Cyclopentadecanone

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 2016 - 16 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 2016 - 16 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

October 2008

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in refrigerator (2-8°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stability for at least 6 hours at room temperature, under normal laboratory light conditions, is confirmed over the concentration range 1 to 200 mg/mL.

OTHER SPECIFICS:
- No correction factor for purity is required

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: not applicable, repro/developmental screening study
- Age at study initiation: approximately 11 weeks
- Weight at study initiation: males 298-345 g, females 185-224 g
- Fasting period before study: no
- Housing:
Pre-mating: in groups of 5 animals/sex/cage in Macrolon plastic cages
Mating: males and females on 1-to-1 basis in Macrolon plastic cages
Post-mating: males: max. 5/cage in their home cages (Macrolon), females individually in Macrolon plastic cages
Lactation: pups with the dam in Macrolon plastic cages, except during locomotor activity monitoring of the dams. The pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
Locomotor activity measurements: individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water
Sterilized sawdust was provided as bedding material and paper as cage-enrichment/nesting material, except during locomotor activity measurements.
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except overnight before sacrifice
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY: Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients were performed according to facility standard procedures. There were no findings that could interfere with the study

ENVIRONMENTAL CONDITIONS (set to maintain):
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 Augusut 2016 To: 16 November 2016

Route of administration:

oral: gavage

Details on route of administration:

This study should provide part of a rational basis for toxicological risk assessment in man. The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. To facilitate homogenization, the test item (in vehicle) was heated to a maximum of 70°C for a maximum duration of 40 minutes. Adjustment was made for specific gravity of the test item (0.973) and vehicle (0.92).

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at Charles River Den Bosch.
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Stability of the test item in formulations was determined as part of the analytical method development and validation study.

Duration of treatment / exposure:

Males: 31 days (2 weeks prior to mating, during mating, and up to and including the day prior to scheduled necropsy)
Females that delivered: 40-54 days (during 2 weeks prior to mating, the variable time to conception, the duration of the pregnancy and during 4-6 days of lactation (up to and including the day before scheduled necropsy)).
Females which failed to deliver healthy offspring: 42 and 54 days, respectively

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Dose / conc.:

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

Remarks:

Concurrent vehicle controls (Group 1)

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 20-day dose range finding study
- Section schedule:
Males: following completion of the mating period (a minimum of 28 days of dose administration).
Females that delivered: PND 5-7
Females that failed to deliver: Post-coitum days 25-27 (females with evidence of mating)

Positive control:

Not applicable.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, at least 3 hours (± 30 min) after treatment (on the peak period of anticipated effects after treatment). Once
prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of exposure (prior to first exposure) and weekly thereafter.
Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.

FOOD CONSUMPTION: yes
- Time schedule for examinations: weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of scheduled sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight (max. 24 hours)
- How many animals: 5/sex/group
- Parameters examined: WBC, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), RBC, reticulocytes, RDW, haemoglobin, haematocrit, MCV, MCH, MCHC, platelets, PT, APTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of scheduled sacrifice
- Animals fasted: Yes, overnight (max. 24 hours)
- How many animals: 5/sex/dose
- Parameters examined: ALAT, ASAT, ALP, total protein, albumin, total bilirubin, bile acids, urea creatinine, glucose, cholesterol, sodium , potassium, chloride, calcium, inorganic phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: selected males: during week 4 of treatment, selected females: from lactation day 4 onwards.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity (hearing, pupillary reflex, static righting reflex), grip strength (fore- and hind-limbs), motor activity (total movements and ambulations)

IMMUNOLOGY: No

OTHER: for details on pup examination see sections 7.8.1 and 7.8.2 of this IUCLID (cross-references)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following organ wegiths were recorded:
Selected 5 animals/sex/group: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostates, seminal vesicles including coagulating glands, spleen, testes, thymus, thyroid (including parathyroid), uterus (including cervix)
All remaining animals: epididymides, testes

HISTOPATHOLOGY: Yes
The following tissues were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex in control and high-dose groups: adrenal glands, brain - cerebellum, mid-brain, cortex (7-levels), caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides, eyes (with optic nerve if detectable and Harderian gland), female mammary gland area, femur including joint, heart, ileum, jejunum, kidneys, liver, lung (infused with formalin), lymph nodex (mandibular, mesenteric0, ovaries, Peyer's patches (jejunum, ileum, if detectable), pituitary gland, preputial gland, prostate gland, rectum, sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, midthoracic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid if detectable, trachea, urinary bladder, uterus, vagina, all gross lesions.
- Additional slides of the testes of the selected 5 males of the control and high-dose group and all males that failed to sire, to examine staging of spermatogenesis
- The preserved organs and tissues of one female euthanized in extremis
- Thyroid glands and liver of all selected 5 animals of low- and mid-dose groups, pituitary gland and kidneys of all selected 5 males of low- and mid-dose groups, and adrenal glands and thymus of all selected 5 females of low- and mid-dose groups, based on (possible) treatment-related changes in these organs in high-dose group
- All gross lesions of all animals (all dose groups).
- The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups (one non-pregnant female in controls, in low-dose and high-dose group and one high-dose group female euthanized in extremis).

Other examinations:

For examinations on reproductive and developmental toxicity see sections 7.8.1 and 7.8.2 of this IUCLID (cross-references).

