Nerol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 422 (diet) in rats:

LOAEL for maternal toxicity = 3000 ppm (equivalent to 227.6 mg/kg bw/day) based on reduced bodyweight gain in females during gestation associated with low food consumption due to the low palatability of the treated diet.
NOAEL for reproductive toxicity = 12000 ppm (equivalent to 720 mg/kg bw/day) based on no adverse effects on fertility and post-natal development of offspring.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 April 2012 - 13 July 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted according to OECD Guideline 422 with minor deviation: relative humidity in the experimental room was outside the range.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

relative humidity in the experimental room was outside the range

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Han Wistar Rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V., NM Horst, Netherlands
- Age at study initiation: 13 weeks
- Weight at study initiation: Males: 350-437 g; females: 197-232 g
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding with paper enrichment.
- Diet: Microgranulated standard Harlan Teklad 2914C rodent maintenance diet (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: Community tap-water from Füllinsdorf, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.4 – 24.0 °C
- Humidity: 23.6 – 73.4 %
- Air changes: 10 - 15 air changes per hour
- Photoperiod: 12 h dark / 12 h light

Route of administration:

oral: feed

Vehicle:

other: 2 % corn oil feed admixtures

Details on exposure:

DIET PREPARATION
- Dietary admixtures were prepared before the treatment start. The amount of admixture necessary for 7 days was stored frozen at -20 °C in small portions. Feed preparations were administered to the animals each day of treatment after defrosting. At first the appropriate amount of corn oil (as supplied) was added to the appropriate amount of test item Nerol which was weighed into a tared glass baker on a suitable precision balance and mixed together. Then this mixture was added to the diet and mixed in a dietary mixer (Schneider HM100) at a constant speed. Feed preparations were performed separately for each test item treated group. The final concentration of corn oil in the dietary admixture was 2 %. Dietary admixture for control animals was prepared the way that the appropriate amount of corn oil (as supplied) was added to the diet, so that the final concentration of corn oil in the dietary admixture was 2 %.

STABILITY OF TEST ITEM IN VEHICLE
- The test item was stable in feed containing 2 % corn oil at 3000, 6000 and 12000 ppm for 7 days when stored at -20 ± 5 °C and it was stable at room temperature (20 ± 5 °C) for 24 h.

Details on mating procedure:

- M/F ratio per cage: 1: 1
- Length of cohabitation: Up to 14 days
- Proof of pregnancy: Presence of sperm within the vaginal smear and/or copulation plug referred to as Day 0 post coitum.
- If mating was not recorded during a pairing period of a maximum of 14 days, the female was sacrificed 7 days after the last day of the pairing period and the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- For assessment of content and homogeneity, a 100 g sample was collected from the top, middle and bottom of dietary admixture of the respective diet preparation (from group 1 admixtures only one sample was drawn from the middle of the preparation). Samples for assessment of content and homogeneity were collected on the day of the first and last preparation and retained frozen at approximately -20 °C prior to analysis. The samples were analyzed by GS-FID method following an analytical procedure provided by the Sponsor and adapted and validated in Harlan Laboratories (Harlan Laboratories study D49257).
- Results: The diet samples investigated during the study were found to comprise Nerol in the range of 99.3 % to 108.9 % and, thus, the required content limit of ±20 % with reference to the nominal concentration was met. The homogeneous distribution of Nerol in the diet preparations was approved because single results found did not deviate more than 2.7% (acceptance criteria: <15 %) from the corresponding mean.

Duration of treatment / exposure:

Main phase groups (all dose groups)
Males: From Day 1 until and including Day 42 of treatment
Females: From Day 1 of pre-pairing throughout pairing and gestation until Day 6 post partum

Toxicity phase groups (all dose groups)
Females: From Day 1 until and including Day 42 of treatment

Recovery phase groups (control and high dose groups only)
Males and females: From Day 1 until and including Day 42 of treatment then was maintained without treated diet for fourteen days.

Frequency of treatment:

Once daily

Details on study schedule:

None

Remarks:

Doses / Concentrations:
0, 3000, 6000 and 12000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
0, 191.2, 374 and 720 mg/kg bw/day
Basis:
other: equivalent to mean achieved dosages

No. of animals per sex per dose:

Main phase groups (all dose groups)
Males: 10 males/dose for 3000 and 6000 ppm dose groups; 5 males for control and high dose (12000 ppm)
Females: 10 females/dose for control and all dose groups

Toxicity phase groups (all dose groups)
Females: 5 females/dose for control and all dose groups

Recovery phase groups (control and high dose groups only)
5 rats/sex/dose for control and high dose group (12000 ppm)

Control animals:

other: basal laboratory diet with 2 % corn oil added

Details on study design:

- Dose selection rationale: Dose levels were selected based on a previous GLP “fourteen day repeat dose oral (dietary) dose range-finding toxicity/palatability study in the Han Wistar rat”, Study No.: D49235, in which study the treatment was extended up to 21 days using dose levels of 0, 5000, 10000 and 20000 ppm initially. After 6 days of administration the high dose level was reduced to 15000 ppm.
- Rationale for animal assignment: Animals were allocated to dose groups using a randomisation procedure based on body weights (recorded on the day of allocation) which were taken into consideration in order to ensure similar mean body weights in all groups.

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed outside the home cage in all animals, in a standard arena. In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, it was preferred once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on Days 0, 6, 13 and 20 of the gestation period and on Day 6 post partum (except for animals assigned for FOB).
- Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: Once during the study relevant parameters were performed with five main phase males (Day 6 after pairing), five main phase females (Day 3 post partum) and five toxicity phase females (Day 41 of treatment) from each group.
- Animals were observed for the following:
Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
Hand-held observations: muscle tone, constituation, skin, pupil size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.
Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
Reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly; mated females were weighed on Days 0, 7, 14 and 21 post coitum and on Days 1, 4 and 7 post partum.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Daily due to the everyday replacement of feed (it was not measured during pairing).

WATER CONSUMPTION: Yes
- Daily visual inspection

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Main phase males and toxicity phase females (5/dose): Day 42; Recovery phase (all animals): After 13 days without treated diet (Day 13 of recovery period)
- Blood samples were drawn sublingually from all animals under light isoflurane anesthesia.
- Haematology parameters: Haemoglobin, Erythrocyte count, Haematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Leukocyte count (total), Differential leukocyte count, Platelet count, Prothrombin time and Activated partial Thromboplastin time
- Blood Chemistry parameters: Urea, glucose, Creatinine, Bilirubin (total), Cholesterol (total), Triglycerides, Sodium, Potassium, Chloride , Calcium, Inorganic phosphorus, Protein (total), Albumin, Globulin, Albumin/Globulin ratio, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl-transferase and Bile acids

PREGNANCY AND PARTURITION:
- All dams were allowed to give birth and rear their litters (F1 pups) up to Day 7 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight, detailed histological examination of the testes and epididymides with special emphasis on stages of spermatogenesis

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- No. of pups alive was recorded daily
- Litters were examined for litter size, live births, still births and any gross anomalies.
- Sex ratio of the pups
- Pups were weighed individually (without identification) on days 0 (if possible), 1, 4 and 7 post partum.

Postmortem examinations (parental animals):

SACRIFICE
- Main phase group males were sacrificed on Day 43 of study when no longer needed for the assessment of reproductive effects. Main phase group dams were sacrificed on Day 7 post partum. Toxicity phase group females were sacrificed on Day 43 of study. Recovery males and females of control and high dose group animals were sacrificed after 14 days (following a 42 days treatment period) without treated diet. If birth did not occur on the expected date (Day 21 post coitum), the dam was sacrificed on Day 25 post coitum.
- At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital. All main animals were exsanguinated.

