Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
06 July to 02 September 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 422 without any deviation. The study was fully reliable (Klimisch score = 1), however the reliability score was lowered to 2 which is the maximum score for read-across. The supporting substance is considered adequate for read-across purpose (see IUCLID section 13 for additional justification).
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
2,2,5-trimethyl-5-pentylcyclopentan-1-one
EC Number:
265-779-3
EC Name:
2,2,5-trimethyl-5-pentylcyclopentan-1-one
Cas Number:
65443-14-3
IUPAC Name:
2,2,5-trimethyl-5-pentylcyclopentanone
Test material form:
other: liquid
Details on test material:
- Physical state: Clear colourless liquid
- Storage condition of test material: At room temperature in the dark (metal container with plastic screw cap)

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
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 (F0): Approximately 10-12 weeks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). 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.
- Diet: Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Tap-water in polycarbonate bottles (Bio Services BV, Uden, The Netherlands), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18-24 °C
- Humidity: 40-70 %
- Air changes: At least 10 room air changes/hour
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Diet: Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Method: The test substance was admixed with the powder diet as follows:
Test substance was mixed with approximately 150 g of powder diet (premix) and stirred with spatula. Powder diet was added to an end weight of approximately 2-2.5 kg and mixed with mixing machine for at least 10 minutes. Powder diet was added to the definitive end weight and mixed with mixing machine for at least 10 minutes.
- Frequency of preparation: Diets were prepared once weekly and divided into portions for use during 1-2 days.
- Storage conditions: Diets were kept frozen (≤ -15 °C) for a maximum of 15 days. When needed, they were taken from the freezer and transferred to the animal room where they were kept at room temperature. Diets were replaced at least every second day.

STABILITY:
- Stability in rat powder diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany): Stability for up to 2 days at room temperature is confirmed over the concentration range 1500 to 20000 ppm (Project 507086). Stability for up to 15 days in the freezer (≤ -15 °C) is confirmed over the concentration range 100 to 20000 ppm (Project 507086).
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- Once mating occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
- Any other deviations: Detection of mating was not confirmed for female no. 46 (Group 1) which did deliver live offspring. The mating date of this female was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
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 (13 July 2015), according to a validated method (Project 507086). Samples of diet preparations were analysed 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 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females were exposed for 43-58 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy for selected females which were deprived of food during the night before necropsy, or up to the day of scheduled necropsy for non-selected females).
Pups were not dosed directly but could have potentially be exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/faeces.
Frequency of treatment:
ad libitum
Doses / concentrations
Remarks:
Doses / Concentrations:
2000, 5000 and 12000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were based on a 14-day dose range finding study (WIL Research Project 505976) in which Wistar Han rats (3/sex/dose level) received diets containing 0, 1500, 5000 or 15000 ppm of the test substance. No dose limiting effects were seen at 1500 and 5000 ppm. At 15000 ppm severe hyaline droplet accumulation accompanied by renal tubular degeneration, increased liver weights, and peribiliary inflammation and bile duct proliferation were observed for one high dose male. The hyaline droplet accumulation at 15000 ppm is considered to represent alpha-2u-globulin and was without additional hallmarks of kidney damage in the 1500 and 5000 ppm groups (males) and therefore at 1500 ppm and 5000 ppm regarded as a non-adverse microscopic finding. Based on these findings, the high dose level in the main study was reduced to 12000 ppm (target intake = 800 mg/kg bw/day). Based on the absence of adverse effects at 5000 ppm and 1500 ppm in the pilot study, dose levels of 5000 ppm and 2000 ppm were selected as the mid and low dose.
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule:
Mortality / Viability: At least twice daily
Clinical signs: At least once daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals. 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: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- During premating, food consumption was measured every 2 days (both sexes).
- In the mating period, food consumption of males were measured per cage starting the day mating was confirmed for the first male in the cage and was continued daily.
- For females, food consumption was measured daily during the post coitum and lactation periods.
- Food consumption was not measured for males and females housed together for mating and for females without evidence of mating, but food was replaced for these animals every day.