Statistics:

The following statistical methods were used to analyze the data:
• If the variables were assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related clinical signs of toxicity were noted at 300 mg/kg bw/day and 1000 mg/kg bw/day (both sexes). Frequent findings included hunched posture, uncoordinated movements, piloerection and/or tremors. Additionally, slight lethargy and/or ptosis (both at a slight degree) were noted for two females at 1000 mg/kg bw/day on 1-2 days.
During the weekly arena observations, no additional treatment-related clinical signs were noted.
There were no toxicologically relevant findings for males and females at 100 mg/kg bw/day. Salivation seen after dosing among males at 100 mg/kg bw/day and higher, and females at 300 and 1000 mg/kg bw/day was not considered toxicologically relevant, taking into account the nature and slight severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Any other clinical signs noted incidentally (wound, scabs on the skin, red discoloration of the skin, alopecia, rales and diarrhoea) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. As they were observed for a control animal only, showed no dose-related trend and/or were isolated findings, they were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One high dose female was euthanized on Day 23 post-coitum due to complications during parturition. Before euthanasia she had a pale appearance, piloerection, ptosis, and dull eyes. She had started to give birth: blood was found in her cage and at least one pup was seen which was missing at a later time point (taken from study daybook). Most likely these pups had been cannibalized by their mother. At necropsy, she had a pale appearance. Next to one dead fetus obstructing the cervix, 10 other dead fetuses were found in her uterus (in total 5 males and 6 females; no external abnormalities). This single death in the high dose group was considered not to be related to treatment with the test item.
All remaining animals survived until their scheduled necropsies.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In females at 1000 mg/kg bw/day, there was a trend towards slightly higher body weight gain during mating and the first two weeks of post-coitum. This resulted in slightly, but statistically significant, higher group mean values for absolute body weight in the high dose group compared to the concurrent control group during Days 4-14 post-coitum. As values remained within the normal range of biological variation, no toxicological relevance was attached to this finding.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The slightly lower food intake noted for males at 300 mg/kg bw/day during the mating period was considered a chance finding, since in the same period males at 1000 mg/kg bw/day had normal values for food consumption. Furthermore, no treatment-related effects on body weights were observed in this study.
When compared to the concurrent control group, a slightly, but statistically significantly, higher food intake (absolute and/or relative to body weight) was recorded for females treated at 1000 mg/kg bw/day during Days 0-4 post-coitum (absolute and relative) and Days 7-11 postcoitum (absolute). As values remained within the normal range of biological variation, no toxicological relevance was attached to this finding.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes occurred in haematology parameters in males and females treated up to 1000 mg/kg bw/day.
Any statistically significant changes noted in haematology parameters in males at the end of the treatment period were considered not toxicologically relevant due to the absence of a dose-related response and/or slight magnitude of the difference (values in treated rats remained within normal limits).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes occurred in clinical biochemistry parameters in males and females treated up to 1000 mg/kg bw/day.
Any statistically significant changes at 300 and 1000 mg/kg bw/day were not considered to be toxicologically significant as they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals.
The variation in motor activity did not indicate a relation with treatment. The relative low mean value of total movements and ambulations noted for low dose males (Group 2) during the first 5 minutes (interval 1) of the motor activity measurement was not considered to be treatment-related as a similar low activity was seen for males of the control (Group 1). From interval 2 onwards, motor activity of Groups 1 and 2 was in the same range as for Groups 3 and 4, and all groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related higher liver, thyroid glands, kidneys and spleen weights (absolute and/or relative to body weights) were noted in males starting at 100 mg/kg bw/day and in females at 1000 mg/kg bw/day.
Liver:
Higher liver weights (absolute and/or relative to body weights) were present in males treated at 100, 300 and 1000 mg/kg bw/day and in females treated at 1000 mg/kg bw/day and there was an apparent, non-significant, increase in females treated at 300 mg/kg bw/day. The microscopic correlate was hepatocellular hypertrophy.
Thyroid gland:
Higher thyroid gland weights (absolute and relative to body weights) were present in males treated at 1000 mg/kg bw/day and there was an apparent, non-significant, increase in males treated at 300 mg/kg bw/day. The microscopic correlate was follicular cell hypertrophy.
Kidneys:
Higher kidney weights (absolute and relative to body weighs) were present in males treated at 1000 mg/kg bw/day. The microscopic correlate was increased hyaline droplet accumulation.
Spleen:
Higher spleen weights (relative to body weights) were present in males treated at 1000 mg/kg bw/day. There was no microscopic correlate.
There were no other test item-related organ weight changes. The higher absolute epididymides weights in males of the 300 and 1000 mg/kg bw/day groups were not test item-related, as no dose-related response could be established and values in treated rats remained within normal limits.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The following test item-related macroscopic findings were noted in males treated at 1000 mg/kg bw/day:
Liver:
Enlarged liver was present in 8/10 males. This corresponded microscopically with hepatocellular hypertrophy.
Thyroid gland:
Enlarged thyroid glands were present in 10/10 males. This corresponded microscopically with increased follicular cell hypertrophy. Background incidences were present in 1/10 males each in the control, 100 and 300 mg/kg bw/day groups.
Kidneys:
Pale discolouration of the kidneys was noted in 9/10 males treated at 1000 mg/kg bw/day. This corresponded microscopically with increased hyaline droplet accumulation.
No test item-related macroscopic abnormalities were noted in males at 100 and 300 mg/kg bw/day and females up to 1000 mg/kg bw/day. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment were noted in the liver and thyroid gland of males and females starting at 100 mg/kg bw/day, in the kidneys of males starting at 100 mg/kg bw/day, in the pituitary gland of males at 1000 mg/kg bw/day, and in the adrenal glands and thymus of females at 1000 mg/kg bw/day.
Liver:
Hepatocellular hypertrophy was present in males and females treated at 100, 300 and 1000 mg/kg bw/day up to moderate degree.
Thyroid gland:
Follicular cell hypertrophy was present at increased incidence and severity in males and females treated at 100, 300 and 1000 mg/kg bw/day up to moderate degree.
Kidneys:
Hyaline droplet accumulation was present at increased incidence and severity in males treated at 100, 300 and 1000 mg/kg bw/day up to marked degree.
Tubular basophilia was present at increased incidence and severity in males treated at 300 and 1000 mg/kg bw/day up to moderate degree.
Granular casts were present in males treated at 300 and 1000 mg/kg bw/day up to moderate degree.
Pituitary gland:
Hypertrophy pars distalis was present in males treated at 1000 mg/kg bw/day at minimal degree.
Adrenal glands:
Vacuolation zona glomerulosa was present at increased incidence and severity in females treated at 1000 mg/kg bw/day.
Thymus:
Lymphoid atrophy was present at increased incidence in females treated at 1000 mg/kg bw/day up to slight degree.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

For reproductive and developmental toxicity findings see Sections 7.8.1 and 7.8.2 of this IUCLID (cross-references).

