Reaction mass of 3,5,6,6-tetramethyl-4-methyleneheptan-2-one and (E)-3,4,5,6,6-pentamethylhept-3-en-2-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

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:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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:

with 10-week premating period

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Wistar strain: Crl:WI(Han)
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at initiation of treatment: 5 (males) - 6 (females) weeks
- Weight at initiation of treatment: about 100 g (males) - 130 g (females)
- Housing: in macrolon cages with wood shavings (Lignocel, type 3/4) as bedding and shreds of paper (Enviro-dri) as environmental enrichment. Premating period: 4 rats of the same sex/cage; mating period: one male and one female per cage; after mating, males were returned to their group cage and females were housed individually during gestation and with their litter during lactation.
- Diet: powdered Rat and Mouse no. 3 breeding diet, RM3 (SDS Special Diets Services, Witham, England), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days
- Allocation: by computer randomization proportionately to body weight

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%):45-65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 hours dark, 12 hours light

IN-LIFE DATES:
From 11 May 2011 to 1-2 August (males) / 15-22 August (females) 2011 (from start treatment to end in-life period)

Route of administration:

oral: feed

Details on exposure:

The test material was mixed into the diet using a mechanical blender. Fresh batches of the experimental diets were prepared about once every 4-5 weeks, divided into daily amounts, packed in plastic bags, and stored in a freezer (<-18°C). Once daily in the afternoon (generally between 3-4 PM on working days), a fresh portion from the freezer was given to the animals. This frequency and timing of feed refreshing was applied because stability analyses showed that the concentration of the test substance in the feed decreased during storage at room temperature (by 18-27% after one day).

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximally 7 days.
- Proof of pregnancy: sperm in vaginal smear (made in the morning), referred to as day 0 of pregnancy.
- After successful mating, each pregnant female was caged individually for the birth and rearing of the pups.
- Sperm positive females that appeared non-pregnant (one control and one high-dose female) were sacrificed 27-28 days after copulation.
- One high-dose female that did not show evidence of copulation was housed individually after the last day of the mating period and sacrificed 29 days after the start of the mating period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose confirmation analyses were conducted using a HPLC method with UV detection (at 247 nm) after extracton with acetonitrile.
Stability under simulated experimental conditions was checked by analysis of samples immediately after diet preparation and after storage in the animal room (open container) for one day or in a freezer (<-18°C) for at least 5 weeks. The test substance was stable in the diet when stored in a freezer. The concentrations measured after one day storage in the animal room were 18% (low dose), 27% (mid dose) and 22% (high dose) lower than those measured at the start of the storage period.
Homogeneity and achieved concentration were confirmed by analysis of samples from diets prepared shortly before initiation of treatment. For homogeneity, 5 samples per concentration (low, mid, high dose) were analysed. The measured concentrations were generally within 90-110% of the nominal concentrations.

Duration of treatment / exposure:

Males: up to 83 days (70 days premating, 7 days mating, 5-6 days until sacrifice)
Females: up to 103 days (70 days premating, 1-4 days mating, 21/22 days gestation, 4-6 days lactation)

Frequency of treatment:

Continuous administration via the diet (fixed dietary concentrations)

Remarks:

Doses / Concentrations:
0, 200, 750 and 2500 mg/kg diet
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
Premating males: 16 (low), 58 (mid), 202 (high) mg/kg body weight/day
Basis:
other: calculated from nominal dietary concentrations; due to instability actual concentrations were lower (loss is maximally 30%)

Remarks:

Doses / Concentrations:
Premating females: 15 (low), 56 (mid), 171 (high) mg/kg body weight/day
Basis:
other: calculated from nominal dietary concentrations; due to instability actual concentrations were lower (loss is maximally 30%)

Remarks:

Doses / Concentrations:
Gestation females: 14 (low), 51 (mid), 165 (high) mg/kg body weight/day
Basis:
other: calculated from nominal dietary concentrations; due to instability actual concentrations were lower (loss is maximally 30%)

Remarks:

Doses / Concentrations:
Lactation females: 14 (low), 45 (mid), 195 (high) mg/kg body weight/day
Basis:
other: calculated from nominal dietary concentrations; due to instability actual concentrations were lower (loss is maximally 30%)

No. of animals per sex per dose:

12 rats/sex/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: selection based on 2-week range-finding study.
- Rationale for animal assignment (if not random): computerised randomisation proportionately to body weight. Animals were allocated one day before initiation of treatment.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: at least once daily.
- Additional checks: daily afternoon check for dead or moribund animals.

DETAILED CLINICAL OBSERVATIONS (in standard arena): Not conducted.

