Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 April to 10 October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 April to 10 October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
November 2015
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River (UK) Limited
- Age of F0 animals at study initiation: Approximately 10 weeks
- Weight at study initiation: Males: 338-383 g; Females: 207-259 g
- Housing: No. of animals per cage - Males: pre pairing and post pairing - up to 5 animals; Females: pre
pairing - up to 5 animals; During pairing - one male and one female;
Toxicity phase and recovery phase - up to five animals of one sex. Cages comprised
of a polycarbonate body with a stainless steel mesh lid. Solid (polycarbonate) bottom cages were use
d during the acclimatisation and treatment period with the exception of pairing periods. Grid bottomed
cages comprising of polypropylene body were used during pairing.
- Diet: SDS VRF1 Certified powdered diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light
Route of administration:
oral: feed
Vehicle:
other: carrier diet, including corn oil
Details on oral exposure:
- Carrier diet: SDS VRF1 certified diet.
- Stabiliser: Corn oil (test material to corn oil ratio 5:1)
- Method of preparation: On each occasion of the preparation of the premix the required amount of test
substance and corn oil were mixed together and stirred. The mixture was added to an equal amount of
plain diet and stirred until visibly homogenous. This doubling up process was repeated until half of the
final weight of premix was achieved or the mixture appeared dry. This mixture was then ground using
a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This
premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the
diet. Aliquots of the premix were then diluted with further quantities of plain diet to produce the required
dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
For Control diet the corn oil was added directly to the diet and then prepared as indicated for the premix.
- Frequency of preparation: Weekly, however, preparations on some occasions were made in advance a
nd stored frozen, but were used within the frozen stability period (22 days when stored frozen nominally
-20 °C).
- Storage of preparation: Frozen (nominally -20 °C).
GROUPS
Groups 1, 2, 3 and 4: 0, 1000, 2500 and 7500 ppm, respectively
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 100 and 20000 ppm were analysed to assess the stability and homogeneity of the test substance in the diet matrix. Stability was confirmed as eight days at ambient temperature and 22 days when stored frozen. However, only one day ambient stability was used.
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analysed for achieved concentration of the test substance.
Duration of treatment / exposure:
Toxicity phase males were treated daily for three weeks before pairing, throughout pairing and up to necropsy after a minimum of five consecutive weeks.
Toxicity phase females were treated daily for a minimum of five consecutive weeks up to necropsy in Week 6.
High dose Recovery phase male and female rats were treated daily for a minimum of six consecutive weeks, followed by a 14 day period without treatment.
Frequency of treatment:
Continuously (fresh diet given daily). During the recovery period, all animals were given untreated diet (without corn oil).
Dose / conc.:
1 000 ppm
Remarks:
nominal in diet
Dose / conc.:
2 500 ppm
Remarks:
nominal in diet
Dose / conc.:
7 500 ppm
Remarks:
nominal in diet
No. of animals per sex per dose:
Toxicity phase : 5 animals/sex/dose for vehicle and high dose groups; 10 males/dose for 1000 and 2500 ppm dose groups; 5 females/dose for 1000 and 2500 ppm dose groups
Recovery phase: 5 animals/sex/dose for vehicle and high dose groups
Control animals:
other: control group was provided with the carrier diet, including corn oil
Details on study design:
- Dose selection rationale: Dose levels were selected in conjunction with the Sponsor following the
completion of the dose range finder study (Study number OAD0022). In the dose range finding study
the doses of 1500, 7500 or 15000 ppm were well tolerated (no mortality), no adverse clinical signs or macroscopic changes in organ appearance were detected at any dose level. Effects of treatment at 15000 ppm included initial body weight loss for the first few days of treatment in males and females, associated with lower food consumption, probably due to low palatability of the treated diet. In males, no effects on body weight and body weight gain were observed up to 7500 ppm but the overall bodyweight change in the high dose group was 23% lower compared to Control group. In females, body weight gain was lower than Control group at 7500 and 15000 ppm, especially during Days 1-7 and 15-22, leading to overall body weight gains 64% and 46% lower than Control group, respectively. The lower bodyweight gain observed in the mid dose group was mainly due to one female showing overall bodyweight loss during the treatment period (-3 g). However, even without this animal, the body weight gain in this group was only 50% of the Control group. Therefore, the high dose to be tested in the main study was considered to be 7500 ppm due to the toxicity observed in females. Then, the lower doses were spaced approximately by a 2.5 to 3-fold factor, i.e. 2500 and 1000 ppm.
Rationale for animal assignment: Randomly allocated on arrival. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day.

NEUROBEHAVIOURAL EXAMINATION: Yes
Detailed physical examination and arena observations: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each adult animal.
Time schedule:
- Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery animals in Groups 1 and 4 and the five lowest numbered surviving toxicity phase males and females in Groups 2 and 3, during Week 6 of treatment.
- Motor activity:
During Week 6 of treatment, the motor activity of all recovery animals in Groups 1 and 4 and the five lowest numbered surviving toxicity phase males and females in Groups 2 and 3 was measured.

BODY WEIGHT: Yes
- Time schedule for examinations:
Toxicity and Recovery phase animals: Before feeding on the day that treatment commenced (Day 1), weekly thereafter and before necropsy.

FOOD CONSUMPTION: Yes
- Time schedule for examinations:
Toxicity and recovery phase males and females: Daily. From these records the mean daily consumption per animal (g/rat/day) was calculated for each cage. Food consumption was not recorded for males during the period when paired for mating (Day 22 to 28). Food consumption recordings recommenced on
Day 29.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
Haematology: Week 6 - Five lowest numbered toxicity phase males and females per group; Recovery Week 2 - All recovery phase animals
Clinical chemistry: Week 6 - Five lowest numbered toxicity phase males and females per group; Recovery Week 2 - All recovery phase males
- Animals fasted: Yes, Blood samples were collected after overnight withdrawal of food.
- Animals were held under light general anaesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count, Percentage reticulocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count, Differential leucocyte count: Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count,
Morphology: Anisocytosis, Macrocytosis, Microcytosis, Hypochromasia, Hyperchromasia, Prothrombin time and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin, Bile acids, Urea, Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus, Total protein, Albumin and Albumin/globulin ratio (A/G Ratio).