Details on results:

Hyaline droplet accumulation occurred at increased incidence and severity in males treated at 100, 300 and 1000 mg/kg bw/day up to marked degree. Correlated necropsy findings in high dose males included pale discolouration of the kidneys (noted in 9/10 males), and a 19% higher mean kidney organ weight (absolute and relative to body weight) as compared to the concurrent control males. The hyaline droplet accumulation recorded was considered to likely represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein is not present in female rats nor in higher mammals, including man. The increased hyaline droplet accumulation in the male kidneys was accompanied by indicators of tubular damage in the form of granular casts and increased tubular basophilia starting at 300 mg/kg bw/day and therefore this was considered to be adverse from 300 mg/kg bw/day onwards.
Significantly higher liver weights (absolute and/or relative to body weights) were present in males treated at 100, 300 and 1000 mg/kg bw/day (up to approximately 53%) and in females treated at 1000 mg/kg bw/day (approximately 45%), and there was an apparent, non-significant, increase in the females treated at 300 mg/kg bw/day. The microscopic correlate was hepatocellular hypertrophy, which was noted in males and females starting at 100 mg/kg bw/day. In the absence of any degenerative changes and corroborative findings in clinical pathology parameters, the treatment-related higher liver weights were considered to be non-adverse by the study director.
In thyroids of males and females starting at 100 mg/kg bw/day an increased incidence and/or severity (up to moderate degree) of follicular cell hypertrophy was observed. In males, this hypertrophy resulted in increased thyroid gland weights at 300 mg/kg bw/day (not statistically significant) and at 1000 mg/kg bw/day. The increased follicular cell hypertrophy was regarded to be an adaptive response to the induction of hepatic enzymes and considered non-adverse by the study director at the incidences and severities recorded.
The minimal hypertrophy in the pars distalis of the pituitary gland seen in 2/5 males treated at 1000 mg/kg bw/day was considered non-adverse by the study director, based on the low severity and incidence and in the absence of any other indicators of toxicity in this organ. The vacuolation of the zona glomerulosa recorded at increased incidence and severity in the adrenal glands of females treated at 1000 mg/kg bw/day was not accompanied by any other indicator of toxicity such as cellular damage, and at the severities observed (up to slight) it was considered non-adverse by the study director.
The increased incidence and severity of lymphoid atrophy observed in the thymus of females at 1000 mg/kg bw/day was considered to be likely secondary to stress and not considered adverse by the study director at this incidence and severity.

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity, set by the study director

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity, used for risk assessment purposes

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Formulation analysis

The concentrations analysed in the formulations of low, mid- and high dose groups were in agreement with target concentrations (i.e. mean accuracies resp. 106% (n = 6), 1095 (n = 2) and 92% (n = 6)).

The formulations of low and high dose groups were homogeneous (i.e. coefficient of variation 6.7% and 7.7%, respectively).

Conclusions:

In a GLP-compliant OECD guideline 422 study with rats, the NOAEL for general toxicity was set at the lowest dose level of 100 mg/kg bw/day by the study director, based on tubular damage in the form of granular casts and increased tubular basophilia in male kidneys starting at 300 mg/kg bw/day. However, for risk assessment purposes the dose level of 100 mg/kg bw/day is considered a LOAEL, based on significantly higher liver weights in males and hepatocellular hypertrophy and follicular cell hypertrophy in the thyroid in both sexes at this dose level.

Executive summary:

In a GLP-compliant OECD guideline 422 study with rats, male and female rats were treated by oral gavage with 100, 300 and 1000 mg/kg bw/day cyclopentadecanone for at least 28 days (males) or at least 40 days (females). There were no treatment-related mortalities. Treatment-related clinical signs of toxicity were noted at 300 and 1000 mg/kg bw/day in both sexes. Frequent findings included hunched posture, uncoordinated movements, piloerection and/or tremors. Adverse test item-related morphologic alterations were present in the kidneys of males. Hyaline droplet accumulation occurred at increased incidence and severity in males treated at 100, 300 and 1000 mg/kg bw/day up to marked degree. Correlated necropsy findings in high dose males included pale discolouration of the kidneys (noted in 9/10 males), and a 19% higher mean absolute and relative kidney weight compared to the concurrent control males. The hyaline droplet accumulation recorded was considered to likely represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein is not present in female rats nor in higher mammals, including man. The increased hyaline droplet accumulation in the male kidneys was accompanied by indicators of tubular damage in the form of granular casts and increased tubular basophilia starting at 300 mg/kg bw/day and therefore this was considered to be adverse from 300 mg/kg bw/day onwards. Furthermore, significantly higher liver weights (absolute and/or relative to body weights) were present in males treated at 100, 300 and 1000 mg/kg bw/day and in females treated at 1000 mg/kg bw/day, and there was an apparent, non-significant, increase in the females treated at 300 mg/kg bw/day. The microscopic correlate was hepatocellular hypertrophy, which was noted in males and females starting at 100 mg/kg bw/day. In thyroids of males and females starting at 100 mg/kg bw/day an increased incidence and/or severity (up to moderate degree) of follicular cell hypertrophy was observed. In males, this hypertrophy resulted in increased thyroid gland weights at 300 (not statistically significant) and at 1000 mg/kg bw/day. Because of the absence of degenerative changes and corroborative findings in clinical pathology the changes in the liver were considered non-adverse by the study director. The observed changes in thyroid were considered to be an adaptive response to the induction of hepatic enzymes and therefore also considered non-adverse. The lowest dose level of 100 mg/kg bw/day was therefore considered a NOAEL by the study director, based on the observed tubular damage and increased tubular basophilia in male rats starting from 300 mg/kg bw/day. However, based on the statistically significant increase in relative liver weight by 18% compared to controls in combination with hepatocellular hypertrophy and follicular cell hypertrophy in the thyroids at the lowest dose level, for risk assessment purposes the lowest dose level of 100 mg/kg bw/day will be considered a LOAEL.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 2016 - 16 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in refrigerator (2-8°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stability for at least 6 hours at room temperature, under normal laboratory light conditions, is confirmed over the concentration range 1 to 200 mg/mL.