BODY WEIGHT:
Males: on day of allocation, at initiation of treatment, weekly thereafter, and on the day of scheduled sacrifice.
Females: on day of allocation, at initiation of treatment, weekly during the premating and mating period, on days 0, 7, 14 and 21 during presumed gestation (mated females), and on days 1 and 4 of lactation. Non-mated females were weighed one per week after the mating period.

FOOD CONSUMPTION:
- Except during the mating period, food consumption was measured from day 0 onwards on the same days as body weight was meausred. Results were expressed as g feed/animal/day and g feed/kg body weight/day.

WATER CONSUMPTION: Not measured.

OPHTHALMOSCOPIC EXAMINATION: Not conducted.

HAEMATOLOGY:
- Time schedule for collection of blood: at end of premating period (week 10)
- Anaesthetic used for blood collection: Yes (carbon dioxide/oxygen)
- Animals fasted: yes (overnight)
- How many animals: 5/sex/group (from different cages)
- Parameters examined:
. Red blood: haemoglobin, red blood cells, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocytes
. White blood: total white blood cells, differential white blood cells (absolute and relative)
. Coagulation: thrombocytes, prothrombin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: at end of premating period (week 10)
- Anaesthetic used for blood collection: Yes (carbon dioxide/oxygen)
- Animals fasted: Yes (overnight)
- How many animals: 5/sex/group (from different cages)
- Parameters examined: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamuyl transferase, total bilirubin, total protein, albumin, albumin/globulin ratio, glucose, total cholesterol, triglycerides, creatinine, urea, electrolytes (calcium, chloride, potassium, sodium, inorganic phosphate).

URINALYSIS: Not conducted.

NEUROBEHAVIOURAL EXAMINATION:
Time schedule for examinations: towards the end of the premating period (in week 9)
Battery of functions tested:
- Grip strength (forelimb and hindlimb)
- Motor activity
- Observational screen including sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive). Screen components:
Autonomic: lacrimation (R), salivation(R), pupil response to light(Q), palpebral closure(R), piloerection(Q), defaecation(C), urination(C);
Neuromuscular: gait(D,R), mobility(R), forelimb and hindlimb gripstrength(I), landing foot splay(I), righting reflex(R);
Sensorimotor: response(R) to tail pinch, click, touch and approach of a visual object;
Convulsive: clonic and tonic movements(D);
Excitability: ease of removal(R), handling reactivity(R), arousal(R), vocalizations(Q);
Activity: rearing(C), posture(D);
Physiological: body temperature(I)
(C=count data; D=descriptive rank data; I=interval or continuous data; Q=quantal data; R=rank order data).

Oestrous cyclicity (parental animals):

Not conducted.

Sperm parameters (parental animals):

The left cauda epididymis and the left testis of 5 males/group were used for sperm examinations.
Sperm parameters examined in all groups (by Hamilton Thorne Integrated Visual Optical System, IVOS):
- sperm motility
- sperm count (cauda epididymal sperm reserve)
Sperm parameters examined in the control and high-dose group:
- sperm morphology (epididymal sperm smears were prepared and stained for all groups; 200 spermatozoa/smear of the control and high-dose animals were examined).
- number of homogenization-resistant spermatids (enumeration by the IVOS)
- daily sperm production (calculated from testicular sperm count)

Litter observations:

PARTURITION AND LITTER EVALUATION:
At the end of the gestation period (gestation day 21), females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.

PARAMETERS EXAMINED:
The following parameters were examined in F1 offspring on days 1 and 4 of lactation:
- total litter size
- numbers of each sex
- numbers of stillbirths, live and dead pups
- grossly malformed pups/clinical observations
- pup weight (pups were weighed individually; mean pup weight was calculated per sex and for both sexes combined)

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes (all male and female parent animals)
Animals were sacrificed by exsanguination from the abdominal aorta under carbondioxide/oxygen anaesthesia.
Males were sacrificed about one week after the mating period. Females were sacrificed at or shortly after day 4 of lactation.

ORGAN WEIGHTS:
All male animals: testes, epididymides
5 Animals/sex/group: adrenals, brain, heart, kidneys, liver, spleen, thymus
Paired organs were weighed together.
Organ weights relative to terminal body weight were calculated.

