between 400 and 500 mg Terpinyl-Acetate-Alpha per kg bodyweight per day. Therefore, the NOAEL was calculated to be higher than 400 mg/kg bw. Based on these results, the read-across approach was applied and the NOAEL of laevo alpha terpineol was determined to be higher than 314 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Key study. Read-across approach. Test method according to OECD Guideline 422 with GLP. Based on the read-across approach from the analogue terpineol, the NOAEL of laevo alpha terpineol for parental reproduction is determined to be 300 mg/kg bw/day in males and 100 mg/kg bw/day in females, and the NOAEL for development toxicity is concluded to be 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Key study with Klimisch score = 2

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

Key study. Read-across approach. A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of terpineol (CAS 8000-41-7) by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. Terpineol was diluted with corn oil and added to Sprague-Dawley SPF rats (12 per sex per dose) at doses of 0, 100, 300 and 1000 mg/kg bw/day, from day 14 before mating up to 30 days after mating for males (total 44 days) and up to 4 days of lactation for females (total 41-51 days). A non-mating female group (10) was exposed at doses of 0 and 1000 mg/kg bw/day for 44 days. Also, after administration period some animals from the control and high dose groups (5 males of mating group, 5 females of control and 3 females of high dose of non-mating group) were kept for further 14 days without treatment to detect delayed occurrence and recovery from toxic effects.

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, haematology, blood chemistry, urinalysis, 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 1000 mg/kg/day dose group of the mating group female, 9/12 cases were infertile, 2/12 cases were pregnant, and 1/12 cases died on day 3 pregnancy. Female infertility was considered to be caused by the effects found in histopathology of testis for the same dose group: Atrophy (moderate) of seminiferous tubules, multinucleated giant cells (minor to moderate) and vacuolization of seminiferous tubules (minor, mild). For epididymis, reduction of sperm (mild to severe) and cell debris content of the lumen (mild, moderate) were observed in the 1000 mg/kg/day dose group, reflecting the influence on the testes by administration of the test substance. No oestrous cycle abnormalities were found, and no significant difference was observed between the control group and each test substance administration group in the sexual cycle abnormality rate, development of oestrous stages and the average cycle duration. The copulation ratio was 100% for control group and each test substance administration group. There was no significant difference in the number of days required for copulation between the control group and each test substance administration group. Insemination index (males) and fertility index (females) were 100% for control group and for administration doses of 300 mg/kg/day or less. However, in the 1000 mg/kg/day dose group, insemination index (males) was 2/11 (18.2%) and fertility index was 2/10 (20%), with significant difference (p<0.01) from control group. No significant differences were found between the control group and each test substance-administered group in terms of gestation ratio, pregnancy period, number of corpora lutea, implantation number, implantation ratio, delivery ratio, number of stillborn babies and number of live-borns. In two female animals all littermate died on 2nd day of lactation in the 300 and 1000 mg/kg/day dose group, respectively. Since these conditions were supported by the histopathological examination, it is suggested that it was influenced by the administration of the test substance.

Based on these results, the read-across approach was applied and the NOAEL of laevo alpha terpineol for parental reproduction was established at 300 mg/kg bw/day for males and 100 mg/kg bw/day for females, and the NOAEL for development toxicity was determined as the highest dose tested, i.e. 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on the available information, the substance is not classified for toxicity to reproduction in accordance with CLP Regulation (EC) no 1272/2008.