OTHER SPECIFICS:
- No correction factor for purity is required

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 11 weeks
- Weight at study initiation: males 298-345 g, females 185-224 g
- Fasting period before study: no
- Housing:
Pre-mating: in groups of 5 animals/sex/cage in Macrolon plastic cages
Mating: males and females on 1-to-1 basis in Macrolon plastic cages
Post-mating: males: max. 5/cage in their home cages (Macrolon), females individually in Macrolon plastic cages
Lactation: pups with the dam in Macrolon plastic cages, except during locomotor activity monitoring of the dams. The pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
Locomotor activity measurements: individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water
Sterilized sawdust was provided as bedding material and paper as cage-enrichment/nesting material, except during locomotor activity measurements.
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except overnight before sacrifice
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY: Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients were performed according to facility standard procedures. There were no findings that could interfere with the study

ENVIRONMENTAL CONDITIONS (set to maintain):
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 Augusut 2016 To: 16 November 2016

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. To facilitate homogenization, the test item (in vehicle) was heated to a maximum of 70°C for a maximum duration of 40 minutes. Adjustment was made for specific gravity of the test item (0.973) and vehicle (0.92).

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at Charles River Den Bosch.
- Amount of vehicle (if gavage): 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until mating, for maximum 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually in Macrolon plastic cages

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Stability of the test item in formulations was determined as part of the analytical method development and validation study.

Duration of treatment / exposure:

Males: 31 days (2 weeks prior to mating, during mating, and up to and including the day prior to scheduled necropsy)
Females that delivered: 40-54 days (during 2 weeks prior to mating, the variable time to conception, the duration of the pregnancy and during 4-6 days of lactation (up to and including the day before scheduled necropsy)).
Females which failed to deliver healthy offspring: 42 and 54 days, respectively

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Details on study schedule:

- Age at mating of the mated animals in the study: 13 weeks

Dose / conc.:

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

Remarks:

Concurrent vehicle controls (Group 1)

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 20-day dose range finding study
- Section schedule:
Males: following completion of the mating period (a minimum of 28 days of dose administration).
Females that delivered: PND 5-7
Females that failed to deliver: Post-coitum days 25-27 (females with evidence of mating)

Positive control:

Not applicable.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, at least 3 hours (± 30 min) after treatment (on the peak period of anticipated effects after treatment). Once
prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of exposure (prior to first exposure) and weekly thereafter.
Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.

FOOD CONSUMPTION: yes
- Time schedule for examinations: weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OTHER:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of scheduled sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight (max. 24 hours)
- How many animals: 5/sex/group
- Parameters examined: WBC, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), RBC, reticulocytes, RDW, haemoglobin, haematocrit, MCV, MCH, MCHC, platelets, PT, APTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of scheduled sacrifice
- Animals fasted: Yes, overnight (max. 24 hours)
- How many animals: 5/sex/dose
- Parameters examined: ALAT, ASAT, ALP, total protein, albumin, total bilirubin, bile acids, urea creatinine, glucose, cholesterol, sodium , potassium, chloride, calcium, inorganic phosphate

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: selected males: during week 4 of treatment, selected females: from lactation day 4 onwards.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity (hearing, pupillary reflex, static righting reflex), grip strength (fore- and hind-limbs), motor activity (total movements and ambulations)

OTHER: for details on general toxicity evaluation see section 7.5.1 of this IUCLID (cross-reference)

Oestrous cyclicity (parental animals):

Not evaluated.

Sperm parameters (parental animals):

Parameters examined in the male parental generation:
testis weight, epididymis weight, stages of spermatogenesis (during testes histopathology)

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no, necropsy of all pups on PND 5-7


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not performed

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not performed

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals following completion of the mating period (a minimum of 28 days of dose administration).
- Maternal animals:
Females that delivered: PND 5-7
Females that failed to deliver: Post-coitum days 25-27 (females with evidence of mating)

GROSS PATHOLOGY: Yes


HISTOPATHOLOGY: Yes
The following tissues were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex in control and high-dose groups: adrenal glands, brain - cerebellum, mid-brain, cortex (7-levels), caecum, cervix, clitoral gland, colon, coagulation gland, duodenum, epididymides, eyes (with optic nerve if detectable and Harderian gland), female mammary gland area, femur including joint, heart, ileum, jejunum, kidneys, liver, lung (infused with formalin), lymph nodex (mandibular, mesenteric0, ovaries, Peyer's patches (jejunum, ileum, if detectable), pituitary gland, preputial gland, prostate gland, rectum, sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, midthoracic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid including parathyroid if detectable, trachea, urinary bladder, uterus, vagina, all gross lesions.
- Additional slides of the testes of the selected 5 males of the control and high-dose group and all males that failed to sire, to examine staging of spermatogenesis
- The preserved organs and tissues of one female euthanized in extremis
- Thyroid glands and liver of all selected 5 animals of low- and mid-dose groups, pituitary gland and kidneys of all selected 5 males of low- and mid-dose groups, and adrenal glands and thymus of all selected 5 females of low- and mid-dose groups, based on (possible) treatment-related changes in these organs in high-dose group
- All gross lesions of all animals (all dose groups).
- The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups (one non-pregnant female in controls, in low-dose and high-dose group and one high-dose group female euthanized in extremis).