TISSUES PRESERVED:
In neutral aqueous phosphate-buffered 4% formaldehyde solution or Bouin's fixative (testes)
All parental animals:
Ovaries (after counting of the corpora lutea)
Uterus (after counting of the implantation sites)
testes
epididymides
seminal vesicles
prostate
all gross lesions

5 Parental animals/sex/group:
adrenals
bone marrow (femur)
brain (3 levels: cerebrum, cerebellum, medulla/pons)
heart
small and large intestine (including Peyer's patches)
kidneys
liver
lung
lymph nodes (mesenterial and axilary)
peripheral nerve (tibial)
spinal cord (3 levels: cervical, mid-thoracic, lumbar)
spleen
stomach
thymus
thyroid
trachea
urinary bladder

HISTOPATHOLOGY:
Control and high-dose: all preserved organs and tissues.
Low- and mid-dose: kidneys (males only) and gross lesions.
Paraffin sections were stained with haematoxylin-eosin or PAS haematoxylin (testes).
Additional kidney sections of the male control and high-dose animals were processed for immunocytochemical staining of alpha 2-microglobuline using a mouse-anti-rat alpha 2-microglobuline antibody (SSI, Copenhagen, Denmark).

Postmortem examinations (offspring):

GROSS NECROPSY
Stillborn pups and pups that died during the study were examined for gross abnormalities (external and internal examination).

Statistics:

- Body weight (gain), food consumption, haematology, clinical chemistry, organ weights, continuous behavioural data, total distance moved in motor activity test: one-way analysis of variance followed by Dunnett's multiple comparison test.
- Habituation of motor activity: repeated measures analysis of variance on time blocks.
- Behaviour - rank data: Kruskal-Wallis non-parametric one-way analysis of variance followed by Dunnett's multiple comparison test.
- Behaviour - categorical data: Pearson's chi-square analysis.
- General clinical signs and histopathology: Fisher's exact probability test.

Reproduction toxicity parameters:
- Fisher's exact probability test was used to evaluate the number of mated and pregnant females, the number of pregnant females with implants but no pups, females with live pups, females with stillborn pups, live and dead fetuses or pups and the numbers of litters lost entirely.
- Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U test was used to evaluate pre-coital time (mean number of days), duration of gestation, number of corpora lutea and implantation sites, total number of pups delivered (mean), mean number of live pups per litter and pre- and post-implantation loss (%).
- Sperm parameters were evaluated by one-way analysis of variance followed by Dunnett’s multiple comparison test (epididymal and testicular sperm count and numerical sperm motility parameters) or by Kruskal-Wallis non parametric analysis of variance followed by the Mann-Whitney U test (motility parameters expressed as a percentage and sperm morphology).

Reproductive indices:

The following parameters were calculated:
- pre-coital time = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- mating index= (number of females mated/number of females placed with males) x 100
- male fertility index = (number of males that became sire/number of males placed with females) x 100
- female fertility index = (number of pregnant females/number of females placed with males) x 100
- female fecundity index =(number of pregnant females/number of females mated) x 100
- gestation index = (number of females with live pups/number of females pregnant) x 100
- pre-implantation loss = [(number of corpora lutea – number of implantation sites)/number of corpora lutea] x 100

Offspring viability indices:

The following parameters were calculated:
- live birth index = (number of pups born alive/number of pups born) x 100
- viability index day n-m = (number of pup surviving m days/number of liveborn on day n) x 100
- pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
- sex ratio day n = (number of live male pups on day n/ number of live pups on day n) x 100
- number of lost implantations = number of implantations sites - number of pups born alive
- post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY
No treatment-related clinical signs or mortality (the death of one low-dose female and one high-dose female on day 4 of lactation was unrelated to treatment).

BODY WEIGHT (GAIN) AND FOOD CONSUMPTION
Body weight, body weight gain and food consumption (expressed as g/animal/day) of male and female rats of the high-dose group were statistically significantly decreased during the entire or substantial parts of the study. Mean terminal body weights of high-dose rats were 11% (males) or 3% (females) lower compared to controls; at the end of the premating period, high-dose females were about 6% lighter than controls. During gestation, mean body weights of high-dose females remained statistically signficantly lower compared with controls but weight gain was not significantly affected. During lactation, no statistically significant intergroup differences in body weight or weight gain occurred.

TEST SUBSTANCE INTAKE
Substance intake in successive weeks was calculated from the nominal dietary concentrations. As the test material was not stable over a 1-day storage period in the animal room, the actual test material intake was lower than indicated below (compared to the concentration measured at the start of the storage period, the concentration measured after storage was decreased by 18%, 27% and 22% at the low-, mid- and high-dose, respectively).
The mean (range) intakes in mg test material/kg bw/day (not corrected for instability) were:
- Premating males: 16 (11-26) at the low-dose, 58 (41-86) at the mid-dose, 202 (144-287) at the high-dose.
- Premating females: 15 (12-20) at the low-dose, 56 (45-73) at the mid-dose, 171 (139-214) at the high-dose.
- Gestation females: 14 (13-16) at the low-dose, 51 (46-59) at the mid-dose, 165 (161-190) at the high-dose.
- Lactation females: 14 at the low-dose, 45 at the mid-dose, 195 at the high-dose.