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

OTHER:
Functional Observations: The following tests were performed on the selected 5 animals/sex/group:
hearing ability, pupillary reflex and static righting reflex; fore- and hind-limb grip strength; locomotor activity
The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (from lactation Day 4 onwards). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).

General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

Clinical laboratory investigations
Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane.
Haematology: The following haematology parameters were determined in blood prepared with K3-EDTA as an anti-coagulant, using the ADVIA® 120 Hematology System (Siemens Healthcare Diagnostics B.V., Den Haag, The Netherlands): White blood cells (WBC), Differential leucocyte count: Neutrophils, Lymphocytes, Monocytes, Eosinophils and Basophils, Red blood cells, Reticulocytes, Red blood cell distribution width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelets
Clotting parameters: Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)
Clinical biochemistry
The following clinical biochemistry parameters were determined using the AU400 (Beckman Coulter Nederland B.V., Woerden, The Netherlands). All parameters were determined in plasma, except for bile acids which were determined in serum: Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP), Total protein, Albumin, Total Bilirubin, Bile acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate (Inorg. Phos)
Litter observations:
Each litter was examined to determine the following:
Mortality / Viability: The numbers of live and dead pups were determined on Day 1 of lactation and daily thereafter.
Clinical signs: At least once daily, detailed clinical observations were made for all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.
Postmortem examinations (parental animals):
NECROPSY
- Male animals: All surviving animals [Following completion of the mating period (a minimum of 28 days of dose administration)]
- Maternal animals:
Females which delivered: Lactation Days 5-7.
Female which failed to deliver (no. 78): Post-coitum Day 27 (female with evidence of mating)
- All males and the selected 5 females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food.
- All animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- The numbers of former implantation sites and corpora lutea were recorded for all paired females.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
- Selected 5 animals/sex/group:
Identification marks: not processed, Adrenal glands, (Aorta), Brain - cerebellum, midbrain, 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, Lacrimal gland - exorbital#, Larynx#, Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, Nasopharynx#, Oesophagus#, Ovaries, Pancreas#, Peyer's patches [jejunum, ileum] if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, Salivary glands - mandibular, sublingual#, Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin#, Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes *, Thymus, Thyroid including parathyroid if detectable, Tongue#, Trachea, Urinary bladder, Uterus, Vagina, All gross lesions
- All remaining animals and female 78 which failed to deliver:
Cervix, Clitoral gland, Coagulation gland, Epididymides*, Mammary gland area, Ovaries, Identification marks: not processed, Preputial gland, Prostate gland, Seminal vesicles, Testes*, Uterus, Vagina, All gross lesions

* Fixed in modified Davidson's solution (prepared at WIL Research Europe using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial) (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.
#Tissues/organs were not examined, since no signs of toxicity were noted at macroscopic examination.

ORGAN WEIGHTS
Terminal body weight was recorded from all males and the selected 5 females/sex/group.
The following organ weights were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus and Uterus (including cervix)
Prostate, Seminal vesicles including coagulating glands and Thyroid including parathyroid (weighed when fixed for at least 24 hours)
All remaining males: Epididymides and Testes

HISTOPATHOLOGY
All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometres. These slides were stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 and of the non-selected male (no. 38) that failed to sire to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The liver and kidneys of the selected 5 males of Groups 2 and 3 and the liver, thymus, spleen and (sternal) bone marrow of the selected 5 females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- The reproductive organs of the couple that failed to deliver healthy pups (female 78 and male 38). Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Postmortem examinations (offspring):
NECROPSY OF PUPS
- Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.
- All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be 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.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Fertility index (%) = (Number of pregnant females/Number of females paired) x 100
Conception index (%) = (Number of pregnant females/Number of females mated) x 100
Gestation index (%) = (Number of females bearing live pups/Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Percentage live males at First Litter Check = (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check = (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
Post-implantation survival index (%) = (Number of live pups at First Litter Check/Number of implantation sites) x 100
Percentage of postnatal loss = (Number of dead pups before planned necropsy/Number of live pups at First Litter Check) x 100
Viability index = (Number of live pups before planned necropsy/Number of pups born alive) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Secondary to palatability-induced reduced food consumption
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Secondary to palatability-induced reduced food consumption
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

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

Details on results (P0)

MORTALITY (PARENTAL ANIMALS)
- No mortality occurred during the study period.