ORGAN WEIGHTS: Yes
The following organ wegiths were recorded:
Selected 5 animals/sex/group: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostates, seminal vesicles including coagulating glands, spleen, testes, thymus, thyroid (including parathyroid), uterus (including cervix)
All remaining animals: epididymides, testes

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on PND 5-7
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS: not performed

Statistics:

The following statistical methods were used to analyze the data:
• If the variables were assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Reproductive indices:

The following indices were calculated:
Precoital time = Number of days between initiation of cohabitation and confirmation of mating
Mating index (%) = 100 x (number of females mated/number of females paired)
Fertility index (%) = 100 x (number of pregnant females/number of females paired)
Conception index (%) = 100 x (number of pregnant females/number of females mated)
Gestation index (%) = 100 x (number of females bearing live pups/number of pregnant females)
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

The following indices were calculated:

Percentage live males at first litter check (%) = 100 x (number of live males at first litter check/number of live pups at first litter check)
Percentage live females at first litter check (%) = 100 x (number of live females at first litter check/number of live pups at first litter check)
Percentage of postnatal loss (%) = 100 x (number of dead pups before scheduled necropsy/number of live pups at first litter check)
Viability index (%) = 100 x (number of live offspring on PND 4/number of liver offspring on day 1 after littering)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related clinical signs of toxicity were noted at 300 mg/kg bw/day and 1000 mg/kg bw/day (both sexes). Frequent findings included hunched posture, uncoordinated movements, piloerection and/or tremors. Additionally, slight lethargy and/or ptosis (both at a slight degree) were noted for two females at 1000 mg/kg bw/day on 1-2 days.
During the weekly arena observations, no additional treatment-related clinical signs were noted.
There were no toxicologically relevant findings for males and females at 100 mg/kg bw/day. Salivation seen after dosing among males at 100 mg/kg bw/day and higher, and females at 300 and 1000 mg/kg bw/day was not considered toxicologically relevant, taking into account the nature and slight severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Any other clinical signs noted incidentally (wound, scabs on the skin, red discoloration of the skin, alopecia, rales and diarrhoea) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. As they were observed for a control animal only, showed no dose-related trend and/or were isolated findings, they were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One high dose female was euthanized on Day 23 post-coitum due to complications during parturition. Before euthanasia she had a pale appearance, piloerection, ptosis, and dull eyes. She had started to give birth: blood was found in her cage and at least one pup was seen which was missing at a later time point (taken from study daybook). Most likely these pups had been cannibalized by their mother. At necropsy, she had a pale appearance. Next to one dead fetus obstructing the cervix, 10 other dead fetuses were found in her uterus (in total 5 males and 6 females; no external abnormalities). This single death in the high dose group was considered not to be related to treatment with the test item.
All remaining animals survived until their scheduled necropsies.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In females at 1000 mg/kg bw/day, there was a trend towards slightly higher body weight gain during mating and the first two weeks of post-coitum. This resulted in slightly, but statistically significant, higher group mean values for absolute body weight in the high dose group compared to the concurrent control group during Days 4-14 post-coitum. As values remained within the normal range of biological variation, no toxicological relevance was attached to this finding.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The slightly lower food intake noted for males at 300 mg/kg bw/day during the mating period was considered a chance finding, since in the same period males at 1000 mg/kg bw/day had normal values for food consumption. Furthermore, no treatment-related effects on body weights were observed in this study.
When compared to the concurrent control group, a slightly, but statistically significantly, higher food intake (absolute and/or relative to body weight) was recorded for females treated at 1000 mg/kg bw/day during Days 0-4 post-coitum (absolute and relative) and Days 7-11 postcoitum (absolute). As values remained within the normal range of biological variation, no toxicological relevance was attached to this finding.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes occurred in haematology parameters in males and females treated up to 1000 mg/kg bw/day.
Any statistically significant changes noted in haematology parameters in males at the end of the treatment period were considered not toxicologically relevant due to the absence of a dose-related response and/or slight magnitude of the difference (values in treated rats remained within normal limits).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes occurred in clinical biochemistry parameters in males and females treated up to 1000 mg/kg bw/day.
Any statistically significant changes at 300 and 1000 mg/kg bw/day were not considered to be toxicologically significant as they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals.
The variation in motor activity did not indicate a relation with treatment. The relative low mean value of total movements and ambulations noted for low dose males (Group 2) during the first 5 minutes (interval 1) of the motor activity measurement was not considered to be treatment-related as a similar low activity was seen for males of the control (Group 1). From interval 2 onwards, motor activity of Groups 1 and 2 was in the same range as for Groups 3 and 4, and all groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment were noted in the liver and thyroid gland of males and females starting at 100 mg/kg bw/day, in the kidneys of males starting at 100 mg/kg bw/day, in the pituitary gland of males at 1000 mg/kg bw/day, and in the adrenal glands and thymus of females at 1000 mg/kg bw/day.
Liver:
Hepatocellular hypertrophy was present in males and females treated at 100, 300 and 1000 mg/kg bw/day up to moderate degree.
Thyroid gland:
Follicular cell hypertrophy was present at increased incidence and severity in males and females treated at 100, 300 and 1000 mg/kg bw/day up to moderate degree.
Kidneys:
Hyaline droplet accumulation was present at increased incidence and severity in males treated at 100, 300 and 1000 mg/kg bw/day up to marked degree.
Tubular basophilia was present at increased incidence and severity in males treated at 300 and 1000 mg/kg bw/day up to moderate degree.
Granular casts were present in males treated at 300 and 1000 mg/kg bw/day up to moderate degree.
Pituitary gland:
Hypertrophy pars distalis was present in males treated at 1000 mg/kg bw/day at minimal degree.
Adrenal glands:
Vacuolation zona glomerulosa was present at increased incidence and severity in females treated at 1000 mg/kg bw/day.
Thymus:
Lymphoid atrophy was present at increased incidence in females treated at 1000 mg/kg bw/day up to slight degree.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Spermatogenic staging profiles were normal for all males examined. No further assessment was performed.