GROSS NECROPSY
- All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death.
- Implantation sites and corpora lutea were counted and recorded for all dams with litters.
- For the parent animals, special attention was directed at the organs of the reproductive system.

ORGAN WEIGHTS
- The following organ weights were recorded for all surviving main phase males and toxicity females and recovery phase animals at terminal kill (bilateral organs were weighed separately): Adrenal glands, Brain, Epididymides, Testes, Heart, Kidneys , Uterus (with Cervix and Oviducts), liver, Ovaries, Prostate, Seminal vesicles, Thymus and Spleen
- The following organ weights were recorded for all surviving main phase females at terminal kill: Ovaries (left and right) and Uterus (with Cervix and Oviducts)

HISTOPATHOLOGY
- Samples of the tissues from the main phase males, toxicity phase females, recovery animals and any decedent animals from these phases were preserved in neutral phosphate buffered 4 % formaldehyde solution. Adrenals, Bone and bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Coagulating gland, Colon, Duodenum, Epididymides**, Eyes*, Gross lesions***, Heart, Aorta, Ileum (including Peyer’s patches), Jejunum, Kidneys, Liver, Lungs (with bronchi)#, Lymph nodes (mandibular and mesenteric), Muscle (skeletal), Oesophagus, Ovaries***, Pituitary, Prostate, Rectum, Seminal vesicles, Skin with mammary gland, Spinal cord (cervical, mid-thoracic and lumbar), Spleen, Stomach, Sciatic nerve, Testes**, Thymus Thyroid/parathyroid, Trachea, Urinary bladder, Uterus (with Cervix and Oviducts) and Vagina***
- All organ and tissue samples were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Histopathology slides of all organs, gross lesions and tissues listed collected at terminal sacrifice from the animals were examined. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

* Eyes fixed in Davidson’s fluid (without the optic nerve and Harderian gland)
** Testes and Epididymides were fixed in Bouin’s solution.
*** Tissues preserved in neutral phosphate buffered 4 % formaldehyde solution.
# Lungs inflated to approximately normal inspiratory volume with 4 % formalin before immersion in fixative

Postmortem examinations (offspring):

SACRIFICE:
- Pups were sacrificed on Day 7 post partum.

GROSS NECROPSY
- All offspring were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

The following statistical methods were used to analyze food consumption, body weights, reproduction data, parental organ weights, grip strength, locomotor activity, body temperature and clinical laboratory investigation:
- Means and standard deviations of various data were calculated.
- Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

Percentage mating = (Females mated / Females paired) * 100
Fertility index = (Females achieving a pregnancy / Females paired) * 100
Conception rate = (Females achieving a pregnancy / Females mated) * 100
Gestation index = (Number of females with living pups / Number of females pregnant) * 100

Offspring viability indices:

Birth index = (number of pups born alive / number of implantations) * 100
Viability index = (number of alive pups on Day 4 p.p. / number of pups born alive) * 100
Weaning index = (number of alive pups on Day 21 p.p. / number of alive pups on Day 4 p.p.) * 100

Clinical signs:

no effects observed

Description (incidence and severity):

One toxicity phase female was killed in extremis due to paralysis of both hind legs. This was considered to be incidental. All other animals survived the scheduled study period. No clinical signs or observations which were considered to be test item-related were noted in males or females at any dose level.

Mortality:

no mortality observed

Description (incidence):

One toxicity phase female was killed in extremis due to paralysis of both hind legs. This was considered to be incidental. All other animals survived the scheduled study period. No clinical signs or observations which were considered to be test item-related were noted in males or females at any dose level.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Reduced overall body weight gain was evident in main phase males, main phase females, toxicity phase females treated with 12000 ppm.
- In males, bodyweight gain was globally similar to controls at 3000 ppm and 6000 ppm. However, in the 12000 ppm group, the reduction in bodyweight gain was particularly high during the first week of treatment (80 % decrease compared to controls) and remained lower than controls throughout the treatment period (17 % decrease compared to controls between Days 8-42). During the recovery period, previously treated males at 12000 ppm showed a 50 % increase in bodyweight gain compared to controls.
- During the pre-pairing period, females showed a decrease in bodyweight gain, especially during the first week of the treatment. From Day 8 to the end of the study, toxicity group females showed similar mean bodyweight gain when compared to controls at 3000 and 6000 ppm but a 25 % decrease in mean bodyweight gain compared to controls at 12000 ppm. During the recovery phase, their mean bodyweight gain was 3 times higher than the concurrent control group.
- During gestation period, main phase females showed an overall decrease in mean bodyweight gain at 3000, 6000 and 12000 ppm (11, 6 and 33 % decrease respectively). This decrease was mainly observed during the last week of gestation. This decrease in bodyweight gains was also observed during lactation period with 50 % decrease in mean bodyweight gain in all treated groups compared to controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Generally, food consumption was reduced during the first days of treatment in all treated groups. This reduction in food intake was maintained at the high dose throughout the treatment period in both males and females but particularly for main phase females. This reduction in food consumption was considered to reflect a reluctance to eat the diet admixture due to its low palatability, particularly at the high dose.
- During the lactation period, the dietary intake of main phase females was reduced (up to 37 % decrease compared to controls at 12000 ppm) in all test item treated groups. In main phase females, the overall reduction of food consumption throughout the study was 7, 11 and 23 % of control group for 3000, 6000 and 12000 ppm groups, respectively.

Food efficiency:

not specified

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

no effects observed

Description (incidence and severity):

WATER CONSUMPTION:
Daily visual inspection of water bottles did not reveal any significant intergroup differences.

TEST SUBSTANCE INTAKE:
Mean Achieved Dosages:
Males:
Main phase males pre-pairing period: 179.8, 360.3 and 671.8 mg/kg bw/day
Main phase males after pairing period: 156.3, 318.2 and 621.3 mg/kg bw/day
Recovery phase males treatment period: 670.4 mg/kg bw/day

Females:
Main phase females pre-pairing period: 221.7, 415.7 and 778.7 mg/kg bw/day
Main phase females gestation period: 227.6, 442.0 and 796.4 mg/kg bw/day
Main phase females lactation period: 328.3, 702.3 and 1152.0 mg/kg bw/day
Toxicity phase females treatment period: 207.0, 401.9 and 784.1 mg/kg bw/day
Recovery phase females treatment period: 793.8 mg/kg bw/day

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

- Treatment with the test item at the dose levels of 12000 ppm caused a statistically significant decrease of total bilirubin, sodium level, globulin and triglycerides, and increase in creatinine, ALP and albumin in males and decreased potassium level in recovery phase females. Similar trends were observed in toxicity female groups without reaching statistical significance.
- No further changes of biochemical blood parameters which were considered to be test item-related were found in males or females at any dose level.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- No findings, which were considered to be test item-related, were noted during functional observational battery testing in males or females at any dose level.
- No test item-related effects on locomotor activity of males or females were observed at any dose level.

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):

- Microscopic examination of liver sections revealed minimal centrilobular and partially reversible liver cell hypertrophy in all males. After recovery, the minimal liver cell hypertrophy in two males was associated with consequent minimal diffuse follicular cell hypertrophy in thyroid gland in one male.
- Microscopic examination also revealed possibly adverse effects on the kidneys of main phase males at 12000 ppm: slight increase in tubular hyaline droplets and minimal increase in tubular basophilia which was partially reversible in two weeks. These findings were the result of an increase in the renal content of alpha-2μglobulin; they are considered to be adverse in the young mature male rats but they are not considered to be predictive for a risk to humans.
- After two weeks of recovery, minimal myeloid hyperplasia in sternum bone marrow with mature myeloid elements occurred in four of the five males at 12000 ppm. This finding was minimal in nature and was also reported in one male of control and high dose groups. Therefore it could be considered as an unspecific reactive change.
- A single high dose toxicity phase female showed a minimal diffuse hypertrophy of adrenal cortices. In the majority of recovery phase females at 12000 ppm, a minimal to slight diffuse hypertrophy of adrenal cortices also occurred. This finding may be due to better feed consumption and a drastic increase in bodyweight gain during the recovery period after a long period of feed restriction, rather than an effect of the test-item.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No treatment related effects on mating, fertility and gestation Length were detected.