Sacrifice and pathology:
SACRIFICE
- Toxicity phase animals: Following completion of Week 6 investigations
after mating.
- Recovery phase animals: After at least 14 days without treatment.
- Method of sacrifice: F0 animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination
Other examinations:
GROSS NECROPSY
- All adult animals were subject to a detailed necropsy. All external features and orifices were examined 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.
HISTOPATHOLOGY / ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together.
Histology
Fixation: Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of Testes in modified Davidson’s fluid; Eyes In Davidson’s fluid.
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required. The five lowest numbered males and females in Groups 1 and 4 at scheduled termination.
Abnormalities only: All animals
- Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid Schiff (PAS) method.
Statistics:
See section "Any other information on materials and methods incl. tables”.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain during Week 1 of treatment was reduced in males and female receiving 7500 ppm with statistical significance.
Overall body weight gain in males was similar to Controls up to 2500 ppm and was lower but not statistically significantly at 7500 ppm.
Overall bodyweight gains in females were statistically significantly lower than Controls in all treated groups, but a dose response was not apparent.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Overall food consumption for males and toxicity phase females receiving Neryl Acetate up to 7500 ppm was unaffected by treatment.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly lower group mean erythrocyte counts in males receiving 7500 ppm, and a slight but not statistically significant lower level for males receiving 2500 or 1000 ppm when compared with Controls was revealed.
Statistically significantly higher mean cell haemoglobin levels for males receiving 7500 ppm and slightly but statistically significant higher mean cell volume levels for males receiving 1000 ppm or above were observed.
Mean cell haemoglobin concentration was slightly but statistically significantly higher for females receiving 1000 ppm or above.
After 2 weeks of recovery haematological examinations were similar to that of the Controls. As a consequence these findings were considered to be of no toxicological significance.
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):
Organ weights for males and toxicity phase females killed after 6 weeks of treatment indicated that the adjusted group mean kidney weights for animals that received 7500 ppm and the adjusted group mean liver weights for females that received 7500 ppm were slightly but statistically significantly higher than that of the Controls.
Organ weights taken following the two-week recovery period for animals that received 7500 ppm, were similar to Controls indicating no persistent affects.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All histological changes were considered to be unrelated to treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY:
There were no decedents in the study. Routine physical examination and arena observations did not reveal any abnormalities that were considered related to treatment with test item.

BODY WEIGHT:
Group mean bodyweight gain during Week 1 of treatment was reduced in males receiving 7500 ppm and females receiving 1000, 2500 and 7500 ppm when compared with Controls, with statistical significance being attained at 7500 ppm for both males and females. Bodyweight gain was slightly, but not statistically significantly lower during Weeks 4-5 for males receiving 2500 ppm and almost throughout treatment period for males receiving 7500 ppm when compared with Controls. Overall bodyweight gain in males was similar to Controls up to 2500 ppm and was lower but not statistically significantly at 7500 ppm (87% of Controls). Bodyweight gain was slightly, but not statistically significantly lower during Weeks 1-2, 3-4 and 4-5 for toxicity phase females receiving Neryl Acetate when compared with Controls, and overall bodyweight gains were statistically significantly lower than Controls in all treated groups, but a dose res
ponse was not apparent.
- Overall bodyweight change during the recovery period was slightly but not statistically significantly lower than Controls for males, and was higher for females (200% of Controls) that had received 7500 ppm, but no statistical significance was attained.

FOOD CONSUMPTION:
Overall food consumption for males and toxicity phase females receiving Neryl Acetate up to 7500 ppm was unaffected by treatment. Food intake was slightly lower on Day 1 of treatment for females receiving 7500 ppm when compared with Controls.

GROSS PATHOLOGY:
- The macroscopic examination performed after the six weeks scheduled treatment period revealed no test substance related lesions.
The macroscopic examination performed after 2 weeks of recovery revealed no test substance related lesions. All findings were similar to the background of changes commonly seen in Sprague Dawley rats seen at these laboratories.

HISTOPATHOLOGY:
No treatment related changes were observed in rats up to 7500 ppm by dietary administration for five weeks.
- Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of various cell types present within the different stages. No cell or stage specific abnormalities were noted.
- Minimal cortical tubular hypertrophy of kidney was observed in one male (4M No. 13) at 7500 ppm. This finding correlated with slight increase in the weight of kidneys. However, considering the single incidence in only one sex, its relationship to treatment is uncertain.
- An increase in the minimal multifocal aggregates of alveolar macrophages in the lung was observed in 4/5 females at 7500 ppm. One of the control females also had this finding. The aggregates of alveolar macrophages were mainly observed in the sub pleural locations or in the parenchyma away from terminal bronchioles, which are considered as common background finding in rats. The increase in the incidence in treated females alone is considered as incidental.

OTHER FINDINGS:
- Sensory reactivity and grip strength: There was no effect of test item on sensory reactivity or grip strength.
- Motor activity: Motor activity was unaffected for females up to 7500 ppm, when compared to Controls and there were no clear effects on either rearing (high beam) or ambulatory (low beam) motor activity for males.
- Haematology: Haematological examination during Week 6 of treatment revealed statistically significantly lower group mean erythrocyte counts in males receiving 7500 ppm, and a slight but not statistically significant lower level for males receiving 2500 or 1000 ppm when compared with Controls. Statistically significantly higher mean cell haemoglobin levels for males receiving 7500 ppm and slightly but statistically significant higher mean cell volume levels for males receiving 1000 ppm or above were observed. Mean cell haemoglobin concentration was slightly but statistically significantly higher for females receiving 1000 ppm or above. - Other inter-group differences from Controls were minor; neutrophil counts were higher for females receiving 7500 ppm when compared to Controls, but lacked a dose-relationship and was confined to one sex and was therefore considered due to normal biological variation.
- In Week 2 of recovery, haematological examinations of erythrocyte counts, mean cell haemoglobin and mean cell volume in males that received 7500 ppm and mean cell haemoglobin concentration,
total leucocyte count and differential leucocyte count (neutrophils, lymphocytes, eosinophils, basophils, monocytes and large unstained cells) in females that received 7500 ppm were similar to that of the
Controls
- Clinical chemistry: The biochemical examination of the blood plasma in Week 6 of treatment did not indicate any clear effects of treatment. When compared with the Controls, plasma levels of bile acids for males receiving 1000 ppm or above were slightly, but statistically significantly decreased, but no dose trend was apparent.
- All other inter-group differences from Controls were minor, were confined to one sex or lacked a dose-relationship. Such differences included minor, transient variations of some plasma electrolyte
concentrations such as sodium levels in males and potassium levels in females receiving 7500 ppm; these values were considered fortuitous and were therefore considered of no toxicological importance.
- Review of bile acid levels in Week 2 of recovery revealed levels similar to Controls for males that received 7500 ppm. Group mean sodium levels in Week 2 of recovery were the same as Controls.
Key result
Dose descriptor:
NOAEL
Effect level:
7 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect observed at the highest concentration tested, corresponding to 440 mg/kg bw/day for males, 465 mg/kg bw/day for toxicity phase females and 484 mg/kg/day for reproductive phase females during pre-mating period.
Critical effects observed:
no

Formulation analysis:

The mean concentrations were within applied limits of +10%/-15%, confirming the accuracy of formulation. The difference from mean values was <3 %, confirming precise analysis. Procedural recovery values remained within 94.2 and 100.8 %, confirming the continued accuracy of the method.

Achieved dose

Achieved intake of animals measured during the toxicity phase generally maintained the intended intervals between treatment groups. As anticipated, achieved intake was higher at the start of the treatment period and declined progressively as the animals matured.

Table 7.8.1/4: Achieved dosage for Toxicity and Recovery phase males:

Level (ppm)

Achieved dosage (mg/kg bw/day)

Mean

Range

1000

61

51-72

2500

150

129-175

7500

440

366-518

Table 7.8.1/5: Achieved dosage for Toxicity and Recovery phase females

Level (ppm)

Achieved dosage (mg/kg bw/day)

Mean

Range

1000

65

55-71

2500

150

127-169

7500

465

436-495

Achieved intake of females measured throughout the treatment period, generally maintained the intended intervals between treatment groups. Achieved intake was relatively stable during gestation, but increased significantly during lactation, reflecting increasing food intake during this period of high physiological demand on the parent female.