Reproductive performance:

no effects observed

Description (incidence and severity):

All females of the control and treated groups showed evidence of mating. Mating index was 100% for all groups.
Fertility and conception index were not affected by treatment. All paired and mated females were pregnant, except for one female each in the control, 100 and 1000 mg/kg bw/day group. In addition, one female treated at 1000 mg/kg bw/day was sacrificed moribund on Day 23 post coitum due to difficulties with parturition. In the absence of a dose-related distribution and since no abnormalities were seen in the reproductive organs which could account for these non-pregnancies, they were considered not to be related to treatment.
Precoital time was not affected by treatment. All females were mated within 5 days, except for two females in the 100 mg/kg bw/day group and one female in the 300 mg/kg bw/day group for which it took 12-14 days before they showed evidence of mating. In the absence of a dose-related trend, this finding was considered not to be caused by the test item.
The number of corpora lutea and implantation sites was considered to be unaffected by treatment. For one female at 100 mg/kg bw/day, the number of implantations was slightly higher than the number of corpora lutea. This was considered to be due to normal resorption of these areas as these enumerations were performed on Day 5 of lactation.
All females in this study had a normal parturition, except for the single high-dose female euthanized preterm due to complications when giving birth. This was considered not to be related to treatment. Examination of cage debris of pregnant females revealed no signs of premature birth.
One female at 100 mg/kg bw/day showed deficiencies in maternal care. All her 10 pups were noted with wounds, scabs and/or blue spots on different parts of their body at the first litter check and during one or more days of lactation. Also body weight of these pups were slightly lower when compared to other litters with comparable size or even bigger. There were no indications for less milk in their stomach. The clinical signs and slightly lower body weights in litter 55 were considered to be unrelated to treatment as it was an isolated finding at the low dose only. All pups in this litter survived until scheduled necropsy on lactation Day 5.

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity, set by the study director

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity, used for risk assessment purposes

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on reproductive performance at the highest tested dose of 1000 mg/kg bw/day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment.
One pup each of the control, 100 and 300 mg/kg bw/day group was found dead at first litter check. In addition, one control pup was found missing on lactation Day 7. It was most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were not affected by treatment up to and including 1000 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment. The three pups found dead at first litter check were noted with (beginning) autolysis. This is a normal finding for dead pups. A few pups surviving until scheduled necropsy were observed with scabs. The nature and incidence of this macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio of the pups was unaffected by treatment up to and including 1000 mg/kg bw/day.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on development at the highest tested dose of 1000 mg/kg bw/day

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Formulation analysis:

The concentrations analysed in the formulations of low, mid- and high dose groups were in agreement with target concentrations (i.e. mean accuracies resp. 106% (n = 6), 1095 (n = 2) and 92% (n = 6)).

The formulations of low and high dose groups were homogeneous (i.e. coefficient of variation 6.7% and 7.7%, respectively).

Conclusions:

In a GLP-compliant OECD guideline 422 study with rats, the NOAEL for general toxicity was set at the lowest dose level of 100 mg/kg bw/day by the study director, based on tubular damage in the form of granular casts and increased tubular basophilia in male kidneys starting at 300 mg/kg bw/day. However, for risk assessment purposes the dose level of 100 mg/kg bw/day is considered a LOAEL, based on statistically significantly higher liver weights in males and hepatocellular hypertrophy and follicular cell hypertrophy in the thyroid in both sexes at this dose level. No adverse effects on reproduction and development were noted at the highest tested dose of 1000 mg/kg bw/day, which therefore was considered to be a NOAEL for reproductive and developmental toxicity.

Executive summary:

In a GLP-compliant OECD guideline 422 study with rats, male and female rats were treated by oral gavage with 100, 300 and 1000 mg/kg bw/day cyclopentadecanone for at least 28 days (males) or at least 40 days (females). There were no treatment-related mortalities. Treatment-related clinical signs of toxicity were noted at 300 and 1000 mg/kg bw/day in both sexes. Frequent findings included hunched posture, uncoordinated movements, piloerection and/or tremors. Adverse test item-related morphologic alterations were present in the kidneys of males. Hyaline droplet accumulation occurred at increased incidence and severity in males treated at 100, 300 and 1000 mg/kg bw/day up to marked degree. Correlated necropsy findings in high dose males included pale discolouration of the kidneys (noted in 9/10 males), and a 19% higher mean absolute and relative kidney weight compared to the concurrent control males. The hyaline droplet accumulation recorded was considered to likely represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein is not present in female rats nor in higher mammals, including man. The increased hyaline droplet accumulation in the male kidneys was accompanied by indicators of tubular damage in the form of granular casts and increased tubular basophilia starting at 300 mg/kg bw/day and therefore this was considered to be adverse from 300 mg/kg bw/day onwards. Furthermore, significantly higher liver weights (absolute and/or relative to body weights) were present in males treated at 100, 300 and 1000 mg/kg bw/day and in females treated at 1000 mg/kg bw/day, and there was an apparent, non-significant, increase in the females treated at 300 mg/kg bw/day. The microscopic correlate was hepatocellular hypertrophy, which was noted in males and females starting at 100 mg/kg bw/day. In thyroids of males and females starting at 100 mg/kg bw/day an increased incidence and/or severity (up to moderate degree) of follicular cell hypertrophy was observed. In males, this hypertrophy resulted in increased thyroid gland weights at 300 (not statistically significant) and at 1000 mg/kg bw/day. Because of the absence of degenerative changes and corroborative findings in clinical pathology the changes in the liver were considered non-adverse by the study director. The observed changes in thyroid were considered to be an adaptive response to the induction of hepatic enzymes and therefore also considered non-adverse. The lowest dose level of 100 mg/kg bw/day was therefore considered a NOAEL by the study director, based on the observed tubular damage and increased tubular basophilia in male rats starting from 300 mg/kg bw/day. However, based on the statistically significant increase in relative liver weight by 18% compared to controls in combination with hepatocellular hypertrophy and follicular cell hypertrophy in the thyroids at the lowest dose level, for risk assessment purposes the lowest dose level of 100 mg/kg bw/day is considered a LOAEL for general toxicity.

No reproduction or developmental toxicity was observed up to and including the highest dose level of 1000 mg/kg bw/day. No treatment-related toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites, spermatogenic profiling and histopathological examination of reproductive organs). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy). Based on this, the NOAEL for reproductive and developmental toxicity was considered to be the highest tested dose of 1000 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

March 1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD guideline 421, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 1995

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cyclopentadecanone

EC Number:

207-951-2

EC Name:

Cyclopentadecanone

Cas Number:

502-72-7

Molecular formula:

C15H28O

IUPAC Name:

cyclopentadecanone

Test material form:

solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in refrigerator (2-8°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stability for at least 6 hours at room temperature, under normal laboratory light conditions, is confirmed over the concentration range 1 to 200 mg/mL.