HAEMATOLOGY
Red blood cell and coagulation parameters were not affected by treatment.
Total white blood cell count and absolute numbers of lymphocytes, neutrophils, eosinophils, basophils and monocytes were statistically significantly decreased in high-dose males. To a lesser extent, total and differential white blood cell values in males of the low- and mid-dose groups were also decreased compared to concurrent controls. As the mean and individual values in the low- and mid-dose groups were within the historical control range, the lower values in the intermediate dose groups were not ascribed to treatment.

CLINICAL CHEMISTRY
Treatment-related changes in clinical chemistry values were limited to higher chloride concentrations in males at all dose levels (statistically significant at the low- and high-dose) and a lower potassium concentration in high-dose males. These changes might be related to the effect on the kidneys observed in males.
A few other statistically significant differences from controls occurred (lower aspartate aminotransferase in females at all dose levels, lower alkaline phosphatase in mid-dose females and lower albumin in low-dose females) but were not ascribed to treatment (reason: lack of dose dependence).

NEUROBEHAVIOUR
Functional observational screen parameters, fore- and hindlimb grip strength, and spontaneous motor activity (total distance moved and habituation of activity) were not affected by treatment.

ORGAN WEIGHTS
Relative kidney weight was statistically significantly increased in high-dose males. This finding was likely to be related to the alpha 2-microglobulin nephropathy observed in males.
Other statistically significant findings were not ascribed to treatment (slightly lower absolute brain weight in high-dose males, higher absolute and relative adrenal weight in mid-dose females and lower testicular parenchyma weight in the high dose males).

GROSS PATHOLOGY
Macroscopic examination revealed no treatment-related changes.

HISTOPATHOLOGY
A dose-dependent increase of hyalin droplet nephropathy was found in the male animals, characterised by an abundant presence of eosinophilic hyalin droplets in the proximal tubular cells, and several dilated tubuli filled with eosinophilic debris in the corticomedullary area. This change was accompanied by a dose-dependent increase in basophilic tubuli. Compared with concurrent controls, the high-dose males showed increased alpha 2-microglobulin staining of the cortical tubular epithelial cells and of the corticomedullary tubular cell debris. Alpha 2-microglobulin nephropathy in male rats is generally regarded as of no relevance for humans.

REPRODUCTIVE FUNCTION: SPERM MEASURES
Testicular sperm count and epididymal sperm motility, count and morphology were not affected by treatment. The weight of the testicular parenchyma in the high-dose group was statistically significantly lower than in the control group. Since no effects were observed on testis weight, this is considered an incidental finding.

REPRODUCTIVE PERFORMANCE
- Fertility was not affected by treatment (pre-coital time, mating index, duration of gestation, gestation index, male and female fertility indices and female fecundity index were comparable in all groups). The number of pregnant females was 11, 12, 12 and 10 in the control, low-, mid- and high-dose group, respectively.
- The number of corpora lutea was statistically significantly decreased in the high-dose group (mean per animal: 10.1 versus in concurrent controls 12.7 ). Consequently, the numbers of implantation sites and pups delivered were also decreased in this group. The number of corpora lutea in the high-dose group was also lower compared to historical controls (range 11.0-14.5) but higher than lower numbers of corpora lutea observed incidentally in intermediate dose groups of other studies conducted at the testing laboratory (lowest value observed: 8.7). Therefore, the lower number of corpora lutea in the present high-dose group is of doubtful toxicololgical significance and probably due to chance.
- Pre- and post-implantation loss were not affected by treatment.

Dose descriptor:

other: NOAEL (general toxicity)

Effect level:

750 other: mg/kg diet (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on reduced body weight (gain) and food consumption in both sexes and decreases in total and differential white blood cells (all cell types) in males at the next dose level.

Dose descriptor:

other: NOAEL (general toxicity)

Effect level:

ca. 42 other: mg/kg bw/day based on dietary concentration corrected for measured decrease upon storage in animal room (-27% after 24 hr)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on reduced body weight (gain) and food consumption and decreases in total and differential white blood cells (all cell types) at the next dose level.

Dose descriptor:

other: NOAEL (general toxicity)

Effect level:

ca. 41 other: mg/kg bw/day based on dietary concentration corrected for measured decrease upon storage in animal room (-27% after 24 hr)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on reduced body weight (gain) and food consumption at the next dose level.

Dose descriptor:

other: NOAEL (reproductive toxicity)

Effect level:

ca. 158 other: mg/kg bw, based on dietary concentration corrected for measured decrease upon storage in animal room (-22% after 24 hr)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No male fertility related effects were seen up to the highest dose level tested (2500 mg/kg diet, providing ca. 158 mg/kg bw/day).