CLINICAL SIGNS (PARENTAL ANIMALS)
- Males showed no clinical signs up to 12000 ppm.
- All females treated at 12000 ppm and one female (no. 65) treated at 5000 ppm showed piloerection starting after about five weeks of treatment. The piloerection generally persisted until the end of the study. The piloerection was likely either a stress-related effect of not being able to eat the diet, or simply that the animals were cold because they are not eating enough calories; these observations suggest it is a secondary effect and can be considered not adverse.
- The only other finding consisted of scabs (left ear) noted on a few days for one female treated at 12000 ppm. This background finding was not related to treatment.
- No additional clinical signs were noted during weekly arena observations.
- No mortality occurred during the study period

BODY WEIGHT (PARENTAL ANIMALS)
- Males treated at 12000 ppm showed significantly reduced growth (several animals lost weight) throughout the treatment period (relative difference from control at the end of the study: 9%). For females at 12000 ppm initial weight loss was noted during Weeks 1-2, followed by partial recovery during the premating period. During gestation and lactation, mean body weight and body weight gain were significantly lower compared to the concurrent control group (not always statistically significant for body weight gain). At the end of the study, mean body for high dose females was 18% lower compared to controls.
- Slightly reduced growth was also noted in males at 5000 ppm but the changes were generally not statistically significant (relative difference from control at the end of the study: 5%). Females treated at 5000 ppm as compared to controls showed slightly reduced growth during the premating period (not statistically significant). Also slightly lower body weights were noted during gestation and lactation (not always statistically significant), but body weight gains at 5000 ppm were comparable to control values.
- No treatment-related changes were seen at the lowest dose level tested (2000 ppm).
- The reduced body weights can be considered as secondary effects to reduced food consumption and so they should not be classified as adverse.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- Food consumption of males at 12000 ppm was markedly reduced (by about 60%) during the first three days of the treatment period and gradually increased thereafter. During the mating period, food intake of males at 12000 ppm was comparable to that of controls. Males treated at 5000 ppm consumed less food than controls during the first week of the premating period. The magnitude of the reduction in food consumption increased with dose.
- During the premating period, food consumption of females was dose-dependently reduced at all dose levels, most markedly in Week 1. As in males, food consumption at 12000 ppm in females was markedly reduced (by about 65%) during the first week of the treatment period, followed by almost complete recovery towards control values thereafter. During gestation and lactation, food consumption was considerably reduced at 5000 and 12000 ppm (overall by about 30% at 5000 ppm (gestation and lactation period) and by about 45% (gestation period) or 60% (lactation period) at 12000 ppm). The differences from control values were generally statistically significant and showed a dose-related response. Food consumption before and after allowance for body weight showed a similar pattern.
- At 2000 ppm relative food consumption of pregnant and lactating females was slightly lower (not statistically significantly) during the first few days of gestation and during lactation.
- In this study the reduced food consumption is a palatability issue and so it should not be classified as adverse.

HAEMATOLOGY
- Haematological parameters of treated rats were not affected by treatment.

CLINICAL CHEMISTRY
- Lower plasma levels of total protein and albumin in females at 12000 ppm. These changes were probably related to the reduced food intake of the females.
- Higher plasma level of potassium in males at 12000 ppm.
Statistically significantly higher plasma levels of bilirubin were noted at all dose levels in both sexes.

NEUROBEHAVIOUR
Functional observations:
- Hearing ability, pupillary reflex, static righting reflex and grip strength were not affected by treatment.