Reproductive performance:

no effects observed

Description (incidence and severity):

No treatment related effects on mating, fertility and gestation Length were detected.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- One toxicity phase female was killed in extremis due to paralysis of both hind legs. This was considered to be incidental. All other animals survived the scheduled study period. No clinical signs or observations which were considered to be test item-related were noted in males or females at any dose level.

BODY WEIGHT (PARENTAL ANIMALS)
- Reduced overall body weight gain was evident in main phase males, main phase females, toxicity phase females treated with 12000 ppm.
- In males, bodyweight gain was globally similar to controls at 3000 ppm and 6000 ppm. However, in the 12000 ppm group, the reduction in bodyweight gain was particularly high during the first week of treatment (80 % decrease compared to controls) and remained lower than controls throughout the treatment period (17 % decrease compared to controls between Days 8-42). During the recovery period, previously treated males at 12000 ppm showed a 50 % increase in bodyweight gain compared to controls.
- During the pre-pairing period, females showed a decrease in bodyweight gain, especially during the first week of the treatment. From Day 8 to the end of the study, toxicity group females showed similar mean bodyweight gain when compared to controls at 3000 and 6000 ppm but a 25 % decrease in mean bodyweight gain compared to controls at 12000 ppm. During the recovery phase, their mean bodyweight gain was 3 times higher than the concurrent control group.
- During gestation period, main phase females showed an overall decrease in mean bodyweight gain at 3000, 6000 and 12000 ppm (11, 6 and 33 % decrease respectively). This decrease was mainly observed during the last week of gestation. This decrease in bodyweight gains was also observed during lactation period with 50 % decrease in mean bodyweight gain in all treated groups compared to controls.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- Generally, food consumption was reduced during the first days of treatment in all treated groups. This reduction in food intake was maintained at the high dose throughout the treatment period in both males and females but particularly for main phase females. This reduction in food consumption was considered to reflect a reluctance to eat the diet admixture due to its low palatability, particularly at the high dose.
- During the lactation period, the dietary intake of main phase females was reduced (up to 37 % decrease compared to controls at 12000 ppm) in all test item treated groups. In main phase females, the overall reduction of food consumption throughout the study was 7, 11 and 23 % of control group for 3000, 6000 and 12000 ppm groups, respectively.

WATER CONSUMPTION (PARENTAL ANIMALS)
- Daily visual inspection of water bottles did not reveal any significant intergroup differences.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Mean Achieved Dosages:
Males:
Main phase males pre-pairing period: 179.8, 360.3 and 671.8 mg/kg bw/day
Main phase males after pairing period: 156.3, 318.2 and 621.3 mg/kg bw/day
Recovery phase males treatment period: 670.4 mg/kg bw/day

Females:
Main phase females pre-pairing period: 221.7, 415.7 and 778.7 mg/kg bw/day
Main phase females gestation period: 227.6, 442.0 and 796.4 mg/kg bw/day
Main phase females lactation period: 328.3, 702.3 and 1152.0 mg/kg bw/day
Toxicity phase females treatment period: 207.0, 401.9 and 784.1 mg/kg bw/day
Recovery phase females treatment period: 793.8 mg/kg bw/day

NEUROBEHAVIOURAL EXAMINATION (PARENTAL ANIMALS)
- No findings, which were considered to be test item-related, were noted during functional observational battery testing in males or females at any dose level.
- No test item-related effects on locomotor activity of males or females were observed at any dose level.

CLINICAL LABORATORY INVESTIGATIONS (PARENTAL ANIMALS)
- Treatment with the test item at the dose levels of 12000 ppm caused a statistically significant decrease of total bilirubin, sodium level, globulin and triglycerides, and increase in creatinine, ALP and albumin in males and decreased potassium level in recovery phase females. Similar trends were observed in toxicity female groups without reaching statistical significance.
- No further changes of biochemical blood parameters which were considered to be test item-related were found in males or females at any dose level.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- No treatment related effects on mating, fertility and gestation Length were detected.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Uterus and oviduct absolute weights of main phase females were statistically significantly lower than controls in all treated groups of main phase females without dose dependency. A decrease in mean left ovary weight, reaching statistical significance at 12000 ppm, was observed in all treated groups of main phase females without dose dependency. However, there were no associated findings at macroscopic and microscopic examinations of ovaries and uterus, and no similar decreased organ weights were reported in toxicity phase females. Therefore, these findings were not considered to be related to the treatment with the test item.
- Recovery phase females had statistically significantly higher absolute and relative adrenal weights (bilateral). Microscopic examination revealed diffuse cortical hypertrophy in a single toxicity phase female and in the majority of recovery phase females. The increase in adrenal weights and minimal diffuse cortical hypertrophy could be more likely related to stressful experimental conditions at 12000 ppm, especially in the recovery females group: these females experienced higher food intake and a 3 times higher increase in bodyweight gain compared to controls during the recovery period.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- During macroscopical examination, at 12000 ppm, three main phase group males revealed an enlarged liver that correlated with slight centrilobular hypertrophy of liver cells which was considered to be test item-related. Furthermore treatment-related irritant effects were present in forestomach of main phase males and toxicity phase females.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- Microscopic examination of liver sections revealed minimal centrilobular and partially reversible liver cell hypertrophy in all males. After recovery, the minimal liver cell hypertrophy in two males was associated with consequent minimal diffuse follicular cell hypertrophy in thyroid gland in one male.
- Microscopic examination also revealed possibly adverse effects on the kidneys of main phase males at 12000 ppm: slight increase in tubular hyaline droplets and minimal increase in tubular basophilia which was partially reversible in two weeks. These findings were the result of an increase in the renal content of alpha-2μglobulin; they are considered to be adverse in the young mature male rats but they are not considered to be predictive for a risk to humans.
- After two weeks of recovery, minimal myeloid hyperplasia in sternum bone marrow with mature myeloid elements occurred in four of the five males at 12000 ppm. This finding was minimal in nature and was also reported in one male of control and high dose groups. Therefore it could be considered as an unspecific reactive change.
- A single high dose toxicity phase female showed a minimal diffuse hypertrophy of adrenal cortices. In the majority of recovery phase females at 12000 ppm, a minimal to slight diffuse hypertrophy of adrenal cortices also occurred. This finding may be due to better feed consumption and a drastic increase in bodyweight gain during the recovery period after a long period of feed restriction, rather than an effect of the test-item.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

6 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

12 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed on fertility and post-natal development of offspring

Dose descriptor:

LOAEL

Remarks:

maternal toxicity

Effect level:

3 000 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on the decrease in bodyweight gain at 3000 ppm during the last week of gestation (-17 % compared to controls) and during early lactation (-67 % compared to controls, associated with a 20 % decrease in food consumption)

Clinical signs:

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup weights were comparable to controls at 12000 ppm on post natal Days 1 and 4 and were slightly reduced on Day 7 without reaching statistical significance. Mean pup weights were comparable to controls at 3000 and 6000 ppm.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

no effects observed

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

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Offspring litter Size and Viability:
- Implantation sites were slightly reduced at 3000 and 12000 ppm without reaching statistical significance. A significant dose-related increase in post-implantation loss was observed with the mean values outside the historical control data at mid and high doses. This change was considered to be test item-related. Fewer offspring was born in all test item-treated groups without dose dependency and without reaching statistical significance and was considered to be related to the other findings.
- Viability of offspring was not affected by the treatment with the test item.