Dose descriptor:

other: NOAEL (reproductive toxicity)

Effect level:

ca. 129 other: mg/kg bw/day based on dietary concentration corrected for measured decrease upon storage in animal room (-22% after 24 hr)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No female fertility related effects were seen up to the highest dose level tested (2500 mg/kg diet, providing ca. 133, 129 and 152 mg/kg bw/day during premating, gestation and lactation, respectively).

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY
Viability was not affected by treatment. The live birth index and the number (stillborn pups) and percentage (pup mortality day 1) of pups found dead on day 1 were comparable between the groups.
The number of pups lost between days 1-4 in the high-dose group was lower than that in the control and intermediate-dose groups. The higher pup loss in the latter groups was mainly due to the total loss of 2 litters in each of these groups and considered unrelated to treatment.

LITTER SIZE AND SEX
The mean number of pups delivered in the high-dose group was statistically significantly lower than in the control group (8.4 at the high-dose versus 11.2 in controls). This was due to the lower number of corpora lutea (probably due to chance) in the high-dose group.
The sex ratio on days 1 and 4 was not affected by treatment.

CLINICAL SIGNS
No treatment-related findings. In the high-dose group on day 1, the number of cold pups and pups with no milk in the stomach was statistically significantly lower than in the control group. This was due to a relatively high incidence of this finding in controls.

BODY WEIGHT
Pup weight on lactation days 1 and 4 and weight gain between days 1-4 were not affected by treatment.

GROSS PATHOLOGY
Macroscopic examination of stillborn pups and pups that died during the study revealed no treatment-related abnormalities.

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Reproductive effects observed:

no

Conclusions:

In an oral (diet) Combined Repeated Dose Toxicity Study with Reproduction / Developmental Toxicity Screening Test in rats (OECD 422; 10-week premating period), a NOAEL for parental toxicity of 750 mg/kg diet (ca. 42 and 41 mg/kg body weight/day in males and females, respectively) was established, based on decreases in body weight gain, food consumption and total and differential white blood cell counts. No adverse effects on fertility of the parents or on development of the offspring were noted in any group (NOAEL 2500 mg/kg diet, which provided ca. 158 and 129 mg/kg bw/day in males and females, respectively). The NOAELs expressed as mg/kg bw/day were based on nominal dietary test material concentrations corrected for loss on storage in the animal room.

Executive summary:

The reproductive / developmental toxicity upon repeated oral exposure was examined in a GLP-compliant study performed according to OECD guideline 422 (TNO Triskelion, 2012). Groups of 12 male and 12 female Wistar rats received the test material via their diet, at concentrations of 0, 200, 750 and 2500 mg/diet during a 10-week premating period and during mating (one week), gestation and lactation until postnatal day 4. The total exposure duration was up to 83 days in male rats and up to 103 days in female rats.

Homogeneity and correct concentration of the test material in the diet were confirmed by analysis. The test substance was not stable in the diet upon storage in the animal room. The decrease in concentration during a 1-day storage period was 18%, 27% and 22% at the low-, mid- and high-dose, respectively. To minimize loss of test material under experimental conditions as much as possible, the feed in the feed hoppers was replaced daily by a fresh portion from the freezer (stability in the freezer was confirmed by analysis). Moreover, the fresh feed was provided in the afternoon instead of in the morning because rats eat most of their feed during the dark period.

The following endpoints were evaluated to assess general toxicity: daily clinical observations, neurobehavioural examination (grip strength; functional observational battery including sensory reactivity to different stimuli; spontaneous motor activity), body weight, food consumption, routine haematology and clinical chemistry (in week 10 of the premating period), organ weights, macroscopic examination and histopathological examination of a wide range of tissues and organs (except for the kidneys which were examined in all groups, only high-dose animals and controls were subjected to histopathology). Endpoints to assess reproductive / developmental toxicity included parental fertility and reproductive performance (sperm analysis, weight and morphology of sex organs, mating index, pre-coital time, male and female fertility indices, female fecundity index, gestation index and length, pre- and post-implantation loss) and litter data (numbers of stillborn, live and dead pups, external abnormalities, pup weight on lactation days 1 and 4, necropsy of stillborn pups and pups that died during the study).

The results showed no treatment-related changes at 200 and 750 mg/kg diet. At 2500 mg/kg diet, body weight (gain) and food consumption were statistically significantly decreased during the entire or substantial parts of the study in rats of both sexes, and total and differential white blood cell counts were decreased in male rats. In addition, male rats showed renal changes consisting of an increased relative kidney weight (at the high-dose level) and a dose-dependent increase of alpha 2-microglobulin nephropathy. This type of nephropathy in male rats is generally regarded as of no relevance for humans. The changes in clinical chemistry values in male rats (higher chloride at all dose levels and lower potassium at the high-dose) might be related to the nephropathy.