 

Table 7.8.1/6: Achieved dosage for Reproductive phase females

Level (ppm)

Achieved dosage (mg/kg bw/day)

Pre-pairing phase

Gestation

Lactation

Average

Range

Range

Range

1000

67

63-70

65-80

98-151

2500

172

161-182

168-189

266-390

7500

484

483-484

471-547

624-1080

Conclusions:
The NOAEL for systemic toxicity was 7500 ppm, the highest dose tested, corresponding to 440 mg/kg bw/day for males, 465 mg/kg bw/day for toxicity phase females and 484 mg/kg/day for reproductive phase females during pre-mating period.
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to guideline OECD 422 and in compliance with GLP, groups of Crl:CD(SD) rats received NERYL ACETATE orally, via the diet, at concentrations of 1000, 2500 or 7500 ppm. Toxicity phase males were treated daily for three weeks before pairing, throughout pairing and up to necropsy after a minimum of five consecutive weeks. Toxicity phase females were treated daily for a minimum of five consecutive weeks up to necropsy in Week 6. Five high dose Recovery phase male and female rats were treated daily for a minimum of five consecutive weeks, followed by a 14 day period without treatment. Recovery phase males were used for pairing with reproductive phase females, but recovery females were not paired. A similarly constituted Control group was assigned to each phase, and received the vehicle in untreated diet, throughout the same relative treatment period.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology, blood chemistry, oestrous cycles, organ weight and macroscopic investigations were undertaken for adult animals.

Microscopic pathology investigations were undertaken for the first five Toxicity phase males and Reproductive and Toxicity phase females in Group 1 (Control) and Group 4 (7500 ppm).

The mean concentrations of the test item in formulations were within applied limits of +10%/-15%, confirming the accuracy of formulation.

Administration of the test item at dose levels of 1000, 2500 and 7500 ppm was well tolerated with no mortalities or effects that could be attributed to treatment on clinical condition, sensory reaction, grip strength, motor activity, oestrous cycles, macroscopic or microscopic pathology among the adult animals.

Mean achieved dosages during the pre-pairing phase were 61, 150 and 440 mg/kg bw/day for males and 65, 150 and 465 mg/kg bw/day for toxicity phase females and 67, 172 and 484 mg/kg/day for reproductive phase females receiving 1000, 2500 and 7500 ppm, respectively.

Group mean bodyweight gain (Week 1 of treatment) was reduced in males receiving 7500 ppm and females receiving 1000, 2500 and 7500 ppm when compared with Controls; and overall bodyweight gain was statistically significantly lower than Controls in females, but a dose response was not apparent. Overall bodyweight change during the recovery period was slightly but not statistically significantly lower for males, but was significantly higher for females that had received 7500 ppm.

Overall food consumption for males and toxicity phase females receiving Neryl Acetate up to 7500 ppm was unaffected by treatment. Food intake was slightly lower on Day 1 of treatment for toxicity when compared with Controls.

Haematological examination during Week 6 of treatment revealed statistically significantly lower group mean erythrocyte counts in males receiving 7500 ppm, and a slight but not statistically significant lower level for males receiving 2500 or 1000 ppm when compared with Controls.

Statistically significantly higher mean cell haemoglobin levels for males receiving 7500 ppm and slightly but statistically significant higher mean cell volume levels for males receiving 1000 ppm or above. Mean cell haemoglobin concentration was slightly but statistically significant higher for females receiving 1000 ppm or above.

Haematological parameters examined in Week 2 of recovery were similar to that of the Controls.

The biochemical examination of the blood plasma in Week 6 of treatment did not indicate any clear effects of treatment. When compared with the Controls, plasma levels of bile acids for males receiving 1000 ppm or above were slightly, but statistically significantly decreased, but no dose trend was apparent.

Review of bile acids levels in Week 2 of recovery revealed similar levels to Controls for males that received 7500 ppm.

Organ weights for males and toxicity phase females killed after 6 weeks of treatment indicated that the adjusted group mean kidney weights for animals that received 7500 ppm and the adjusted group mean liver weights for females that received 7500 ppm were slightly but statistically significantly higher than that of the Controls.

Organ weights taken following the two week recovery period for animals that received 7500 ppm, were similar to Controls indicating no persistent effects.

All histological changes were considered to be unrelated to treatment.

Therefore, the NOAEL for systemic toxicity was 7500 ppm, the highest dose tested, corresponding to 440 mg/kg bw/day for males, 465 mg/kg bw/day for toxicity phase females and 484 mg/kg/day for reproductive phase females during pre-mating period.

Reason / purpose:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
21 May to 18 June 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Preliminary study used as range-finder experiment for a OECD 422 screening test, performed in GLP laboratory.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Principles of method if other than guideline:
Range-finder experiment for an OECD guideline 422 study, where clinical condition, bodyweight, food consumption, water consumption, oestrous cycles, blood chemistry, organ weight and macropathology investigations were undertaken in male and female rats exposed to test substance by dietary administration for 21 days.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Approximately 67 days
- Weight at study initiation: Males: 362-407 g; Females: 233-267 g
- Housing: Animals were housed in groups of 4/sex in polycarbonate cages
- Diet: SDS VRF1 Certified Diet; ad libitum
- Water: Potable water from public supply; ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70%
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light
Route of administration:
oral: feed
Vehicle:
other: carrier diet, including corn oil
Details on oral exposure:
DIET PREPARATION
- Carrier diet: SDS VRF1 certified diet.
- Stabiliser: Corn oil (test material to corn oil ratio 5:1)
- Method of preparation: On each occasion of the preparation of the premix the required amount of test substance and corn oil were mixed together and stirred. The mixture was added to an equal amount of plain diet and stirred until visibly homogenous. This doubling up process was repeated until half of the final weight of premix was achieved or the mixture appeared dry. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
For Control diet the corn oil was added directly to the diet and then prepared as indicated for the premix.

- Frequency of preparation: Weekly
- Storage of preparation: Refrigerated (nominally 4 °C).