- The variation in motor activity did not indicate a relation with treatment.
- It was noted that the group mean values for total movements and ambulations were higher (not statistically significantly) for females at 2000 ppm.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The mean daily intake of the test substance per kg body weight during the different phases of the study is given in table 7.8.1/1.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- No toxicologically relevant effects on reproductive parameters were noted.
- The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. The trend towards lower group means of the number of corpora lutea and implantation sites noted at 12000 ppm was considered a chance finding. Changes as compared to controls did not reach statistical significance and values at the individual level remained within the normal range of biological variation.
- All females at all dose levels were pregnant.
- For females no. 49 (Group 1), no. 60 (Group 2), no. 68 (Group 3) and nos. 72 and 77 (Group 4), the number of pups born was slightly higher than the number of implantations and corpora lutea. This was considered to be due to normal resorption of these areas as these enumerations were performed on Day 7 of lactation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Males and females at 12000 ppm had statistically significantly lower terminal body weights (relative differences from controls: 8% and 15%, respectively).
- There was a dose-related increase in liver weight in both sexes. In males, absolute and relative liver weight were statistically significantly higher at all dose levels (absolute and relative liver weight at 12000 ppm was 34% and 51% higher compared to controls, respectively). In females, relative liver weight was statistically significantly higher at 12000 ppm (relative difference from controls: 38%). Relative liver weight in females at 5000 ppm was 21% higher compared to controls but this difference was not statistically significant. These effects on liver can be considered as adaptive as it is often observed after dosing with high levels of xenobiotics/organic substances/chemicals.
- Females treated at 12000 ppm had lower thymus weights (relative differences from controls: 44% and 35% for absolute and relative weight, respectively); the statistical significance was attained to the absolute thymus weight only.
- Males at 2000 and 12000 ppm had statistically significantly higher relative kidney weights (111% and 114%, respectively, when compared to the control group). In the absence of a dose related response and corroborative histopathological changes, the higher weight at 2000 ppm was not attributed to treatment.
- The statistically significantly higher relative weights of the brain in both sexes, the higher relative weight of the epididymides in males, and the lower absolute heart and spleen weights in females at 12000 ppm were considered to be secondary to the lower body weights at this dose level.
- Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- Test item-related macroscopic findings were present in 1/10 females (no. 74) treated at 12000 ppm and consisted of the general observation emaciated and reduce size of the thymus. The microscopic correlate was lymphoid atrophy of the thymus.
Other incidental findings among control and treated animals were within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be unrelated to treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Test item-related microscopic findings after treatment with 2,5,5-trimethyl-2-pentylcyclopentanone were noted in the liver of both sexes, the kidneys of males, and the spleen, thymus and (sternal) bone marrow of females.
Liver
Test item-related liver lesions in both sexes consisted of minimal degrees of hepatocellular hypertrophy in 2/5 females at 5000 ppm and in 5/5 males and 4/5 females at 12000 ppm. These effects on liver can be considered as adaptive as it is often observed after dosing with high levels of xenobiotics/organic substances/chemicals.
Kidneys: Test item-related kidney lesions in males consisted of:
- A dose related increase in incidence and severity of hyaline droplet accumulation up to incidences of 5/5 and severities of moderate degrees.
- An increase in incidence (5/5) and/or severity (up to slight) of basophilic tubules at 12000 ppm.
- The presence of granular casts up to moderate degree in 5/5 males at 12000 ppm.
- The presence of hyaline casts at minimal degree in 2/5 males at 12000 ppm.

In the kidneys of males a dose-related increase in incidence and severity of hyaline droplet accumulation was noted. In the absence of this finding in females, the hyaline droplet accumulation in males was considered to represent alpha 2u globulin, a normal protein in male rats, not present in female rats or in higher mammals, including man, and at 2000 and 5000 ppm considered to be non-adverse in nature. At 12000 ppm there was additional evidence of kidney degeneration. In the cortex, the epithelial cells of the proximal tubules showed degeneration, and at the transition outer/inner stripe of the outer medulla degenerated cells were found in dilated (basophilic) tubular lumens. This was recorded as granular casts. Based on the locations, these findings were regarded to be related to the high amount of hyaline droplet accumulation noted in all examined males treated at 12000 ppm. This spectrum of findings is sometimes diagnosed as alpha 2u globulin nephropathy (Hard, G.C., 2008). In addition, increased plasma levels of creatinine were noted for males at 12000 ppm. The increase in incidence and/or severity of basophilic tubule (up to slight) and presence of hyaline casts (minimal) were considered to be non-adverse in nature based on their low severities.