Based on these results the NOAEL for general toxicity was 750 mg/kg diet. This dietary level provided ca. 42 mg/kg bw/day in male rats and ca. 41 mg/kg bw/day in female rats (based on the overall mean substance intake during the premating period). The intake of the test material per kg body weight was calculated from the nominal dietary concentration corrected for the loss measured after storage in the animal room for one day.

 

Testicular sperm count and epididymal sperm motility, count and morphology were not affected by treatment. The weight of the testicular parenchyma in the high-dose group was statistically significantly lower than in the control group. Since no effects were observed on testis weight, this is considered an incidental finding. No female reproductive toxicity parameters were affected except for a slight statistically significant decrease in corpora lutea compared to the historical controls.The observed decrease in corpora lutea is of doubtful toxicological relevance and probably due to chance.Pre- and post-implantation loss were not affected by treatment.

The viability of pups was not affected by treatment The mean number of pups delivered in the high-dose group was statistically significantly lower than in the control group. This was due to the lower number of corpora lutea (probably due to chance) in the high-dose group. No treatment-related clinical findings or body weight changes were observed. Macroscopic examination of stillborn pups and pups that died during the study revealed no treatment-related abnormalities.

The NOAEL for fertility and reproductive performance of the male and female parental rats and the NOAEL for developmental toxicity were 2500 mg/kg diet based on the absence of reproductive effects up to the highest dose tested. This dietary level provided ca. 158 mg/kg bw/day in male parental rats and ca. 129 mg/kg bw/day in female parental rats.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

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:

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

Deviations:

yes

Remarks:

with 10-week premating period

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at initiation of treatment: 6 weeks (females)
- Weight at initiation of treatment: about 130 g (females)
- Housing: in macrolon cages with wood shavings (Lignocel, type 3/4) as bedding and shreds of paper (Enviro-dri) as environmental enrichment. Premating period: 4 rats of the same sex/cage; mating period: one male and one female per cage; after mating females were housed individually during gestation and with their litter during lactation.
- Diet: powdered Rat and Mouse no. 3 breeding diet, RM3 (SDS Special Diets Services, Witham, England), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%):45-65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: feed

Details on exposure:

The test material was mixed into the diet using a mechanical blender. Fresh batches of the experimental diets were prepared about once every 4-5 weeks, divided into daily amounts, packed in plastic bags, and stored in a freezer (<-18°C). Once daily in the afternoon (generally between 3-4 PM on working days), a fresh portion from the freezer was given to the animals. This frequency and timing of feed refreshing was applied because stability analyses showed that the concentration of the test substance in the feed decreased during storage at room temperature (by 18-27% after one day).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose confirmation analyses were conducted using a HPLC method with UV detection (at 247 nm) after extracton with acetonitrile. Stability under simulated experimental conditions was checked by analysis of samples immediately after diet preparation and after storage in the animal room (open container) for one day or in a freezer (<-18°C) for at least 5 weeks. The test substance was stable in the diet when stored in a freezer. The concentrations measured after one day storage in the animal room were 18% (low dose), 27% (mid dose) and 22% (high dose) lower than those measured at the start of the storage period. Homogeneity and achieved concentration were confirmed by analysis of samples from diets prepared shortly before initiation of treatment. For homogeneity, 5 samples per concentration (low, mid, high dose) were analysed. The measured concentrations were generally within 90-110% of the nominal concentrations.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximally 7 days.
- Proof of pregnancy: sperm in vaginal smear (made in the morning), referred to as day 0 of pregnancy.
- After successful mating, each pregnant female was caged individually for the birth and rearing of the pups.
- Sperm positive females that appeared non-pregnant (one control and one high-dose female) were sacrificed 27-28 days after copulation.
- One high-dose female that did not show evidence of copulation was housed individually after the last day of the mating period and sacrificed 29 days after the start of the mating period.

Duration of treatment / exposure:

Up to 103 days (70 days premating, 1-4 days mating, 21/22 days gestation, 4-6 days lactation)

Frequency of treatment:

Continuous administration via the diet (fixed dietary concentrations)

Dose / conc.:

200 mg/kg diet

Remarks:

Low dose group; Dietary equivalent to 15, 14, 14 mg/kg bw/day for premating females, gestation females and lactation females, respectively. Calculated from nominal dietary concentrations; due to instability actual concentrations were lower (loss is maximally 30% ).

Dose / conc.:

750 mg/kg diet

Remarks:

Mid dose group; Dietary equivalent to 56, 51, 45 mg/kg bw/day for premating females, gestation females and lactation females, respectively. Calculated from nominal dietary concentrations; due to instability actual concentrations were lower (loss is maximally 30% ).