Stability and homogeneity
Homogeneity and stability of the test material in the vehicle from Study No. OAD0021. Formulations have been demonstrated to be stable and homogenous at 100 and 20000 ppm for eight days when stored frozen (-20 °C) or at ambient temperature.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
21 days
Frequency of treatment:
Continuously (preparations replaced daily although stability was demonstrated up to 8 days).
Dose / conc.:
1 500 ppm
Remarks:
nominal in diet
Dose / conc.:
7 500 ppm
Remarks:
nominal in diet
Dose / conc.:
15 000 ppm
Remarks:
nominal in diet
No. of animals per sex per dose:
4
Control animals:
other: control group was provided with the carrier diet, including corn oil
Details on study design:
- Dose selection rationale: Doses used in this study (0, 1500, 7500 and 15000 ppm) were selected in conjunction with the Sponsor.
Neryl Acetate has very low acute toxicity: oral and dermal LD50 were reported to be higher than 5000 mg/kg bw in rats and rabbits respectively.
The dose levels in this study were therefore set at 1500, 7500 and 15000 ppm, with achieved intake at 15000 ppm expected to equate to approximately 1000 mg/kg bw/day.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants.
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 1, 8, 15 and 22 to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded during the acclimatisation period, on the day that treatment commenced (Day 1), daily throughout the study and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded daily throughout the study.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 3 from all animals
- Animals were held under light general anaesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein.
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Urea and Creatinine.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; Animals were killed following 21 days of treatment by carbon dioxide asphyxiation and subjected to detailed necropsy.
ORGAN WEIGHTS: Yes; organ weights of adrenals, epididymides, heart, kidneys, liver, ovaries, prostate, spleen and testes were recorded. For bilateral organs, left and right organs were weighed together.
HISTOPATHOLOGY: No; but tissues with macroscopic abnormalities were preserved for microscopic examinations in 10% neutral buffered formalin for microscopic examination (except testes: In modified Davidson’s fluid).
Other examinations:
None
Statistics:
No data
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males receiving 15000 ppm showed marginally low weight gains during Weeks 2 and 3 of treatment and the overall bodyweight gain in the high dose group was 23% lower than in Controls.
In females, bodyweight gain was lower than Control group at 7500 and 15000 ppm, leading to overall bodyweight gains 64% and 46% lower than Control group, respectively.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 15000 ppm food consumption in males was generally lower when compared with Controls.
Food consumption in females was lower for the first two days of treatment when compared with Controls; thereafter differences to the control group were small.
No difference with Controls could be observed in food consumption for both sexes treated at 1500 ppm
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:
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):
There was a slight dose-dependant increase in absolute, adjusted and body weight relative kidney weights in males and liver weights in females.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- There were no signs observed at routine examination that could be associated to treatment.

BODY WEIGHT AND WEIGHT GAIN
- Following the administration of test diet, bodyweight loss was recorded in males receiving 15000 and 7500 ppm and females receiving 15000, 7500 and 1500 ppm, but slight gain or stasis was recorded from Day 3 of treatment. Bodyweight gain during the first week of treatment was lower for animals receiving 15000 or 7500 ppm compared with Controls.
- In males, no further effects on body weight and weight gain were observed up to 7500 ppm, but males receiving 15000 ppm showed marginally low weight gains during Weeks 2 and 3 of treatment and the overall bodyweight gain in the high dose group was 23 % lower than in Controls.
In females, bodyweight gain was lower than Control group at 7500 and 15000 ppm, especially during Days 1-7 and 15-22, leading to overall bodyweight gains 64 and 46 % lower than Control group, respectively. The lower bodyweight gain observed in the mid dose group was mainly due to one female showing overall bodyweight loss during the treatment period (-3 g). However, even when this female was excluded, the bodyweight gain in this group was only 50% of the Control group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- At 15000 ppm food consumption in males was generally lower throughout the three weeks of treatment when compared with Controls, leading to an overall decrease in food consumption of 12 % compared to Controls. Food consumption in females was lower for the first two days of treatment when compared with Controls; thereafter differences to the control group were small, leading to an overall decrease in food consumption of 10 % compared to Controls.
- Food intake was reduced for animals receiving 7500 ppm only on Day 1 when compared with Controls, therefore overall food consumption at this dose level could be considered as similar to Controls.
- No difference with Controls could be observed in food consumption for both sexes treated at 1500 ppm.

WATER CONSUMPTION
- No effects were observed throughout the study.

CLINICAL CHEMISTRY
- Blood chemistry investigations conducted in Week 3 did not reveal any clear findings that were considered related to treatment with Neryl Acetate.
- Plasma creatinine levels slightly dose-dependently increased in all treated females when compared with Controls. These levels were also slightly higher in males from 7500 ppm.
- Urea levels were higher than Control for females receiving 15000 ppm. This isolated observation in one sex only is of uncertain relationship to treatment with Neryl Acetate.
- All other differences from Control were minor or lacked dose-relationship and were therefore considered due to normal biological variation.

ORGAN WEIGHTS
- There was a slight dose-dependent increase in absolute, adjusted and bodyweight-relative kidneys weights in males and liver weights in females. There was also a marginal decrease in spleen weights for males that received Neryl Acetate and higher spleen weights for females that received 15000 ppm when compared with Controls.
- Testes weights were slightly higher for males that received 15000 ppm and epididymides weights were slightly higher in males that received 1500 or 15000 ppm when compared with Controls, but without dose-dependency for both organs.

GROSS PATHOLOGY
- There were no macroscopic abnormalities detected in animals receiving Neryl Acetate.

OTHER FINDINGS
OESTROUS CYCLES:
- There was no effect of treatment on oestrus cycle activity of females receiving Neryl Acetate at dietary levels up to 15000 ppm.
Critical effects observed:
not specified

Overall mean achieved dose levels for animals receiving 1500, 7500 or 15000 ppm were 100, 485 or 916 mg/kg bw/day for males and 104, 509 or 939 mg/kg bw/day for females, respectively.

Conclusions:
Due to the bodyweight effects observed in females at 7500 ppm, a suitable high dose to be tested in the subsequent OECD 422 screening study was selected to be 7500 ppm.
Executive summary:

In a repeated dose toxicity range-finding study, groups of Crl:CD(SD) rats (4/sex/dose) received NERYL ACETATE orally, via the diet at concentrations of 1500, 7500 or 15000 ppm. A similarly constituted control group received untreated diet throughout the same treatment period. During the study, clinical condition, body weight, food consumption, oestrous cycles, blood chemistry, organ weight and macropathology investigations were undertaken. 

Overall mean achieved dose levels for animals receiving 1500, 7500 or 15000 ppm were 100, 485 or 916 mg/kg/day for males and 104, 509 or 939 mg/kg/day for females, respectively.

All animals survived the dosing period, with no adverse clinical signs or macroscopic changes in organ appearance at any dose level. There were no visual water consumption effects or effects on female oestrus cycle activity.

Following the administration of the test item in diet, bodyweight loss was recorded in males and females, but slight gain or stasis was recorded from Day 3 of treatment. At 1500 ppm there was no overall effect on bodyweight gain or food consumption for either sex at this level when compared with Controls.

At 7500 ppm, in females, bodyweight gain was lower than Control group, especially during Days 1-7 and 15-22, leading to overall bodyweight gains of 64% lower than Control group. The lower bodyweight gain was mainly due to one female showing overall bodyweight loss during the treatment period (-3 g). However, even when this female was excluded, the bodyweight gain in this group was only 50% of the Control group. Food intake was reduced in both sexes on Day 1 when compared with Controls.

At 15000 ppm, bodyweight gain during the first week of treatment was lower than Controls at this dose level. After the first week of treatment, males showed marginally low weight gains during Weeks 2 and 3 of treatment, while females showed low weight gains during Week 3 of treatment. Food consumption in males was generally lower throughout the treatment when compared with Controls. Food consumption in females was lower for the first two days of treatment when compared with Controls; thereafter differences to the Control were small.