OTHER SPECIFICS:
- No correction factor for purity is required

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: not applicable, repro/developmental screening study
- Age at study initiation: approximately 11 weeks
- Weight at study initiation: males 298-345 g, females 185-224 g
- Fasting period before study: no
- Housing:
Pre-mating: in groups of 5 animals/sex/cage in Macrolon plastic cages
Mating: males and females on 1-to-1 basis in Macrolon plastic cages
Post-mating: males: max. 5/cage in their home cages (Macrolon), females individually in Macrolon plastic cages
Lactation: pups with the dam in Macrolon plastic cages, except during locomotor activity monitoring of the dams. The pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
Locomotor activity measurements: individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water
Sterilized sawdust was provided as bedding material and paper as cage-enrichment/nesting material, except during locomotor activity measurements.
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except overnight before sacrifice
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY: Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients were performed according to facility standard procedures. There were no findings that could interfere with the study

ENVIRONMENTAL CONDITIONS (set to maintain):
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 Augusut 2016 To: 16 November 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. To facilitate homogenization, the test item (in vehicle) was heated to a maximum of 70°C for a maximum duration of 40 minutes. Adjustment was made for specific gravity of the test item (0.973) and vehicle (0.92).

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at Charles River Den Bosch.
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Stability of the test item in formulations was determined as part of the analytical method development and validation study.

Details on mating procedure:

M/F ratio per cage: 1:1
- Length of cohabitation: until mating, for maximum 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually in Macrolon plastic cages

Duration of treatment / exposure:

Males: 31 days (2 weeks prior to mating, during mating, and up to and including the day prior to scheduled necropsy)
Females that delivered: 40-54 days (during 2 weeks prior to mating, the variable time to conception, the duration of the pregnancy and during 4-6 days of lactation (up to and including the day before scheduled necropsy)).
Females which failed to deliver healthy offspring: 42 and 54 days, respectively

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Duration of test:

Males: 31 days
Females that delivered: until necropsy on PND 5-7
Females which failed to deliver healthy offspring: 42 and 54 days, respectively

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Concurrent vehicle controls (Group 1)

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 20-day dose range finding study

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, at least 3 hours (± 30 min) after treatment (on the peak period of anticipated effects after treatment). Once
prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of exposure (prior to first exposure) and weekly thereafter.
Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.

FOOD CONSUMPTION: yes
- Time schedule for examinations: weekly, except for females which were housed together for mating and females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1 and 4.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OTHER:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of scheduled sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight (max. 24 hours)
- How many animals: 5/sex/group
- Parameters examined: WBC, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), RBC, reticulocytes, RDW, haemoglobin, haematocrit, MCV, MCH, MCHC, platelets, PT, APTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of scheduled sacrifice
- Animals fasted: Yes, overnight (max. 24 hours)
- How many animals: 5/sex/dose
- Parameters examined: ALAT, ASAT, ALP, total protein, albumin, total bilirubin, bile acids, urea creatinine, glucose, cholesterol, sodium , potassium, chloride, calcium, inorganic phosphate

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on selected females from lactation day 4 onwards.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity (hearing, pupillary reflex, static righting reflex), grip strength (fore- and hind-limbs), motor activity (total movements and ambulations)

OTHER: for details on general toxicity evaluation see section 7.5.1 of this IUCLID (cross-reference)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No, females were allowed to litter naturally
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

- External examinations: Yes, all per litter
- Soft tissue examinations: no specific examinations were performed, but gross necropsy with attention paid to all anomalies was conducted on all pups
- Skeletal examinations: no specific examinations were performed, but gross necropsy with attention paid to all anomalies was conducted on all pups
- Head examinations: no specific examinations were performed, but gross necropsy with attention paid to all anomalies was conducted on all pups

Statistics:

The following statistical methods were used to analyze the data:
• If the variables were assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Indices:

The following indices were calculated:

Percentage live males at first litter check (%) = 100 x (number of live males at first litter check/number of live pups at first litter check)
Percentage live females at first litter check (%) = 100 x (number of live females at first litter check/number of live pups at first litter check)
Percentage of postnatal loss (%) = 100 x (number of dead pups before scheduled necropsy/number of live pups at first litter check)
Viability index (%) = 100 x (number of live offspring on PND 4/number of liver offspring on day 1 after littering)

Historical control data:

Available from the same testing laboratory for the study conducting period.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related clinical signs of toxicity were noted at 300 mg/kg bw/day and 1000 mg/kg bw/day. Frequent findings included hunched posture, uncoordinated movements, piloerection and/or tremors. Additionally, slight lethargy and/or ptosis (both at a slight degree) were noted for two females at 1000 mg/kg bw/day on 1-2 days.
During the weekly arena observations, no additional treatment-related clinical signs were noted.
There were no toxicologically relevant findings for females at 100 mg/kg bw/day. Salivation seen after dosing among females at 300 and 1000 mg/kg bw/day was not considered toxicologically relevant, taking into account the nature and slight severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Any other clinical signs noted incidentally (wound, scabs on the skin, red discoloration of the skin, alopecia, rales and diarrhoea) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. As they were observed for a control animal only, showed no dose-related trend and/or were isolated findings, they were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One high dose female was euthanized on Day 23 post-coitum due to complications during parturition. Before euthanasia she had a pale appearance, piloerection, ptosis, and dull eyes. She had started to give birth: blood was found in her cage and at least one pup was seen which was missing at a later time point (taken from study daybook). Most likely these pups had been cannibalized by their mother. At necropsy, she had a pale appearance. Next to one dead fetus obstructing the cervix, 10 other dead fetuses were found in her uterus (in total 5 males and 6 females; no external abnormalities). This single death in the high dose group was considered not to be related to treatment with the test item.
All remaining animals survived until their scheduled necropsies.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In females at 1000 mg/kg bw/day, there was a trend towards slightly higher body weight gain during mating and the first two weeks of post-coitum. This resulted in slightly, but statistically significant, higher group mean values for absolute body weight in the high dose group compared to the concurrent control group during Days 4-14 post-coitum. As values remained within the normal range of biological variation, no toxicological relevance was attached to this finding.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