Offspring Growth and Development:
- Mean pup weights were comparable to controls at 12000 ppm on post natal Days 1 and 4 and were slightly reduced on Day 7 without reaching statistical significance. Mean pup weights were comparable to controls at 3000 and 6000 ppm.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

3 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Increased post-implantation loss at 6000 ppm and above

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

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:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Reproductive effects observed:

not specified

None

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 6000 ppm based on the decrease in bodyweight gain in toxicity phase females and in males. The NOAEL on reproductive toxicity was 12000 ppm (highest dose tested) because no adverse effects were observed on fertility and post-natal development of offspring. The NOAEL for developmental toxicity was 3000 ppm based on increased post-implantation loss at 6000 ppm and above. The LOAEL for maternal toxicity was 3000 ppm based on the decrease in bodyweight gain at 3000 ppm during the last week of gestation.

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, nerol was administered by dietary admixture (initially mixed with 2% corn oil to avoid evaporation) to three groups of Han Wistar rats for up to 42 consecutive days for main phase males, toxicity females and recovery animals, and between 41 and 53 days (including three weeks exposure phase, pairing, gestation and early lactation) for main phase females, at dietary concentrations of 3000, 6000 and 12000 ppm (equivalent to a global mean achieved dosage of 191.2, 374 and 720 mg/kg bw/day, respectively). Each dose group was subdivided into following phases: main phase (males: 10 animals/dose for 3000 and 6000 ppm dose groups; 5 males/dose for control and high dose (12000 ppm); females: 10 animals/dose for control and all dose groups) and toxicity phase (5 female/dose). Two recovery groups (5 rats/sex/dose) were treated with 12000 ppm or basal laboratory diet alone for 42 consecutive days and then maintained without treatment for a further 14 days. A control group was treated with basal laboratory diet (with 2% corn oil). During the study, data was recorded on mortality, clinical signs, behavioural assessments, body weight change, food and water consumption, haematology, blood chemistry, mating performance, fertility and gestation length. All animals were subjected to a gross necropsy examination, selected organs were weighed and histopathological evaluation of selected tissues was performed. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.

No unscheduled deaths or treatment-related clinical signs were noted. No treatment-related effects were noted on behavioural, sensory reactivity and functional performance parameters. Reduced overall body weight gain was evident in main phase males, main phase females, toxicity phase females treated with 12000 ppm. In males, bodyweight gain was similar to controls at 3000 and 6000 ppm. However, at 12000 ppm, the reduction in bodyweight gain was particularly high during the first week of treatment (80% decrease compared to controls) and remained lower than controls throughout the treatment period (17% decrease compared to controls between Days 8-42). During the recovery period, previously treated males at 12000 ppm showed a 50% increase in bodyweight gain compared to controls. During the pre-pairing period, females showed a decrease in bodyweight gain, especially during the first week of the treatment. From Day 8 to the end of the study, toxicity group females showed similar mean bodyweight gain when compared to controls at 3000 and 6000 ppm but a 25% decrease in mean bodyweight gain compared to controls at 12000 ppm. During the recovery phase, their mean bodyweight gain was 3 times higher than the concurrent control group. During gestation period, main phase females showed an overall decrease in mean bodyweight gain at 3000, 6000 and 12000 ppm (11, 6 and 33% decrease, respectively). This decrease was mainly observed during the last week of gestation. This decrease in bodyweight gains was also observed during lactation period (but without dose dependency) with about 50 % decrease in mean bodyweight gain in all treated groups compared to controls. Generally, food consumption was reduced during the first days of treatment in all treated groups. This reduction in food intake was maintained at the high dose throughout the treatment period in both males and females but particularly for main phase females. This reduction in food consumption was considered to reflect a reluctance to eat the diet admixture due to its low palatability, particularly at the high dose. During the lactation period, the dietary intake of main phase females was reduced (up to 37% decrease compared to controls at 12000 ppm) in all test item treated groups. In main phase females, the overall reduction of food consumption throughout the study was 7, 11 and 23% of control group for 3000, 6000 and 12000 ppm groups, respectively. Water consumption did not reveal any significant intergroup differences. At 12000 ppm, a statistically significant decrease of total bilirubin, sodium level, globulin and triglyceride, and increase in creatinine, ALP and albumin in males and decreased potassium level in recovery phase females. Similar trends were observed in toxicity female groups without reaching statistical significance. Uterus and oviduct absolute weights of main phase females were statistically significantly lower than controls in all treated groups, without dose dependency. A decrease in mean left ovary weight, reaching statistical significance at 12000 ppm, was observed in all treated groups of main phase females without dose dependency. However, there were no associated findings at macroscopic and microscopic examinations of ovaries and uterus, and no similar decreased organ weights were reported in toxicity phase females. Therefore, these findings were not considered to be related to the treatment with the test item. Recovery phase females had statistically significantly higher absolute and relative adrenal weights (bilateral). Microscopic examination revealed diffuse cortical hypertrophy in a single toxicity phase female and in the majority of recovery phase females. The increase in adrenal weights and minimal diffuse cortical hypertrophy could be more likely related to stressful experimental conditions at 12000 ppm, especially in the recovery females group: these females experienced higher food intake and a 3 times higher increase in bodyweight gain compared to controls during the recovery period. During macroscopical examination, at 12000 ppm, three main phase group males revealed an enlarged liver that correlated with slight centrilobular hypertrophy of liver cells which was considered to be test item-related. Furthermore treatment-related irritant effects were present in forestomach of main phase males and toxicity phase females. Histopathology revealed microscopic abnormalities in liver (minimal centrilobular and partially reversible liver cell hypertrophy in all males) and thyroid (minimal diffuse follicular cell hypertrophy in males) at 12000 ppm. At 12000 ppm, partly reversible changes in kidney (slight increase in tubular hyaline droplets and minimal increase in tubular basophilia) were observed in main phase and recovery males. These kidney effects were considered to be related to alpha-2µglobulin nephropathy and of no relevance to humans. No treatment-related significant effects were noted on offspring litter size, sex ratio, viability, growth and development.

No treatment-related effects were detected in mating performance, fertility and gestation length. Implantation sites were slightly reduced at 3000 and 12000 ppm without reaching statistical significance and without dose dependency. A significant dose-related increase in post-implantation loss was observed with the mean values outside the historical control data at mid and high doses. Fewer offspring was born in all test item-treated groups without dose dependency and without reaching statistical significance. This was considered to be related to the other findings. Mean pup weights were comparable to controls at 12000 ppm on post natal Days 1 and 4 and were slightly reduced on Day 7 without reaching statistical significance. Mean pup weights were comparable to controls at 3000 and 6000 ppm.

Therefore, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 6000 ppm based on the decrease in bodyweight gain in toxicity phase females and in males at the highest dose tested. The LOAEL for maternal toxicity was 3000 ppm based on the decrease in bodyweight gain at 3000 ppm during the last week of gestation. The NOAEL on reproductive toxicity was 12000 ppm (highest dose tested) because no adverse effects were observed on fertility and post-natal development of offspring. The NOAEL for developmental toxicity was 3000 ppm based on increased post-implantation loss at 6000 ppm and above.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

720 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP study conducted according to OECD Guideline 422 without any deviation (Klimisch score = 1).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, nerol was administered by dietary admixture (initially mixed with 2 % corn oil to avoid evaporation) to three groups of Han Wistar rats for up to 42 consecutive days for main phase males, toxicity females and recovery animals, and between 41 and 53 days (including three weeks exposure phase, pairing, gestation and early lactation) for main phase females, at dietary concentrations of 3000, 6000 and 12000 ppm (equivalent to a global mean achieved dosage of 191.2, 374 and 720 mg/kg bw/day, respectively). Each dose group was subdivided into the following phases: main phase (males: 10 animals/dose for 3000 and 6000 ppm dose groups; 5 males/dose for control and high dose (12000 ppm); females: 10 animals/dose for control and all dose groups) and toxicity phase (5 female/dose). Two recovery groups (5 rats/sex/dose) were treated with 12000 ppm or basal laboratory diet alone for 42 consecutive days and then maintained without treatment for a further 14 days. A control group was treated with basal laboratory diet (with 2 % corn oil). During the study, data was recorded on mortality, clinical signs, behavioural assessments, body weight change, food and water consumption, haematology, blood chemistry, mating performance, fertility and gestation length. All animals were subjected to a gross necropsy examination, selected organs were weighed and histopathological evaluation of selected tissues was performed. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.