Spleen: Test item-related spleen lesions in females consisted of:
- A decrease in incidences and severity of extramedullary haematopoiesis at 12000 ppm (only at minimal degree in 1/5 females of the 12000 ppm group, compared to incidences of 4/5 to 5/5 and severities up to moderate degree) in the other groups. This was not accompanied by relevant changes in haematology parameters or spleen weight. Furthermore, there was no correlation with litter sizes. Therefore the decreased extramedullary haematopoesis in the spleen was regarded to be non-adverse

Thymus: Test item-related thymus lesions in females consisted of:
- Lymphoid atrophy at minimal degree in 2/5 females of the 12000 ppm group. This was related to the lower thymus weight and considered to be non-adverse at the noted low incidence and severity (minimal). In one female the lymphoid atrophy was related to reduced size of the thymus and the general necropsy finding of emaciation.

Bone marrow: Test item-related (sternal) bone marrow lesions in females consisted of:
- An increase in incidence (5/5) and/or severity (up to slight) of increased adipocytes in females of the 12000 ppm group, compared to minimal degree in a single female of the 2000 ppm group.
- Cellular depletion at minimal degree in 1/5 females of the 12000 ppm group.
The noted increase in incidence and/or severity of increased adipocytes in the bone marrow (sternal) of females at 12000 ppm was in one female accompanied by minimal cellular depletion. Based on this isolated incidence and low severity and absence of relevant haematologic changes, these findings in the bone marrow were regarded to be non-adverse in nature.
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.
There was one 12000 ppm treated couple (male 38 and female 78) without offspring. No abnormalities were seen in the reproductive organs, which could account for their lack of offspring. The female showed proof of a former pregnancy noted as the presence of implantation sites in the uterus.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and spermatogenic staging profiles were normal for all males examined.

OTHER FINDINGS
Reproductive results:
- No reproduction toxicity was observed up to the highest dose level tested (12000 ppm). There was one 12000 ppm treated couple (male 38 and female 78) without offspring. No abnormalities were seen in the reproductive organs, which could account for their lack of offspring. The female showed proof of a former pregnancy noted as the presence of implantation sites in the uterus.
- No treatment-related or toxicologically relevant changes were noted in any of the reproductive parameters investigated in this study.

Developmental results:
- No developmental toxicity was observed at 2000 and 5000 ppm.
- At 12000 ppm body weights of male and female pups were lower at Days 1 and 4 of lactation. The difference from controls increased from about 10% at Day 1 to about 20% at Day 4, indicating reduced postnatal body weight gain of these pups. As no developmental effects were observed at dose levels at which no effects were induced in the parental rats, this is most likely secondary to the maternal toxicity seen in this study and therefore should not be considered as adverse.
- No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study

OTHER FINDINGS (PARENTAL ANIMALS)
Functional observations
- Hearing ability, pupillary reflex, static righting reflex and grip strength were not affected by treatment.
The mean value for fore limb grip strength in males at 12000 ppm was higher than that in controls. As the difference was not statistically significant and individual values at 12000 ppm were in the normal range, this finding was not attributed to treatment. In the absence of a dose-related response, the lower mean fore limb grip strength in females at 5000 ppm (not statistically significant) was not attributed to treatment either.
- The variation in motor activity did not indicate a relation with treatment. Motor activity data showed a similar habituation profile for all groups with high activity in the first interval that decreased over the duration of the test period.
- It was noted that the group mean values for total movements and ambulations were higher (not statistically significantly) for females at 2000 ppm. This finding was not attributed to treatment because there was no dose-related response.