Plasma creatinine levels were slightly higher for females receiving 1500 ppm and animals receiving 7500 ppm or above when compared with Controls. Urea levels were higher than Control for females receiving 15000 ppm.

 

There was a slight dose-dependent increase in absolute, adjusted and bodyweight-relative kidneys weights in males and liver weights in females. There was also a marginal decrease in spleen weights for males that received NERYL ACETATE and higher spleen weights for females that received 15000 ppm when compared with Controls. As there were no observed macroscopic findings and as no histopathological examination was conducted, the aetiology of these findings is unclear.

Therefore, due to the bodyweight effects observed in females at 7500 ppm, a suitable high dose to be tested in the subsequent OECD 422 screening study was concluded to be 7500 ppm.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2016

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:
no
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
Test material form:
liquid
Details on test material:
Batch No.: 156211
Purity: 90.1%
Name of test material (as cited in study report): neryl acetate
Physical state: colourless to slightly amber liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 21 April 2016

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited
- Age of F0 animals at study initiation: Approximately 10 weeks
- Weight at study initiation: Males: 338-383 g; Females: 207-259 g
- Housing: No. of animals per cage - Males: pre pairing and post pairing - up to 5 animals; Females: pre pairing - up to 5 animals; During pairing - one male and one female; Gestation - one female; Lactation: one female + litter. Toxicity phase and recovery phase - up to five animals of one sex. Cages comprised of a polycarbonate body with a stainless steel mesh lid. Solid (polycarbonate) bottom cages were used during the acclimatisation and treatment period with the exception of pairing periods. Grid bottomed cages comprising of polypropylene body were used during pairing.
- Diet: SDS VRF1 Certified powdered diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: carrier diet, including corn oil
Details on exposure:
DIET PREPARATION
- Carrier diet: SDS VRF1 certified diet.
- Stabiliser: Corn oil (test material to corn oil ratio 5:1)
- Method of preparation: On each occasion of the preparation of the premix the required amount of test substance and corn oil were mixed together and stirred. The mixture was added to an equal amount of plain diet and stirred until visibly homogenous. This doubling up process was repeated until half of the final weight of premix was achieved or the mixture appeared dry. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
For Control diet the corn oil was added directly to the diet and then prepared as indicated for the premix.
- Frequency of preparation: Weekly, however, preparations on some occasions were made in advance and stored frozen, but were used within the frozen stability period (22 days when stored frozen nominally -20 °C).
- Storage of preparation: Frozen (nominally -20 °C).

GROUPS
Groups 1, 2, 3 and 4: 0, 1000, 2500 and 7500 ppm, respectively
Details on mating procedure:
- Toxicity phase and recovery phase males were paired with reproductive phase females. Toxicity and recovery phase females were not paired for mating.
- M/F ratio per cage: 1:1 from within the same treatment groups
- Pairing commenced: After three weeks of treatment
- Length of cohabitation: Up to 3 weeks
- Proof of pregnancy: Presence of sperm within the vaginal smear and/or ejected copulation plugs referred to as Day 0 of pregnancy.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 100 and 20000 ppm were analysed to assess the stability and homogeneity of the test substance in the diet matrix. Stability was confirmed as eight days at ambient temperature and 22 days when stored frozen. However, only one day ambient stability was used.
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analysed for achieved concentration of the test substance.
Duration of treatment / exposure:
Toxicity phase males were treated daily for three weeks before pairing, throughout pairing and up to necropsy after a minimum of five consecutive weeks.
Toxicity phase females were treated daily for a minimum of five consecutive weeks up to necropsy in Week 6.
Reproductive females were treated daily for three weeks before pairing, throughout pairing, gestation and until Day 6 of lactation.
High dose Recovery phase male and female rats were treated daily for a minimum of six consecutive weeks, followed by a 14 day period without treatment.
Frequency of treatment:
Continuously (fresh diet given daily). During the recovery period, all animals were given untreated diet (without corn oil).
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 ppm
Remarks:
nominal in diet
Dose / conc.:
2 500 ppm
Remarks:
nominal in diet
Dose / conc.:
7 500 ppm
Remarks:
nominal in diet
No. of animals per sex per dose:
Reproduction phase: 10 females/dose for all dose groups
Toxicity phase : 5 animals/sex/dose for vehicle and high dose groups; 10 males/dose for 1000 and 2500 ppm dose groups; 5 females/dose for 1000 and 2500 ppm dose groups
Recovery phase: 5 animals/sex/dose for vehicle and high dose groups
Control animals:
other: control group was provided with the carrier diet, including corn oil
Details on study design:
- Dose selection rationale: Dose levels were selected in conjunction with the Sponsor following the completion of the dose range finder study (Study number OAD0022). In the dose range finding study the doses of 1500, 7500 or 15000 ppm were well tolerated (no mortality), no adverse clinical signs or macroscopic changes in organ appearance were detected at any dose level. Effects of treatment at 15000 ppm included initial body weight loss for the first few days of treatment in males and females, associated with lower food consumption, probably due to low palatability of the treated diet. In males, no effects on body weight and body weight gain were observed up to 7500 ppm but the overall bodyweight change in the high dose group was 23% lower compared to Control group. In females, body weight gain was lower than Control group at 7500 and 15000 ppm, especially during Days 1-7 and 15-22, leading to overall body weight gains 64% and 46% lower than Control group, respectively. The lower bodyweight gain observed in the mid dose group was mainly due to one female showing overall bodyweight loss during the treatment period (-3 g). However, even without this animal, the body weight gain in this group was only 50% of the Control group. Therefore, the high dose to be tested in the main study was considered to be 7500 ppm due to the toxicity observed in females. Then, the lower doses were spaced approximately by a 2.5 to 3-fold factor, i.e. 2500 and 1000 ppm.

- Rationale for animal assignment: Randomly allocated on arrival. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
Positive control:
Not applicable

Examinations

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

NEUROBEHAVIOURAL EXAMINATION: Yes
Detailed physical examination and arena observations: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each adult animal.
- Time schedule:
- Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery animals in Groups 1 and 4 and the five lowest numbered surviving toxicity phase males and females in Groups 2 and 3, during Week 6 of treatment.
- Motor activity:
During Week 6 of treatment, the motor activity of all recovery animals in Groups 1 and 4 and the five lowest numbered surviving toxicity phase males and females in Groups 2 and 3 was measured.

BODY WEIGHT: Yes
- Time schedule for examinations:
Toxicity and Recovery phase animals: Before feeding on the day that treatment commenced (Day 1), weekly thereafter and before necropsy.
Reproductive females: Before feeding on the day that treatment commenced (Day 1), weekly before pairing, on Days 0, 6, 13 and 20 after mating, on Days 1, 4 and 7 of lactation and before necropsy.