No unscheduled deaths or treatment-related clinical signs were noted. No treatment-related effects were noted on behavioural, sensory reactivity and functional performance parameters. Reduced overall body weight gain was evident in main phase males, main phase females, toxicity phase females treated with 12000 ppm. In males, bodyweight gain was similar to controls at 3000 and 6000 ppm. However, at 12000 ppm, the reduction in bodyweight gain was particularly high during the first week of treatment (80 % decrease compared to controls) and remained lower than controls throughout the treatment period (17 % decrease compared to controls between Days 8-42). During the recovery period, previously treated males at 12000 ppm showed a 50 % increase in bodyweight gain compared to controls. During the pre-pairing period, females showed a decrease in bodyweight gain, especially during the first week of the treatment. From Day 8 to the end of the study, toxicity group females showed similar mean bodyweight gain when compared to controls at 3000 and 6000 ppm but a 25 % decrease in mean bodyweight gain compared to controls at 12000 ppm. During the recovery phase, their mean bodyweight gain was 3 times higher than the concurrent control group. During gestation period, main phase females showed an overall decrease in mean bodyweight gain at 3000, 6000 and 12000 ppm (11, 6 and 33 % decrease, respectively). This decrease was mainly observed during the last week of gestation. This decrease in bodyweight gains was also observed during lactation period (but without dose dependency) with about 50 % decrease in mean bodyweight gain in all treated groups compared to controls. Generally, food consumption was reduced during the first days of treatment in all treated groups. This reduction in food intake was maintained at the high dose throughout the treatment period in both males and females but particularly for main phase females. This reduction in food consumption was considered to reflect a reluctance to eat the diet admixture due to its low palatability, particularly at the high dose. During the lactation period, the dietary intake of main phase females was reduced (up to 37 % decrease compared to controls at 12000 ppm) in all test item treated groups. In main phase females, the overall reduction of food consumption throughout the study was 7, 11 and 23 % of control group for 3000, 6000 and 12000 ppm groups, respectively. Water consumption did not reveal any significant intergroup differences. At 12000 ppm, a statistically significant decrease of total bilirubin, sodium level, globulin and triglyceride, and increase in creatinine, ALP and albumin in males and decreased potassium level in recovery phase females. Similar trends were observed in toxicity female groups without reaching statistical significance. Uterus and oviduct absolute weights of main phase females were statistically significantly lower than controls in all treated groups, without dose dependency. A decrease in mean left ovary weight, reaching statistical significance at 12000 ppm, was observed in all treated groups of main phase females without dose dependency. However, there were no associated findings at macroscopic and microscopic examinations of ovaries and uterus, and no similar decreased organ weights were reported in toxicity phase females. Therefore, these findings were not considered to be related to the treatment with the test item. Recovery phase females had statistically significantly higher absolute and relative adrenal weights (bilateral). Microscopic examination revealed diffuse cortical hypertrophy in a single toxicity phase female and in the majority of recovery phase females. The increase in adrenal weights and minimal diffuse cortical hypertrophy could be more likely related to stressful experimental conditions at 12000 ppm, especially in the recovery females group: these females experienced higher food intake and a 3 times higher increase in bodyweight gain compared to controls during the recovery period. During macroscopical examination, at 12000 ppm, three main phase group males revealed an enlarged liver that correlated with slight centrilobular hypertrophy of liver cells which was considered to be test item-related. Furthermore treatment-related irritant effects were present in forestomach of main phase males and toxicity phase females. Histopathology revealed microscopic abnormalities in liver (minimal centrilobular and partially reversible liver cell hypertrophy in all males) and thyroid (minimal diffuse follicular cell hypertrophy in males) at 12000 ppm. At 12000 ppm, partly reversible changes in kidney (slight increase in tubular hyaline droplets and minimal increase in tubular basophilia) were observed in main phase and recovery males. These kidney effects were considered to be related to alpha-2µglobulin nephropathy and of no relevance to humans. No treatment-related significant effects were noted on offspring litter size, sex ratio, viability, growth and development.

No treatment-related effects were detected in mating performance, fertility and gestation length. Implantation sites were slightly reduced at 3000 and 12000 ppm without reaching statistical significance and without dose dependency. A significant dose-related increase in post-implantation loss was observed with the mean values outside the historical control data at mid and high doses. Fewer offspring was born in all test item-treated groups without dose dependency and without reaching statistical significance. This was considered to be related to the other findings. Mean pup weights were comparable to controls at 12000 ppm on post natal Days 1 and 4 and were slightly reduced on Day 7 without reaching statistical significance. Mean pup weights were comparable to controls at 3000 and 6000 ppm.

Therefore, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 6000 ppm based on the decrease in bodyweight gain in toxicity phase females and in males at the highest dose tested. The LOAEL for maternal toxicity was 3000 ppm based on the decrease in bodyweight gain at 3000 ppm during the last week of gestation. The NOAEL on reproductive toxicity was 12000 ppm (highest dose tested) because no adverse effects were observed on fertility and post-natal development of offspring. The NOAEL for developmental toxicity was 3000 ppm based on increased post-implantation loss at 6000 ppm and above.

Effects on developmental toxicity

Description of key information

OECD 414 (gavage) in rats:

NOAEL for maternal toxicity = 750 mg/kg bw/day (highest dose tested)

NOAEL for developmental toxicity = 750 mg/kg bw/day (highest dose tested)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

August - September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

The purpose of this study was to assess the influence of Nerol on embryo-fetal survival and development in the Crl:CD(SD) rat in order to establish suitable doses for a main embryo fetal toxicity study.

GLP compliance:

no

Remarks:

but generally followed Good Laboratory Practice principles.

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited
- Age at study initiation: 69 to 76 days old
- Weight at study initiation: 243 to 275 g
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods. Up to 4 animals were in the same cage during acclimatization period, and animla were house alone during the gestation.
Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing. There were only one male and one female in the same cage during paring.
- Diet (ad libitum): SDS VRF1 Certified pelleted diet
- Water (ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes.
- Acclimation period: Five days before commencement of pairing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%.
- Air changes : Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light):12 hours light : 12 hours dark

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The oral gavage route of administration was chosen to simulate the conditions of potential human exposure.
A dose of 5 mL/kg body weight was used.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
- M/F ratio per cage: 1:1 with identified stock males.
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm. Day 0 of gestation was considered when positive evidence of mating was detected.

Frequency of treatment:

Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 female per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest dose of 1000 mg/kg bw/day was selected, as this is the limit dose for a study of this type and as diets containing 12000 ppm were tolerated by female Han Wistar rats on an OECD 422. In addition, the oral LD50 is documented as 4500 mg/kg bw in the rat. Intermediate and low dose levels of 500 and 250 mg/kg bw/day, respectively, were selected in order to investigate the response at lower doses.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s). During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 0, 5, 12, 18, and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

FOOD CONSUMPTION : Yes
- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Animals were killed on Day 20 after mating.
- Organs examined:Uterus, and each ovary / uterine horn. The liver and kidneys from each adult female were weighed.