Haematology:
- Haematological parameters of treated rats were not affected by treatment.
- There were a few statistically significant differences among the dose groups which occurred in the absence of a dose-related response (lower haemoglobin in males at 5000 ppm, lower mean corpuscular volume (MCV) in females at 2000 and 12000 ppm, and higher activated partial thromboplastin time (APTT) in females at 2000 ppm) These findings were not attributed to treatment.
- Further it was noted that the number of platelets was statistically significantly higher in females at 12000 ppm. Values at 12000 ppm were in the range of historical control values for rats of this strain and age whereas concurrent control values were lower than observed historically. Therefore, this finding was considered not to be toxicologically relevant.

Clinical biochemistry
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher plasma level of creatinine in males at 5000 and 12000 ppm. The differences showed a dose-related response.
- Lower plasma levels of total protein and albumin in females at 12000 ppm.
- Higher plasma level of potassium in males at 12000 ppm.
Statistically significantly higher plasma levels of bilirubin were noted at all dose levels in both sexes. In the absence of a dose-related response, these findings were not attributed to treatment. For the same reason, the statistically significantly lower plasma level of chloride in males at 12000 ppm was considered unrelated to treatment.
A statistically significantly higher plasma level of urea was noted in males at 12000 ppm. Values at 12000 ppm were in the normal range for rats of this strain and age whereas concurrent control values were below this range. Therefore, this finding was considered not to be toxicologically relevant.

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
parental and reproductive
Effect level:
12 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant effects that were attributed to the administration of test item were noted in reproductive parameters in parental animals

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Considered to be secondary to reduced food consumption of the dams
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
- The numbers of females with living pups on Day 1 of lactation were 10, 10, 10 and 9 at 0, 2000, 5000 and 12000 ppm, respectively.
- No pups were found dead at the first litter check. During lactation, one pup of the control group, seven pups at 2000 ppm, and two pups at 12000 ppm were missing or found dead. Five out of the 7 pups in the low dose group that did not survive until scheduled necropsy came from the same litter (no. 53). The reason for this relatively high postnatal loss is not known. Evaluation of the available data did not indicate an impaired condition of this dam. As a consequence of the relatively high number of dead pups in the low dose group (n=7), Group 2 had a statistically significantly lower viability index as compared to the concurrent control group. In the absence of a dose-related trend no toxicological significance was attached to this finding. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these missing/dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS (OFFSPRING)
- Clinical signs noted in pups that went missing included scabs on the head, wound on the cheek, blue spot and scabs on the snout, blue discolouration of or blue spot on the abdomen, pale appearance and absence of milk in the stomach. Incidental clinical signs seen in surviving pups consisted of blue discolouration of or blue spot on the snout, absence of milk in the stomach, pale appearance, scabs on the snout, wound on the cheek, blue discolouration of or blue spot on the abdomen, blue spot on the head, and greenish discoloured tail apex. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHT (OFFSPRING)
- At 12000 ppm mean body weights of pups (male and female) were lower at lactation Day 1 (relative difference from control: 8%, not statistically significant) and lactation Day 4 (relative difference from control: 19%, statistically significant). This was considered to be secondary to reduced food consumption in the dams.
- Pup weights at 2000 and 5000 ppm were not affected by treatment

GROSS PATHOLOGY (OFFSPRING)
- Macroscopic findings were limited to a greenish discoloured tail apex in one surviving pup at 5000 ppm. This incidental finding was not related to treatment.

OTHER FINDINGS (OFFSPRING)
- Gestation: The gestation index and duration of gestation were unaffected by treatment up to 12000 ppm.
- Parturition/maternal care: No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
- Early postnatal pup development: Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, and external macroscopy did not reveal treatment-related findings.
The mean number of living pups at first litter check was slightly lower at 12000 ppm. The difference from the concurrent control group was not statistically significant and values at 12000 ppm were within normal limits. Therefore, this finding was not attributed to treatment.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
12 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No developmental toxicity was observed at dose levels which were non-toxic to the parental rats.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 7.8.1/1: Test substance intake

 