FOOD CONSUMPTION: Yes
- Time schedule for examinations:
Toxicity and recovery phase males and females: Daily. From these records the mean daily consumption per animal (g/rat/day) was calculated for each cage. Food consumption was not recorded for males during the period when paired for mating (Day 22 to 28). Food consumption recordings recommenced on Day 29.
- Reproductive phase females: Daily until paired for mating. From these records the mean daily consumption per animal (g/rat/day) was calculated for each cage.
Following pairing, on Days 0-5, 6-12 and 13-19 after mating and Days 1-3 and 4-6, of lactation. From these records the mean daily consumption (g/rat/day) was calculated for each animal.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Haematology: Week 6 - Five lowest numbered toxicity phase males and females per group; Recovery Week 2 - All recovery phase animals
Clinical chemistry: Week 6 - Five lowest numbered toxicity phase males and females per group; Recovery Week 2 - All recovery phase males
- Animals fasted: Yes, Blood samples were collected after overnight withdrawal of food.
- Animals were held under light general anaesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count, Percentage reticulocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count, Differential leucocyte count: Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count, Morphology: Anisocytosis, Macrocytosis, Microcytosis, Hypochromasia, Hyperchromasia, Prothrombin time and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin, Bile acids, Urea, Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus, Total protein, Albumin and Albumin/globulin ratio (A/G Ratio).

PARTURITION OBSERVATIONS AND GESTATION LENGTH:
- Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
- Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.
Oestrous cyclicity (parental animals):
- Dry smears: Daily smears were taken for 15 days before pairing, using cotton swabs moistened with saline. Smears were subsequently examined to establish the duration and regularity of the oestrous cycle.
- Wet smears: After pairing with the male, daily smearing was continued using pipette lavage, until evidence of mating was observed.
Litter observations:
PARAMETERS EXAMINED
- Clinical observations: Examined at approximately 24 h after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-7 of age.
- Sex ratio: The sex ratio of each litter was recorded on Days 1, 4 and 7 of age.
- Individual offspring bodyweights: F1 offspring - Days 1, 4 and 7 of age.
Postmortem examinations (parental animals):
SACRIFICE
- Toxicity phase animals: Following completion of Week 6 investigations
- Reproductive phase females: Scheduled kill - Day 7 of lactation; Failing to produce viable litter - Day 25 after mating.
- Recovery phase animals: After at least 14 days without treatment.
- Method of sacrifice: F0 animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination.

GROSS NECROPSY
- All adult animals were subject to a detailed necropsy. All external features and orifices were examined 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. In addition the number of uterine implantation sites recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together.
Histology
Fixation: Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of Testes in modified Davidson’s fluid; Eyes In Davidson’s fluid.
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
The five lowest numbered males and females in Groups 1 and 4 at scheduled termination.
Abnormalities only: All animals
- Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid Schiff (PAS) method.
Postmortem examinations (offspring):
SACRIFICE:
- F1 offspring scheduled kill: Day 7 of age
- Method of sacrifice: Intraperitoneal injection of sodium pentobarbitone

GROSS NECROPSY
- Offspring at scheduled termination: All F1 offspring were examined externally; any offspring found to be externally abnormal were also examined internally.
- Premature deaths (before Day 7 of age): Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before Day 7 of age were subject to fresh macroscopic examination, where possible (external and internal); this also included an assessment for the presence of milk in the stomach, where this was possible.
Statistics:
See section "Any other information on materials and methods incl. tables”.
Reproductive indices:
Mating Performance and Fertility:
- Percentage mating = (Number animals mating / Animals paired) x 100
- Conception rate (%) = (Number animals achieving pregnancy / Animals mated) x 100
- Fertility index (%) = (Number animals achieving pregnancy / Animals pairing) x 100

Gestation length
- Gestation index (%) = (Number of live litters born / Number pregnant) x 100
Offspring viability indices:
Survival indices:
- Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100
- Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
- Viability index (%) = (Number of live offspring on Day 7 after littering / Number live offspring on Day 1 after littering) x 100

Sex ratio:
- Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain during Week 1 of treatment was reduced in males and female receiving 7500 ppm with statistical significance.
Overall body weight gain in males was similar to Controls up to 2500 ppm and was lower but not statistically significantly at 7500 ppm.
Overall bodyweight gains in female were statistically significantly lower than Controls in all treated groups, but a dose response was not apparent.
Overall mean bodyweight gain during gestation and lactation was considered unaffected by treatment with Neryl Acetate up to 7500 ppm, when compared with Controls although isolated differences could be observed.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Overall food consumption for males and toxicity phase females receiving Neryl Acetate up to 7500 ppm was unaffected by treatment.
Overall food consumption for reproductive phase females was unaffected during the pre-pairing phase of treatment when compared with Controls.
Food intake was slightly but statistically significantly lower during Days 2-5 of lactation for females receiving 7500 ppm, leading to an overall reduction on food consumption of 20% when compared with Controls.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly lower group mean erythrocyte counts in males receiving 7500 ppm, and a slight but not statistically significant lower level for males receiving 2500 or 1000 ppm when compared with Controls.
Statistically significantly higher mean cell haemoglobin levels for males receiving 7500 ppm and slightly but statistically significant higher mean cell volume levels for males receiving 1000 ppm or above were observed.
Mean cell haemoglobin concentration was slightly but statistically significantly higher for females receiving 1000 ppm or above.
After 2 weeks of recovery haematological examinations were similar to that of the Controls. As a consequence these findings were considered to be of no toxicological significance.
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):
Organ weights for males and toxicity phase females killed after 6 weeks of treatment indicated that the adjusted group mean kidney weights for animals that received 7500 ppm and the adjusted group mean liver weights for females that received 7500 ppm were slightly but statistically significantly higher than that of the Controls.
Organ weights taken following the two-week recovery period for animals that received 7500 ppm, were similar to Controls indicating no persistent affects.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All histological changes were considered to be unrelated to treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- There were no decedents in the study. Routine physical examination and arena observations did not reveal any abnormalities that were considered related to treatment with test item.