OTHER:
- Hematology, Peripheral Blood : Blood samples were collected prior to dosing on Day 17 after mating on all animals. Blood samples were examined for the following characteristics using a Bayer Advia 120 analyzer: Hematocrit (Hct), Hemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell hemoglobin (MCH), Mean cell hemoglobin concentration (MCHC), Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count: (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M)), Large unstained cells (LUC), Platelet count (Plt), Prothrombin time (PT) and Activated partial thromboplastin time (APTT) .
- Reproductive assessment : see Any other information on materials and methods.

Ovaries and uterine content:

The following were recorded for all animals:
- Uterus: Gravid uterine weight (including cervix and ovaries).
- For each ovary/uterine horn: Number of: corpora lutea, implantation sites, resorption sites (classified as early or late) and fetuses (live and dead).

Fetal examinations:

- External examinations: Yes: All fetuses and placentae were dissected from the uterus and weighed individually. The sex of each fetus was recorded with grossly normal fetuses discarded.

Statistics:

Where appropriate, group mean values, each with standard deviation (SD), were calculated from individual data.

Indices:

Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:
Pre-implantation loss (%) = ((Number of corpora lutea - Number of implantations) / Number of corpoea lutea) X 100

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).
Post-implantation loss was calculated from the formula:

Post-implantation loss (%) = ((Number of implantations - Number of live fetuses)/ Number of implantations) X 100

All group values and SD (as appropriate) were calculated from the individual litter values.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation was observed after dosing in all animals receiving 500 or 1000 mg/kg bw/day, and in four out of five females receiving 250 mg/kg/day. Subsequent post-salivation staining was observed in three animals at 500 mg/kg/day and two animals receiving 1000 mg/kg bw/day. Associated chin rubbing was also observed in a single female receiving 500 mg/kg bw/day and in two females at 1000 mg/kg bw/day. These signs are considered to be due to general distaste, and the corn oil vehicle component of the formulation rather than an effect of treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During Days 6-7 of gestation slight mean body weight loss was observed for females receiving 1000 mg/kg bw/day, and females at 500 mg/kg bw/day showed mean body weight stasis, compared to weight gain in the Controls. At 250 mg/kg bw/day females mean body weight gain during Days 6-7 of gestation was slightly lower than Controls. Subsequent mean body weight gain during gestation was generally similar to Controls at all dose levels investigated and unaffected by treatment.
The magnitude of these effects is unlikely to be considered adverse on a main study of embryo-fetal development.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean adjusted kidney and liver weights of adult females appear to increase across all treated groups when compared to Controls, with a dose response evident. The magnitude of these effects is unlikely to be considered adverse on a main study of embryo-fetal development.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

At 1000 or 500 mg/kg bw/day, the mean percentage of pre implantation loss was higher than in Controls while the mean numbers of resorptions (post implantation loss) was lower than in Controls. At 1000 mg/kg bw/day, the overall difference between the mean number of corpora lutea and live fetuses (a total crude measure of survival of the conceptuses) was 1.4 compared with 2.0 in Controls. At 250 mg/kg bw/day pre-implantation loss was similar to Controls.
Mean post-implantation loss, numbers of early, late and total resorptions, and percentages sex ratio were similar to or lower than Control values at all dose levels.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean litter weight and male, female and overall fetal weights at 500 or 1000 mg/kg bw/day were lower than Controls, although there was no dose response evident.
Mean litter weight and male, female and overall fetal weights at 250 mg/kg bw/day were considered to be similar to Controls and unaffected by treatment.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

not examined

Skeletal malformations:

not examined

Visceral malformations:

not examined

Treatment of pregnant female Sprague Dawley rats with nerol daily from Day 6 to Day 19 after mating, inclusive, at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated, with no deaths nor any toxicity-related changes in the clinical condition of the adult females observed. Signs of chin rubbing and salivation were considered to relate to general distaste of the formulation rather than any effect of toxicity.

At 1000 mg/kg bw/day, there was evidence that some females were more susceptible to dosing with Nerol towards the end of gestation, with two females showing signs including piloerection and/or underactivity and flat posture on Day 19 of gestation.

Treatment elicited slight body weight loss or reduced maternal weight gain at all dose levels, and slightly lower food consumption at 500 or 1000 mg/kg bw/day, following the first dose. Adjusted kidney and liver weights of adult females were also increased across all treated groups, with a dose response evident. The magnitude of these effects is unlikely to be considered adverse on a main study of embryo-fetal development.

Therefore, there was considered to be no effect of treatment on embryo-fetal survival at any of the dose levels investigated. Fetal weights at 500 or 1000 mg/kg bw/day appeared low, although there was no dose response evident.

Conclusions:

At the high dose of 1000 mg/kg bw/day in this preliminary study, there was evidence that some females were more susceptible to dosing with Nerol towards the end of gestation with two females showing signs including piloerection, and/or under-activity and flat posture. It was considered possible that susceptible females could show marked post dosing signs towards the end of gestation on a main study with a greater group size, which could necessitate humane kill of affected females. At dose levels of 500 or 1000 mg/kg bw/day on the TS59TC study, slightly low overall body weight gain and transient body weight loss at 1000 mg/kg bw/day, as well as lower food consumption than in Controls, were observed during the first few days of treatment. A lower high dose of 750 mg/kg bw/day was therefore selected for the main study to reduce the possibility of marked signs being observed during late gestation; this dose was anticipated to fulfil regulatory expectations for some signs of toxicity at the high dose, manifest as reduced body weight gain and food consumption, following the start of treatment.

Executive summary:

The purpose of this study was to assess the influence of nerol on embryo-fetal survival and development in the Crl:CD (SD) rat in order to establish suitable doses for a main embryo‑fetal toxicity study.

Three groups of five females received Nerol at doses of 250, 500 or 1000 mg/kg bw/day by oral gavage administration at 5 mL/kg, daily from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil at the same volume-dose as the treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Blood samples were obtained on Day 17 of gestation for hematology investigations. Adult females were examined macroscopically at necropsy on Day 20 after mating and organ weights were recorded. All fetuses were examined macroscopically at necropsy.

Treatment was well tolerated, with no deaths nor any toxicity related changes in the clinical condition of the adult females observed. Signs of chin rubbing and salivation at 1000 or 500 mg/kg bw/day, and to a lesser extent 250 mg/kg bw/day, were considered to relate to general distaste of the formulation rather than any effect of toxicity. At 1000 mg/kg bw/day, there was evidence that some females were more susceptible to dosing with nerol towards the end of gestation with two females showing signs including piloerection, and/or under-activity and flat posture. It was considred possible that susceptible females could show marked post-dosing signs towards the end of gestation on a main study with a greater group size, which could necessitate human kill of affected females. 

After the first dose (during Days 6-7 of gestation), mean bodyweight loss was observed at 1000 mg/kg bw/day, body weight stasis was observed at 500 mg/kg bw/day and reduced body weight gain was observed at 250 mg/kg bw/day, compared to weight gain in the Controls. Thereafter, body weight gain at all dose levels was similar to Controls. Body weight gain after adjustment for the contribution of the gravid uterine weight was lower than Controls in all treated groups, indicating a slight effect of treatment upon the maternal portion of gestational body weight gain at all dose levels, primarily due to the effect observed following the first dose.

Mean food consumption during Days 6-9 of gestation were slightly low for females receiving 500 or 1000 mg/kg bw/day. Thereafter, mean values of food consumption were similar to Controls. Food consumption during gestation at 250 mg/kg bw/day was unaffected.

