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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
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
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
other: audited draft report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
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. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No. : 122207
Purity : 98.7%
Name of test material (as cited in study report): NEROL (CAS 106-25-2)
Physical state: colourless - slightly yellow liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 21 July 2013

Test animals

Species:
rat
Strain:
other: Han Wistar Rat
Sex:
male/female
Details on test animals 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

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: 2 % corn oil feed admixtures
Details on oral 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.
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
Doses / concentrationsopen allclose all
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: 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.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
No

Examinations

Observations and examinations performed and frequency:
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.
Sacrifice and pathology:
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
Other examinations:
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.

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.

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.

Results and discussion

Results of examinations

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
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 (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
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
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
Description (incidence and severity):
- 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 pathological findings:
effects observed, treatment-related
Description (incidence and severity):
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.
Neuropathological findings:
not examined
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
Description (incidence and severity):
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- No treatment related effects on mating, fertility and gestation Length were detected.

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.

Effect levels

Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
6 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 6000 ppm in rats.
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 with 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. No treatment-related effects were detected in mating performance, fertility and gestation lengths. 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.

Therefore, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 6000 ppm in rats.