BODY WEIGHT (PARENTAL ANIMALS)
- Group mean bodyweight gain during Week 1 of treatment was reduced in males receiving 7500 ppm and females receiving 1000, 2500 and 7500 ppm when compared with Controls, with statistical significance being attained at 7500 ppm for both males and females. Bodyweight gain was slightly, but not statistically significantly lower during Weeks 4-5 for males receiving 2500 ppm and almost throughout treatment period for males receiving 7500 ppm when compared with Controls. Overall bodyweight gain in males was similar to Controls up to 2500 ppm and was lower but not statistically significantly at 7500 ppm (87% of Controls). Bodyweight gain was slightly, but not statistically significantly lower during Weeks 1-2, 3-4 and 4-5 for toxicity phase females receiving Neryl Acetate when compared with Controls, and overall bodyweight gains were statistically significantly lower than Controls in all treated groups, but a dose response was not apparent.
- Overall bodyweight change during the recovery period was slightly but not statistically significantly lower than Controls for males, and was higher for females (200% of Controls) that had received 7500 ppm, but no statistical significance was attained.
- During the first week of treatment (Weeks 0-1) bodyweight gain for reproductive phase females prior to pairing, was moderately but not statistically significantly lower for females receiving 7500 ppm when compared to Controls, resulting in the overall group mean bodyweight gain prior to pairing being slightly lower than Controls for this group. In the 2500 ppm group, 2 females had particularly low bodyweight gain prior to pairing but had normal bodyweight gain thereafter. When excluding those females from calculations, mean bodyweight gain of the group 2 reproductive phase females prior to pairing is similar to Controls (24 g vs. 26 g, for groups 2 and Controls, respectively). Therefore, it is considered that performance of females receiving 1000 or 2500 ppm was globally similar to Controls.
- Overall mean bodyweight gain during gestation and lactation was considered unaffected by treatment with Neryl Acetate up to 7500 ppm, when compared with Controls although isolated significant differences could be observed.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- Overall food consumption for males and toxicity phase females receiving Neryl Acetate up to 7500 ppm was unaffected by treatment. Food intake was slightly lower on Day 1 of treatment for females receiving 7500 ppm when compared with Controls.
- Overall food consumption for reproductive phase females was unaffected during the pre-pairing phase of treatment when compared with Controls. Food intake was slightly lower on Day 1 of treatment for females receiving 7500 ppm when compared with Controls.
- Group mean food consumption on Day 0 of gestation was statistically significantly lower for females receiving 2500 or 7500 ppm when compared with Controls.
- Food intake was slightly but statistically significantly lower during Days 2-5 of lactation for females receiving 7500 ppm, leading to an overall reduction in food consumption of 20% when compared with Controls.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- Oestrous cycles, pre-coital interval and mating performance and fertility were considered unaffected by treatment with Neryl Acetate, when compared with Controls.
- Two females receiving 2500 ppm had irregular cycles. One Control female and one female receiving 7500 ppm had a pre-coital interval of 13-16 days; all other animals had an interval of 1-4 days.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Gestation length was within the expected time frame 22-23 days for all animals; gestation length and gestation index were considered unaffected by treatment at any dose level.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Organ weights for males and toxicity phase females killed after 6 weeks of treatment indicated that the adjusted group mean kidney weights for animals that received 7500 ppm and the adjusted group mean liver weights for females that received 7500 ppm were slightly but statistically significantly higher than that of the Controls.
- Organ weights taken following the two week recovery period for animals that received 7500ppm, were similar to Controls indicating no persistent affects.
- Group mean unadjusted and adjusted organ weights for reproductive phase females killed on Day 7 of lactation were essentially similar to Controls, showing no adverse effects of treatment.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- The macroscopic examination performed after the six weeks scheduled treatment period revealed no test substance related lesions. Macroscopic examination of reproductive female on Day 7 of lactation did not reveal any findings that could be attributed to treatment with test item.
The macroscopic examination performed after 2 weeks of recovery revealed no test substance related lesions. All findings were similar to the background of changes commonly seen in Sprague Dawley rats seen at these laboratories.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- No treatment related changes were observed in rats up to 7500 ppm by dietary administration for five weeks.
- Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of various cell types present within the different stages. No cell or stage specific abnormalities were noted.
- Minimal cortical tubular hypertrophy of kidney was observed in one male (4M No. 13) at 7500 ppm. This finding correlated with slight increase in the weight of kidneys. However, considering the single incidence in only one sex, its relationship to treatment is uncertain.
- An increase in the minimal multifocal aggregates of alveolar macrophages in the lung was observed in 4/5 females at 7500 ppm. One of the control females also had this finding. The aggregates of alveolar macrophages were mainly observed in the sub pleural locations or in the parenchyma away from terminal bronchioles, which are considered as common background finding in rats. The increase in the incidence in treated females alone is considered as incidental.

OTHER FINDINGS (PARENTAL ANIMALS)
Sensory reactivity and grip strength: There was no effect of test item on sensory reactivity or grip strength.
Motor activity: Motor activity was unaffected for females up to 7500 ppm, when compared to Controls and there were no clear effects on either rearing (high beam) or ambulatory (low beam) motor activity for males.
Haematology:
- Haematological examination during Week 6 of treatment revealed statistically significantly lower group mean erythrocyte counts in males receiving 7500 ppm, and a slight but not statistically significant lower level for males receiving 2500 or 1000 ppm when compared with Controls. Statistically significantly higher mean cell haemoglobin levels for males receiving 7500 ppm and slightly but statistically significant higher mean cell volume levels for males receiving 1000 ppm or above were observed. Mean cell haemoglobin concentration was slightly but statistically significantly higher for females receiving 1000 ppm or above. - Other inter-group differences from Controls were minor; neutrophil counts were higher for females receiving 7500 ppm when compared to Controls, but lacked a dose-relationship and was confined to one sex and was therefore considered due to normal biological variation.
- In Week 2 of recovery, haematological examinations of erythrocyte counts, mean cell haemoglobin and mean cell volume in males that received 7500 ppm and mean cell haemoglobin concentration, total leucocyte count and differential leucocyte count (neutrophils, lymphocytes, eosinophils, basophils, monocytes and large unstained cells) in females that received 7500 ppm were similar to that of the Controls.
Clinical chemistry:
- The biochemical examination of the blood plasma in Week 6 of treatment did not indicate any clear effects of treatment. When compared with the Controls, plasma levels of bile acids for males receiving 1000 ppm or above were slightly, but statistically significantly decreased, but no dose trend was apparent.
- All other inter-group differences from Controls were minor, were confined to one sex or lacked a dose-relationship. Such differences included minor, transient variations of some plasma electrolyte concentrations such as sodium levels in males and potassium levels in females receiving 7500 ppm; these values were considered fortuitous and were therefore considered of no toxicological importance.
- Review of bile acid levels in Week 2 of recovery revealed levels similar to Controls for males that received 7500 ppm. Group mean sodium levels in Week 2 of recovery were the same as Controls.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
7 500 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed up to the highest dose tested, corresponding to 440 mg/kg bw/day for males, 471-547 mg/kg bw/day during the gestation period and 624-1080 mg/kg bw/day during the lactation period for reproductive females.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean bodyweight on Days 1, 4 and 7 of age and mean bodyweight gain were lower in male (statistically significant lower bodyweight) and female (statistically significant lower bodyweight on Day 1 of age) offspring from parents treated at 7500 ppm when compared with Controls.
Male and female offspring bodyweights from parents that received 1000 or 2500 ppm were considered unaffected by treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

CLINICAL SIGNS (OFFSPRING)
There were no clinical signs observed for offspring that were considered related to treatment with test item.

LITTER SIZE, SURVIVAL INDICES AND SEX RATIO
- Litter size was not affected by treatment with test item. The number of females raising their litters to Day 7 of age was 9, 10, 10 and 10 for Control, 1000, 2500 and 7500 ppm respectively. One Control female (1F 100) was not pregnant.
- Group mean post implantation survival index, mean live birth index, and viability index were unaffected by treatment with test item.
- The mean percentage of males was statistically significantly higher than in Controls at 1000 or 7500 ppm but there was no dose response. No effect of treatment was inferred since the magnitude of difference from the ideal sex ratio of 50 % at 7500 ppm was similar to the magnitude of difference from 50 % in the Control group.