Stages

Test substance intake (mg/kg bw/day)1

Group 2

2000 ppm

Group 3

5000 ppm

Group 4

12000 ppm

Males

Pre-mating

140 (132 – 154)

323 (272 – 348)

704 (328 – 856)

Mating

133 (115 – 143)

314 (278 – 349)

795 (742 – 927)

Females

Premating

147 (137 – 154)

324 (228 – 371)

683 (359 – 889)

Gestation

244 (199 – 276)

430 (296 – 546)

891 (715 – 1185)

Lactation

333 (245 – 402)

670 (516 – 818)

1079 (924 – 1291)

1Values are the overall group means in the periods indicated and, in brackets, the lowest and highest group mean value in the pertaining period.

 

Chemical analysis of dose preparations

Accuracy of preparation: The concentrations analysed in the diets of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%). 

No test substance was detected in the Group 1 diet.

Homogeneity: The diets of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤10%).

Residue samples: No test substance was detected in the residue diet.

Applicant's summary and conclusion

Conclusions:
This study was performed on an analogue of the substance, further information on the read-across justification may be seen in section 13. Under the test conditions, the systemic parental and reproductive NOAEL was considered to be 12000 ppm in male and female rats (i.e. 704 mg/kg bw/day in male rats and 683 mg/kg bw/day in female rats), based on the absence of significant effects that could be considered to be adverse at the highest dose tested and below and since no toxicologically relevant effects attributed to the administration of the test item were noted in reproductive parameters in parental animals up to 12000 ppm. The developmental NOAEL was also considered to be 12000 ppm in male and female rats, as the reduced body weight and postnatal growth of pups observed at high doses was most likely secondary to the maternal effects seen in this study (reduced food consumption and consequent reduced weight gain) and therefore should not be considered as adverse.
Executive summary:
In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, test substance, formulated in powdered diet, was fed to SPF-bred Wistar Han rats at dose levels of 2000, 5000 and 12000 ppm (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the basal powder diet without test substance. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43-58 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), clinical pathology (end of treatment), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Diet preparations were analysed once during the study to assess accuracy and homogeneity. At 12000 ppm food consumption of males and females was markedly reduced (by approximately 60%) during the first three days of the treatment period. This resulted in severely reduced growth in males, and even 4% body weight loss during the first week in females. Thereafter, food consumption gradually increased, suggesting that poor palatability of the test diet was a cause of the reduced food intake. In males food consumption was normal during the mating period. Food consumption of females at 12000 ppm, however, remained considerably reduced throughout gestation and lactation, which was associated with reduced body weight gain. At the end of the study, mean body weights at 12000 ppm were 9% (males) or 18% (females) lower compared to controls. Males at 12000 ppm did not show clinical signs of toxicity but females showed piloerection starting after about five weeks of treatment. The piloerection was likely either a stress-related effect of not being able to eat the diet, or simply that the animals were cold because they are not eating enough calories; these observations suggest it is a secondary effect and so it can be considered not adverse. Initial reductions in food consumption were also noted at 5000 ppm (both sexes) and 2000 ppm (females only) though less markedly than at 12000 ppm. This was associated with slightly lower body weights at 5000 ppm (differences from control were generally less than 5%). Body weight gain was slightly reduced in the pre-mating period but not during gestation or lactation. As the effect on body weight was slight and not accompanied by clinical signs of toxicity (except for piloerection in one female at 5000 ppm), the reductions in food consumption and/or body weight at 5000 and 2000 ppm were considered not to be toxicologically relevant. Microscopic examination revealed minimal hepatocellular hypertrophy in the liver of males and females treated at 12000 ppm. This change was accompanied by a substantial increase in liver weight (relative liver weights were 51% and 38% higher in males and females, respectively).These effects on liver can be considered as adaptive as it is often observed after dosing with high levels of xenobiotics/organic substances/chemicals. The minimal hypertrophy noted in a few females at 5000 ppm occurred without a statistically significant increase in liver weight (absolute and relative liver weight 11% and 21% higher, respectively, as compared to controls) and