When compared to the concurrent control group, a slightly, but statistically significantly, higher food intake (absolute and/or relative to body weight) was recorded for females treated at 1000 mg/kg bw/day during Days 0-4 post-coitum (absolute and relative) and Days 7-11 postcoitum (absolute). As values remained within the normal range of biological variation, no toxicological relevance was attached to this finding.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes occurred in haematology parameters in females treated up to 1000 mg/kg bw/day.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes occurred in clinical biochemistry parameters in females treated up to 1000 mg/kg bw/day.
Any statistically significant changes at 300 and 1000 mg/kg bw/day were not considered to be toxicologically significant as they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher liver weights (absolute and/or relative to body weights) were present in females treated at 1000 mg/kg bw/day and there was an apparent, non-significant, increase in females treated at 300 mg/kg bw/day. The microscopic correlate was hepatocellular hypertrophy.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related macroscopic abnormalities were noted in females up to and including 1000 mg/kg bw/day. The recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment were noted in the liver and thyroid gland of females starting at 100 mg/kg bw/day and in the adrenal glands and thymus of females at 1000 mg/kg bw/day.
Liver:
Hepatocellular hypertrophy was present in females treated at 100, 300 and 1000 mg/kg bw/day up to moderate degree.
Thyroid gland:
Follicular cell hypertrophy was present at increased incidence and severity in females treated at 100, 300 and 1000 mg/kg bw/day up to moderate degree.
Adrenal glands:
Vacuolation zona glomerulosa was present at increased incidence and severity in females treated at 1000 mg/kg bw/day.
Thymus:
Lymphoid atrophy was present at increased incidence in females treated at 1000 mg/kg bw/day up to slight degree.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

Examination of cage debris of pregnant females revealed no signs of abortions.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

For one female at 100 mg/kg bw/day, the number of implantations was slightly higher than the number of corpora lutea. This was considered to be due to normal resorption of these areas as these enumerations were performed on Day 5 of lactation. No further effects were noted.

Early or late resorptions:

not examined

Dead fetuses:

no effects observed

Description (incidence and severity):

One pup each of the control, 100 and 300 mg/kg bw/day group was found dead at first litter check. In addition, one control pup was found missing on lactation Day 7. It was most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The duration of gestation was unaffected by treatment.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All paired and mated females were pregnant, except for one female each in the control, 100 and 1000 mg/kg bw/day group. In the absence of dose response, this was considered to be a chance finding, unrelated to treatment.

Other effects:

no effects observed

Details on maternal toxic effects:

Hepatocellular hypertrophy, observed in females from the lowest dose level of 100 mg/kg bw/day was considered to be non-adverse by the study director in the absence of any degenerative changes and corroborative findings in clinical pathology parameters. The increased follicular cell hypertrophy of the thyroid observed in females at 100 mg/kg bw/day and above was regarded to be an adaptive response to the induction of hepatic enzymes and considered non-adverse by the study director at the incidences and severities recorded. However, using precautionary principle, for risk assessment purposes the lowest dose level of 100 mg/kg bw/day will be considered a LOAEL.

Effect levels (maternal animals)

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Maternal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: liver, thyroid

Description (incidence and severity):

Hepatocellular hypertrophy in the liver and increased follicular cell hypertrophy in the thyroid starting from 100 mg/kg bw/day.

Results (fetuses)

Fetal body weight changes:

not examined

Description (incidence and severity):

Fetal weights were not examined, as pregnant females were allowed to litter naturally.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio of the pups was unaffected by treatment up to and including 1000 mg/kg bw/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter size and body weight of the pups were not affected by treatment up to and including 1000 mg/kg bw/day.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

There were no changes in postnatal survival up to and including 1000 mg/kg bw/day.

External malformations:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No specific examinations were performed, but gross necropsy conducted on all pups revealed no gross malformations at any treatment level.

Visceral malformations:

no effects observed

Description (incidence and severity):

No specific examinations were performed, but gross necropsy conducted on all pups revealed no gross malformations at any treatment level.

Other effects:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on deveopment up to and including the highest dose level of 1000 mg/kg bw/day.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In a GLP-compliant OECD guideline 422 study with rats, hepatocellular hypertrophy and follicular cell hypertrophy in the thyroid was observed at the lowest dose level of 100 mg/kg bw/day in maternal animals. Although these findings were considered non-adverse by the study director, for risk assessment purposes this dose level is considered to be a LOAEL for general toxicity. No adverse effects on development were noted at the highest tested dose of 1000 mg/kg bw/day, which therefore was considered to be a NOAEL for reproductive and developmental toxicity.

Executive summary:

In a GLP-compliant OECD guideline 422 study with rats, treatment-related clinical signs of toxicity were noted at 300 and 1000 mg/kg bw/day in maternal animals, which included hunched posture, uncoordinated movements, piloerection and/or tremors. Starting from the lowest dose level of 100 mg/kg bw/day, hepatocellular hypertrophy and an increased incidence and/or severity (up to moderate degree) of follicular cell hypertrophy in the thyroids

was observed in the treated females. Significantly higher liver weights (absolute and/or relative to body weights) were present in females treated at 1000 mg/kg bw/day, and there was an apparent, non-significant, increase in the females treated at 300 mg/kg bw/day. Because of the absence of degenerative changes and corroborative findings in clinical pathology the changes in the liver were considered non-adverse by the study director. The observed changes in thyroid were considered to be an adaptive response to the induction of hepatic enzymes and therefore also considered non-adverse. The lowest dose level of 100 mg/kg bw/day was therefore considered a NOAEL by the study director. However, based on the statistically significant increase in relative liver weight by 18% compared to controls in combination with hepatocellular hypertrophy and follicular cell hypertrophy in the thyroids at the lowest dose level, for risk assessment purposes the lowest dose level of 100 mg/kg bw/day is considered to be a LOAEL for general toxicity.

No developmental toxicity was observed up to and including the highest dose level of 1000 mg/kg bw/day. No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy). Based on this, the NOAEL for rdevelopmental toxicity was considered to be the highest tested dose of 1000 mg/kg bw/day.