Dose / conc.:

2 500 mg/kg diet

Remarks:

High dose group; Dietary equivalent to 171, 165, 195 mg/kg bw/day for premating females, gestation females and lactation females, respectively. Calculated from nominal dietary concentrations; due to instability actual concentrations were lower (loss is maximally 30% ).

No. of animals per sex per dose:

12 rats/sex/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: selection based on 2-week range-finding study.
- Rationale for animal assignment: computerised randomisation proportionately to body weight. Animals were allocated one day before initiation of treatment.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: at least once daily.
- Additional checks: daily afternoon check for dead or moribund animals.

BODY WEIGHT:
On day of allocation, at initiation of treatment, weekly during the premating and mating period, on days 0, 7, 14 and 21 during presumed gestation (mated females), and on days 1 and 4 of lactation.

FOOD CONSUMPTION:
- Except during the mating period, food consumption was measured from day 0 onwards on the same days as body weight was meausred. Results were expressed as g feed/animal/day and g feed/kg body weight/day.

HAEMATOLOGY:
- Time schedule for collection of blood: at end of premating period (week 10)
- Anaesthetic used for blood collection: Yes (carbon dioxide/oxygen)
- Animals fasted: yes (overnight)
- How many animals: 5/sex/group (from different cages)
- Parameters examined: Red blood: haemoglobin, red blood cells, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocytes. White blood: total white blood cells, differential white blood cells (absolute and relative). Coagulation: thrombocytes, prothrombin time.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: at end of premating period (week 10)
- Anaesthetic used for blood collection: Yes (carbon dioxide/oxygen)
- Animals fasted: Yes (overnight)
- How many animals: 5/sex/group (from different cages)
- Parameters examined: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamuyl transferase, total bilirubin, total protein, albumin, albumin/globulin ratio, glucose, total cholesterol, triglycerides, creatinine, urea, electrolytes (calcium, chloride, potassium, sodium, inorganic phosphate).

NEUROBEHAVIOURAL EXAMINATION:
Time schedule for examinations: towards the end of the premating period (in week 9)
Battery of functions tested:
- Grip strength (forelimb and hindlimb)
- Motor activity
- Observational screen including sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive). Screen components: Autonomic: lacrimation (R), salivation(R), pupil response to light(Q), palpebral closure(R), piloerection(Q), defaecation(C), urination(C); Neuromuscular: gait(D,R), mobility(R), forelimb and hindlimb gripstrength(I), landing foot splay(I), righting reflex(R); Sensorimotor: response(R) to tail pinch, click, touch and approach of a visual object; Convulsive: clonic and tonic movements(D); Excitability: ease of removal(R), handling reactivity(R), arousal(R), vocalizations(Q); Activity: rearing(C), posture(D); Physiological: body temperature(I) (C=count data; D=descriptive rank data; I=interval or continuous data; Q=quantal data; R=rank order data).

GROSS PATHOLOGY: Yes (all parental animals)
Animals were sacrificed at or shortly after day 4 of lactation by exsanguination from the abdominal aorta under carbondioxide/oxygen anaesthesia.

ORGAN WEIGHTS:
5 Animals/sex/group: adrenals, brain, heart, kidneys, liver, spleen, thymus
Paired organs were weighed together.
Organ weights relative to terminal body weight were calculated.

TISSUES PRESERVED:
In neutral aqueous phosphate-buffered 4% formaldehyde solution
All parental animals: ovaries (after counting of the corpora lutea), uterus (after counting of the implantation sites), all gross lesions

5 Parental animals/sex/group: adrenals, bone marrow (femur), brain (3 levels: cerebrum, cerebellum, medulla/pons), heart, small and large intestine (including Peyer's patches), kidneys, liver, lung , lymph nodes (mesenterial and axillary), peripheral nerve (tibial), spinal cord (3 levels: cervical, mid-thoracic, lumbar), spleen, stomach, thymus, thyroid, trachea, urinary bladder

HISTOPATHOLOGY:
Control and high-dose: all preserved organs and tissues.
Low- and mid-dose: gross lesions.

Fetal examinations:

PARTURITION AND LITTER EVALUATION:
At the end of the gestation period (gestation day 21), females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.

PARAMETERS EXAMINED:
The following parameters were examined in F1 offspring on days 1 and 4 of lactation:
- total litter size
- numbers of each sex
- numbers of stillbirths, live and dead pups
- grossly malformed pups/clinical observations
- pup weight (pups were weighed individually; mean pup weight was calculated per sex and for both sexes combined)

GROSS NECROPSY
Stillborn pups and pups that died during the study were examined for gross abnormalities (external and internal examination).