BODY WEIGHT (OFFSPRING)
- Group mean bodyweight on Days 1, 4 and 7 of age and mean bodyweight gain were lower in male (statistically significant lower bodyweight) and female (statistically significant lower bodyweight on Day 1 of age) offspring from parents treated at 7500 ppm when compared with Controls.
- Male and female offspring bodyweights from parents that received 1000 or 2500 ppm were considered unaffected by treatment.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic examination of offspring that either died prematurely or at scheduled termination on Day 7 of age did not reveal any findings that could be attributed to treatment with test item.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
7 500 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed up to the highest dose tested

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Formulation analysis:

The mean concentrations were within applied limits of +10%/-15%, confirming the accuracy of formulation. The difference from mean values was <3 %, confirming precise analysis. Procedural recovery values remained within 94.2 and 100.8 %, confirming the continued accuracy of the method.

Achieved dose

Achieved intake of animals measured during the toxicity phase generally maintained the intended intervals between treatment groups. As anticipated, achieved intake was higher at the start of the treatment period and declined progressively as the animals matured.

Table 7.8.1/4: Achieved dosage for Toxicity and Recovery phase males

 

Level (ppm)

Achieved dosage (mg/kg bw/day)

Mean

Range

1000

61

51-72

2500

150

129-175

7500

440

366-518

 

Table 7.8.1/5: Achieved dosage for Toxicity and Recovery phase females

 

Level (ppm)

Achieved dosage (mg/kg bw/day)

Mean

Range

1000

65

55-71

2500

150

127-169

7500

465

436-495

Achieved intake of females measured throughout the treatment period, generally maintained the intended intervals between treatment groups. Achieved intake was relatively stable during gestation, but increased significantly during lactation, reflecting increasing food intake during this period of high physiological demand on the parent female.

Table 7.8.1/6: Achieved dosage for Reproductive phase females

 

Level (ppm)

Achieved dosage (mg/kg bw/day)

Pre-pairing phase

Gestation

Lactation

Average

Range

Range

Range

1000

67

63-70

65-80

98-151

2500

172

161-182

168-189

266-390

7500

484

483-484

471-547

624-1080

Applicant's summary and conclusion

Conclusions:
The NOAEL for systemic and reproductive / developmental toxicity was 7500 ppm, corresponding to 440 mg/kg bw/day for males, 471-547 mg/kg bw/day during the gestation period and 624-1080 mg/kg bw/day during the lactation period for reproductive females.
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to guideline OECD 422 and in compliance with GLP, groups of Crl:CD(SD) rats received NERYL ACETATE orally, via the diet, at concentrations of 1000, 2500 or 7500 ppm. Toxicity phase males were treated daily for three weeks before pairing, throughout pairing and up to necropsy after a minimum of five consecutive weeks. Toxicity phase females were treated daily for a minimum of five consecutive weeks up to necropsy in Week 6. Reproductive females were treated daily for three weeks before pairing, throughout pairing, gestation and until Day 6 of lactation. Five high dose Recovery phase male and female rats were treated daily for a minimum of five consecutive weeks, followed by a 14 day period without treatment. Recovery phase males were used for pairing with reproductive phase females, but recovery females were not paired. A similarly constituted Control group was assigned to each phase, and received the vehicle in untreated diet, throughout the same relative treatment period.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology, blood chemistry,oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic investigations were undertaken for adult animals. Microscopic pathology investigations were undertaken for the first five Toxicity phase males and Reproductive and Toxicity phase females in Group 1 (Control) and Group 4 (7500 ppm). The clinical condition of offspring, litter size and survival, sex ratio and body weight were assessed, and macroscopic pathology investigations were undertaken. The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk.

The mean concentrations of the test item in formulations were within applied limits of +10%/-15%, confirming the accuracy of formulation. The difference from mean values was <3 %, confirming precise analysis. Procedural recovery values remained within 94.2 and 100.8 %, confirming the continued accuracy of the method.

Administration of the test item at dose levels of 1000, 2500 and 7500 ppm was well tolerated with no mortalities or effects that could be attributed to treatment on clinical condition, sensory reaction, grip strength, motor activity, oestrous cycles, macroscopic or microscopic pathology among the adult animals. In addition there were no effects on pre‑coital interval, mating performance, fertility, gestation length, offspring survival or development.

Mean achieved dosages during the pre-pairing phase were 61, 150 and 440 mg/kg bw/day for males and 65, 150 and 465 mg/kg bw/day for toxicity phase females and 67, 172 and 484 mg/kg/day for reproductive phase females receiving 1000, 2500 and 7500 ppm, respectively. For F0 females, the ranges of achieved dosages at 1000, 2500 and 7500 ppm were 65-80, 168-189 and 471-547 mg/kg bw/day during the gestation period and 98-151, 266-390 and 624-1080 mg/kg bw/day during the lactation period. 

Group mean bodyweight gain (Week 1 of treatment) was reduced in males receiving 7500 ppm and females receiving 1000, 2500 and 7500 ppm when compared with Controls; and overall bodyweight gain was statistically significantly lower than Controls in females, but a dose response was not apparent. Overall bodyweight change during the recovery period was slightly but not statistically significantly lower for males, but was significantly higher for females that had received 7500 ppm. Overall food consumption for males and toxicity phase females receiving NERYL ACETATE up to 7500 ppm was unaffected by treatment. Food intake was slightly lower on Day 1 of treatment for toxicity and reproductive females receiving 7500 ppm when compared with Controls. Group mean food consumption during early gestation was low for females receiving 2500 or 7500 ppm and during Days 2-5 of lactation for females receiving 7500 ppm, when compared with Controls.

Haematological examination during Week 6 of treatment revealed statistically significantly lower group mean erythrocyte counts in males receiving 7500 ppm, and a slight but not statistically significant lower level for males receiving 2500 or 1000 ppm when compared with Controls. Statistically significantly higher mean cell haemoglobin levels for males receiving 7500 ppm and slightly but statistically significant higher mean cell volume levels for males receiving 1000 ppm or above. Mean cell haemoglobin concentration was slightly but statistically significant higher for females receiving 1000 ppm or above. Haematological parameters examined in Week 2 of recovery were similar to that of the Controls.

The biochemical examination of the blood plasma in Week 6 of treatment did not indicate any clear effects of treatment. When compared with the Controls, plasma levels of bile acids for males receiving 1000 ppm or above were slightly, but statistically significantly decreased, but no dose trend was apparent. Review of bile acids levels in Week 2 of recovery revealed similar levels to Controls for males that received 7500 ppm.

Organ weights for males and toxicity phase females killed after 6 weeks of treatment indicated that the adjusted group mean kidney weights for animals that received 7500 ppm and the adjusted group mean liver weights for females that received 7500 ppm were slightly but statistically significantly higher than that of the Controls. Organ weights taken following the two week recovery period for animals that received 7500 ppm, were similar to Controls indicating no persistent effects. Group mean unadjusted and adjusted organ weights for reproductive phase females killed on Day 7 of lactation were essentially similar to Controls, showing no adverse effects of treatment.

Group mean offspring bodyweight on Days 1, 4 and 7 of age and mean bodyweight gain were slightly lower in male (statistically significant lower bodyweight) and female (statistically significant lower bodyweight on Day 1 of age), offspring from parents treated at 7500 ppm when compared with Controls, but their growth curves were essentially parallel to those of the Controls.

Therefore, the NOAEL for systemic and reproductive / developmental toxicity was 7500 ppm.