There was no clear or consistent effect of treatment upon haematology parameters at any of the dose levels investigated.

Adjusted kidney and liver weights of adult females were increased across all treated groups, with a dose response evident.

There were no maternal or fetal findings at macroscopic examination that were considered to relate to treatment.

Therefore, there was no clear or conclusive effect of treatment on embryo-fetal survival at any of the dose levels investigated. Mean litter weight and male, female and overall fetal weights at 500 or 1000 mg/kg bw/day appeared low, although there was no dose response evident. Litter and fetal weight at 250 mg/kg bw/day, and placental weight at all dose levels, were unaffected by treatment.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July-November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 68 to 75 days old
- Weight at study initiation: 240 to 288 g.
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; Solid (polycarbonate) bottom cages were used during the acclimatisation and gestation periods; Grid bottomed cages were used during pairing
- Diet ( ad libitum): SDS VRF1 Certified pelleted diet.
- Water ( ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes.
- Acclimation period: Five days before pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%
- Air: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12 hours light : 12 hours dark

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Method of preparation: Approximately 50 % of the final volume of vehicle was added to the required amount of test substance and magnetically stirred to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser. A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test substance.
- Frequency of preparation: Weekly
- Storage of preparation: Refrigerated (nominally 2-8 °C)

VEHICLE
- Concentration in vehicle: 20, 60 and 150 mg/mL
- Amount of vehicle (if gavage): 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 Week 1 and the last week were analyzed for achieved concentration of the test item.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1
- Proof of pregnancy: Ejected copulation plug / sperm in vaginal smear referred to as Day 0 of pregnancy.
- A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals

Duration of treatment / exposure:

14 days (Days 6-19 p.c.)

Frequency of treatment:

Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same
time each day

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

20 mated females/dose

Control animals:

yes

Details on study design:

Dose levels were selected in conjunction with the Sponsor based on the results of a preliminary embryo-fetal toxicity study in Sprague Dawley rats (Envigo Study No. TS59TC). At the high dose of 1000 mg/kg bw/day in the preliminary study there was evidence that some females were more susceptible to dosing with Nerol towards the end of gestation with two females showing signs including piloerection, and/or under-activity and flat posture. It was considered possible that susceptible females could show marked post dosing signs towards the end of gestation on a main study with a greater group size, which could necessitate humane kill of affected females. At dose levels of 500 or 1000 mg/kg bw/day on the TS59TC study, slightly low overall body weight gain and transient body weight loss at 1000 mg/kg bw/day, as well as lower food consumption than in Controls, were observed during the first few days of treatment. A lower high dose of 750 mg/kg bw/day was therefore selected for this main study to reduce the possibility of marked signs being observed during late gestation; this dose was anticipated to fulfil regulatory expectations for some signs of toxicity at the high dose, manifest as reduced body weight gain and food consumption, following the start of treatment.
Low and intermediate dose levels were selected as 100 and 300, respectively, in order to assess the response at lower dose levels.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s).
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.
Detailed observations were recorded daily at the following times in relation to dose administration:
At the end of dosing of each group; one to two hours after completion of dosing of all groups; as late as possible in the working day.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and daily from Days 6 to 20 after mating.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Animals were killed by carbon dioxide asphyxiation on Day 20 after mating.
- All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examin ed visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes; Gravid uterine weight (including cervix and ovaries)
- Number of corpora lutea: Yes
- Number of implantation sites: Yes
- Number of early resorption sites: Yes
- Number of late resorption sites: Yes
- Number of live fetuses: Yes
- Number of dead fetuses: Yes

Fetal examinations:

Fetuses were killed by chilling on a cool plate (approximately 0 °C).
Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded, sampled as appropriate and retained in appropriate fixative. The sex of each fetus was recorded.
Examination of nominally 50% of fetuses in each litter: Sexed internally and eviscerated.
Fixation: Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.
Processing: Bouin’s fixed fetuses were subject to free-hand serial sectioning for visceral abnormalities. IMS fixed fetuses were processed and stained with Alizarin Red for skeletal development and abnormalities.

Statistics:

See "Any other information on materials and methods incl. tables"

Indices:

Reproductive assessment
Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100
Post-implantation loss (%) = [(Number of implantations – Number of live fetuses)/ Number of implantations] x 100

Historical control data:

Historical background data was used to compare the incidences of developmental toxicity.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Signs associated with dose administration were limited to a dose related incidence of increased salivation for animals receiving Nerol, with associated chin rubbing for females receiving 750 mg/kg bw/day. These signs were transient and for the majority of animals were no longer apparent 1 to 2 hours after completion of dosing all groups. Increased salivation and chin rubbing is often observed in association with gavage administration and is considered to relate to the palatability of the test formulations rather than a behavioural effect of treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Following the onset of treatment animals receiving 300 or 750 mg/kg bw/day showed a slight but statistically significant mean weight loss during Days 6 to 7 of gestation compared with a Control weight gain of 2g; thereafter body weight gain from Days 7 to 19 of gestation was similar to the Controls. Body weight gain at 100 mg/kg bw/day was unaffected by treatment. (see attached results tables)
Maternal body weight gain following adjustment for the gravid uterine weight was unaffected by treatment with Nerol. (see attached results tables)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females receiving 750 mg/kg bw/day showed statistically significantly low food consumption from Days 6-9 (p<0.01) which marginally improved over Days 10-13 (p<0.05). From Day 14 onwards, food consumption at 750 mg/kg bw/day was similar to Control values.
Food consumption at 100 and 300 mg/kg bw/day was unaffected by treatment. (see attached results tables)

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

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

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Litter data as assessed by the post-implantation loss showed no adverse effect of maternal treatment with Nerol.
The extent of the pre-implantation loss was high in the treated groups when compared with concurrent controls. A dose response was apparent in mean values but the mean pre-implantation loss in the high dose group was mainly due to one female (No. 79) with an incidental pre-implantation loss of 75%; the mean pre-implantation loss without this outlier is 6.17% which is comparable to control value and within historical control data. In addition to the absence of a real dose response, the difference did not attain statistical significance and the resultant mean live litter size was considered unaffected by treatment. (See attached results tables)

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Details on maternal toxic effects:

One Group 4 animal (No. 78) was not pregnant. The assessment of litter data is therefore based on a total of 20, 20, 20 and 19 litters at 0, 100, 300 and 750 mg/kg bw/day.
Placental, litter and fetal weights were essentially similar to Controls at all dose levels and were considered to be unaffected by maternal treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

Description (incidence and severity):

not relevant for OECD 414

External malformations:

no effects observed

Description (incidence and severity):

The incidence of major and minor abnormalities and skeletal variants showed no relationship to maternal treatment. (see attached results tables)

Skeletal malformations:

no effects observed

Description (incidence and severity):

The incidence of major and minor abnormalities and skeletal variants showed no relationship to maternal treatment. (see attached results tables)

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of major and minor abnormalities and skeletal variants showed no relationship to maternal treatment. (see attached results tables)
An increase of abdominal cavity haemorrhages in fetuses, but not in litters, slightly above historical control data was observed. However, it is an isolated effect, that was considered to have no toxicological significance and to be an artefact of the necropsy process. (see attached results tables)

Key result

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for the maternal and fetal toxicity was considered to be 750 mg/kg bw/day.

Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, Nerol was administered by oral (gavage) to groups of mated female Crl:CD(SD) rats (20/dose) at the dose levels of 0, 100, 300 and 750 mg/kg bw/day from Days 6 to 19 after mating.

A similarly constituted Control group received the vehicle, corn oil, at the same dose volume. Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

 

There were no unscheduled deaths and clinical condition was unaffected by treatment.