was, therefore, regarded to be non-adverse in nature. In the absence of corroborative findings indicative of hepatotoxicity, the increases in liver weight in males at 2000 and 5000 ppm were also considered to be non-adverse. In the kidneys of males a dose-related increase in incidence and severity of hyaline droplet accumulation was noted. In absence of this finding in females, the hyaline droplet accumulation in males was considered to represent alpha 2u globulin, a normal protein in male rats, not present in female rats or in higher mammals, including man, and at 2000 and 5000 ppm considered to be non-adverse in nature (Sahota et al., 2013). At 12000 ppm there was additional evidence of kidney degeneration. In the cortex, the epithelial cells of the proximal tubules showed degeneration, and at the transition outer/inner stripe of the outer medulla degenerated cells were found in dilated (basophilic) tubular lumens. This was recorded as granular casts. Based on the locations, these findings were regarded to be related to the high amount of hyaline droplet accumulation noted in all examined males treated at 12000 ppm. This spectrum of findings is sometimes diagnosed as alpha 2u globulin nephropathy (Hard, G.C., 2008). In addition, increased plasma levels of creatinine were noted for males at 12000 ppm. The increase in incidence and/or severity of basophilic tubule (up to slight) and presence of hyaline casts (minimal) were based on their low severities considered to be non-adverse in nature. Further non-adverse, treatment-related microscopic changes were seen in the spleen, thymus and (sternal) bone marrow of females at 12000 ppm. A decrease in incidence and severity of extramedullary haematopoiesis in the spleen was not accompanied by relevant changes in haematology parameters or spleen weight. Furthermore, there was no correlation with litter sizes. Therefore the decreased extramedullary haematopoiesis in the spleen was regarded to be non-adverse. The lymphoid atrophy noted in the thymus of 2/5 females was related to the lower thymus weight and considered to be non-adverse at the noted low incidence and severity (minimal). In one female the lymphoid atrophy was related to reduced size of the thymus and the general necropsy finding of emaciation. The noted increase in incidence (5/5) and/or severity (up to slight) of increased adipocytes in the bone marrow (sternal) of females at 12000 ppm was in one female accompanied by minimal cellular depletion. Based on this isolated incidence and low severity and absence of relevant haematologic changes, these findings in the bone marrow were regarded to be non-adverse in nature based on their low severities. Clinical chemistry showed decreases in the plasma levels of total protein and albumin in females at 12000 ppm. These changes were probably related to the reduced food intake of these females. No treatment-related or toxicologically relevant changes were noted in the remaining parental parameters investigated in this study (i.e. mortality and functional observations). No reproduction toxicity was observed up to the highest dose level tested (12000 ppm). No treatment-related or toxicologically relevant 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 developmental toxicity was observed at 2000 and 5000 ppm. At 12000 ppm body weights of male and female pups were lower at Days 1 and 4 of lactation. The difference from controls increased from about 10% at Day 1 to about 20% at Day 4, indicating reduced postnatal body weight gain of these pups. As no developmental effects were observed at dose levels at which no effects were induced in the parental animals, this is most likely secondary to the maternal effects seen in this study (reduced food consumption and consequent reduced weight gain), and therefore should not be considered as adverse. No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and the early postnatal pup developmental endpoints mortality, clinical signs and macroscopy). In conclusion, the reduced food consumption is a palatability issue and all the other effects (except liver, which was adaptive) are secondary to reduced food consumption and consequent reduced weight gain, and so they should not be classified as adverse. Under the test conditions, the systemic parental and reproductive NOAEL was considered to be 12000 ppm in male and female rats (i.e. 704 mg/kg bw/day in male rats and 683 mg/kg bw/day in female rats), based on the absence of significant effects that could be considered to be adverse at the highest dose tested and below and since no toxicologically relevant effects attributed to the administration of the test item were noted in reproductive parameters in parental animals up to 12000 ppm. The developmental NOAEL was also considered to be 12000 ppm in male and female rats, as the reduced body weight and postnatal growth of pups observed at high doses is most likely secondary to the maternal effects seen in this study and therefore should not be considered as adverse.