Signs associated with dose administration were limited to a dose related incidence of increased salivation and chin rubbing; these were considered to relate to palatibility rather than an effect of treatment.  

During Days 6 to 7 of gestation animals receiving 300 or 750 mg/kg bw/day showed a slight but statistically significant mean weight loss compared with a Control weight gain; thereafter body weight gain from Days 7 to 19 of gestation was similar to the Controls. Body weight gain at 100 mg/kg bw/day was unaffected by treatment.

Females receiving 750 mg/kg bw/day showed statistically significantly low food consumption from Days 6-13 of gestation. From Day 14 onwards, food consumption at 750 mg/kg bw/day was similar to Control values. Food consumption at 100 and 300 mg/kg bw/day was unaffected by treatment.

There were no maternal macroscopic findings that could be related to treatment.

Litter data as assessed by the number of corpora lutea, implantations, resorptions, live embryos and the extent of pre- and post-implantation loss showed no adverse effect of maternal treatment with Nerol.

Placental, litter and fetal weights were unaffected by maternal treatment.

Detailed fetal examination did not reveal any skeletal or visceral findings that could be related to maternal treatment.

 

Oral gavage administration of Nerol to pregnant Sprague-Dawley rats at dose levels of 100, 300 and 750 mg/kg bw/day during the organogenesis and fetal phase of gestation was well tolerated with no unscheduled deaths, no adverse effect on clinical condition and no treatment-related maternal macropathology. There was a slight initial effect on body weight gain and food consumption however there was no overall effect on maternal bodyweight gain.

Maternal treatment did not affect fetal survival, weight or development.

It was therefore concluded that in this study both the maternal and fetal no observed adverse effect level was 750 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP study conducted according to OECD Guideline 414 without any deviation (Klimisch score = 1).

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

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, nerol was administered by oral (gavage) to groups of mated female Crl:CD(SD) rats (20/dose) at the dose levels of 0, 100, 300 and 750 mg/kg bw/day from Days 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil, at the same dose volume. Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

There were no unscheduled deaths and clinical condition was unaffected by treatment.

Signs associated with dose administration were limited to a dose-related incidence of increased salivation and chin rubbing; these were considered to relate to palatibility rather than an effect of treatment.  

During Days 6 to 7 of gestation animals receiving 300 or 750 mg/kg bw/day showed a slight but statistically significant mean weight loss compared with a Control weight gain; thereafter body weight gain from Days 7 to 19 of gestation was similar to the Controls. Body weight gain at 100 mg/kg/day was unaffected by treatment.

Females receiving 750 mg/kg bw/day showed statistically significantly low food consumption from Days 6-13 of gestation. From Day 14 onwards, food consumption at 750 mg/kg bw/day was similar to Control values. Food consumption at 100 and 300 mg/kg bw/day was unaffected by treatment.

There were no maternal macroscopic findings that could be related to treatment.

Litter data as assessed by the number of corpora lutea, implantations, resorptions, live embryos and the extent of pre- and post-implantation loss showed no adverse effect of maternal treatment with Nerol.

Placental, litter and fetal weights were unaffected by maternal treatment.

Detailed fetal examination did not reveal any skeletal or visceral findings that could be related to maternal treatment.

Oral gavage administration of Nerol to pregnant Sprague-Dawley rats at dose levels of 100, 300 and 750 mg/kg bw/day during the organogenesis and fetal phase of gestation was well tolerated with no unscheduled deaths, no adverse effect on clinical condition and no treatment-related maternal macropathology. There was a slight initial effect on body weight gain and food consumption however there was no overall effect on maternal bodyweight gain.

Maternal treatment did not affect fetal survival, weight or development.

It was therefore concluded that in this study both the maternal and fetal no observed adverse effect level was 750 mg/kg bw/day.

In a Combined Repeated Dose Toxicity Study with the Reproduction/developmental Toxicity Screening Test according to OECD Guideline 422 and in compliance with GLP, nerol was administered by dietary admixture to three groups of Han Wistar rats for up to 42 consecutive days for main phase males, toxicity females and recovery animals, and between 41 and 53 days (including three weeks exposure phase, pairing, gestation and early lactation) for main phase females, at dietary concentrations of 3000, 6000 and 12000 ppm (equivalent to a global mean achived dosage of 191.2, 374 and 720 mg/kg bw/day, respectively).

No unscheduled deaths or treatment-related clinical signs were noted.

During the pre-pairing period, females showed a decrease in bodyweight gain, especially during the first week of the treatment. Form Day 8 to the end of the study, toxicity group females showed similar mean bodyweight gain when compared to controls at 3000 and 6000 ppm but a 25% decrease in mean bodyweight gain compared to controls at 12000 ppm.

During the recovery phase, their mean bodyweight gain was 3 times higher than the concurrent control group. During gestation period, main phase females showed an overall decrease in mean bodyweight gain at 3000, 6000 and 12000 ppm (11, 6 and 33% decrease, respectively). This decrease was mainly observed during the last week of gestation. This decrease in bodyweight gains was also observed during lactation period (but without dose dependancy) with about 50% decrease in mean bodyweight gain in all treated groups compared to controls. Generally, foos consumption was reduced during the first days of treatment in all treated groups. This reduction in food intake was maintained at the high dose throughout the treatment period in both males and females but particularly for main phase females. This reduction in food consumption was considered to reflect a relunctance to eat the diet admixture due to its low palatability, particularly at the high dose. During the lactation period, the dietary intake of main phase females was reduced (up to 37% decrease compared to controls at 12000 ppm) in all test item treated groups.

No treatment-related effects were detected in mating performance, fertility and gestation length. Implantation sites were slightly reduced at 3000 and 12000 ppm without reaching statistical significance and without dose dependency. A significant dose-related increase in post-implantation loss was observed with the mean values outside the historical control data at mid and high doses. Fewer offspring was born in all test item-treated groups without dose dependency and without reaching statistical significance. This was considered to be related to the other findings. Mean pup weights were comparable to controls at 12000 ppm on post natal Days 1 and 4 and were slightly reduced on Day 7 without reaching statistical significance. Mean pup weights were comparable to controls at 3000 and 6000 ppm.

Therefore, the Lowest Observed Adverse Effect Level (LOAEL) for maternal toxicity was 3000 ppm based on the decrease in bodyweight gain at 3000 ppm during the last week of gestation. The NOAEL for reproductive toxicity was 12000 ppm (highest dose tested) beacuse no adverse effects were observed on fertility and post-natal development of offspring.

The absence of adverse effects observed up to 750 mg/kg bw/day on the OECD 414 study in rats treated by oral gavage with nerol enabled to demonstrate that the adverse effects observed in the gestation females in the OECD 422 by diet (especially the increase in post-implantation loss and the decrease in bodyweight gain), were due to the reduction in food consumption because of the low palatability of the treated diet, leading to an adverse decrease in bodyweight gain at the end of gestation and during lactation, rather than a direct adverse toxic effect of nerol. Therefore, these effects were not considred for the determination of the maternal and developmental NOAEL of the registered substance.

Justification for classification or non-classification

In a recent GLP combined repeated dose toxicity and reproduction / developmental toxicity screening test conducted according to OECD guideline 422, no adverse signs of toxicity to reproduction (mating performance, fertility and post-natal development of offspring until Day 7) that could be attributable to the test item were identified in male and female rats.

Increased post-implantation loss was observed in gestating female rats but it was associated with reduction in food consumption and decreased bodyweight gain therefore a direct effect of the test item could not be clearly identified.

In a recent GLP prenatal developmental toxicity test conducted according to OECD guideline 414, no adverse signs of toxicity were identified. Maternal treatment did not affect fetal survival, weight or development at the highest dose tested.

Therefore, the registered substance does not need to be classified according to CLP Regulation (EC) n° 1272/2008.