Statistics:

- Body weight (gain), food consumption, haematology, clinical chemistry, organ weights, continuous behavioural data, total distance moved in motor activity test: one-way analysis of variance followed by Dunnett's multiple comparison test.
- Habituation of motor activity: repeated measures analysis of variance on time blocks.
- Behaviour - rank data: Kruskal-Wallis non-parametric one-way analysis of variance followed by Dunnett's multiple comparison test.
- Behaviour - categorical data: Pearson's chi-square analysis.
- General clinical signs and histopathology: Fisher's exact probability test.

Reproduction toxicity parameters:
- Fisher's exact probability test was used to evaluate the number of mated and pregnant females, the number of pregnant females with implants but no pups, females with live pups, females with stillborn pups, live and dead fetuses or pups and the numbers of litters lost entirely.
- Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U test was used to evaluate pre-coital time (mean number of days), duration of gestation, number of corpora lutea and implantation sites, total number of pups delivered (mean), mean number of live pups per litter and pre- and post-implantation loss (%).

Indices:

REPRODUCTIVE INDICES
The following parameters were calculated:
- pre-coital time = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- mating index= (number of females mated/number of females placed with males) x 100
- female fertility index = (number of pregnant females/number of females placed with males) x 100
- female fecundity index =(number of pregnant females/number of females mated) x 100
- gestation index = (number of females with live pups/number of females pregnant) x 100
- pre-implantation loss = [(number of corpora lutea – number of implantation sites)/number of corpora lutea] x 100

OFFSPRING VIABILITY INDICES
The following parameters were calculated:
- live birth index = (number of pups born alive/number of pups born) x 100
- viability index day n-m = (number of pup surviving m days/number of liveborn on day n) x 100
- pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
- sex ratio day n = (number of live male pups on day n/ number of live pups on day n) x 100
- number of lost implantations = number of implantations sites - number of pups born alive
- post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The death of one low-dose female and one high-dose female on day 4 of lactation was unrelated to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight and body weight gain of the high-dose group were statistically significantly decreased during the entire or substantial parts of the study. Mean terminal body weights of high-dose females were 3% lower compared to controls; at the end of the premating period, high-dose females were about 6% lighter than controls. During gestation, mean body weights of high-dose females remained statistically signficantly lower compared with controls but weight gain was not significantly affected. During lactation, no statistically significant intergroup differences in body weight or weight gain occurred.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption (expressed as g/animal/day) of the high-dose group was statistically significantly decreased during the entire or substantial parts of the study.
Substance intake in successive weeks was calculated from the nominal dietary concentrations. As the test material was not stable over a 1-day storage period in the animal room, the actual test material intake was lower than indicated below (compared to the concentration measured at the start of the storage period, the concentration measured after storage was decreased by 18%, 27% and 22% at the low-, mid- and high-dose, respectively).
The mean (range) intakes in mg test material/kg bw/day (not corrected for instability) were:
- Premating females: 15 (12-20) at the low-dose, 56 (45-73) at the mid-dose, 171 (139-214) at the high-dose.
- Gestation females: 14 (13-16) at the low-dose, 51 (46-59) at the mid-dose, 165 (161-190) at the high-dose.
- Lactation females: 14 at the low-dose, 45 at the mid-dose, 195 at the high-dose.

Haematological findings:

no effects observed

Description (incidence and severity):

Female rats showed no significant changes in total and differential white blood cell counts, red blood cell variables or clotting potential.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Aspartate aminotransferase (ASAT) was decreased in females of all dose groups but there was no dose-response relationship. The toxicological significance of a decrease in ASAT activity may be doubtful, the more so as this finding was not corroborated by changes in related parameters. Changes in ALP activity (mid-dose females) and albumin level (low-dose females), were not confirmed at the higher dose level(s) and are considered incidental findings.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observational screen parameters, fore- and hindlimb grip strength, and spontaneous motor activity (total distance moved and habituation of activity) were not affected by treatment.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The absolute and relative weights of the adrenals were statistically increased in mid-dose females, but this finding was not confirmed at the high-dose level.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination revealed no treatment-related changes.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathologic examination revealed no treatment-related changes.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre- and post-implantation loss were not affected by treatment.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

A total loss of 2 litters in each of the intermediate dose groups was considered unrelated to treatment.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Duration of gestation were comparable in all groups.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The number of pregnant females was 11, 12, 12 and 10 in the control, low-, mid- and high-dose group, respectively.

Description (incidence and severity):

REPRODUCTIVE PERFORMANCE
- Fertility was not affected by treatment (pre-coital time, mating index, duration of gestation, gestation index, fertility indices and female fecundity index were comparable in all groups).
- The number of corpora lutea was statistically significantly decreased in the high-dose group (mean per animal: 10.1 versus in concurrent controls 12.7 ). Consequently, the numbers of implantation sites and pups delivered were also decreased in this group. The number of corpora lutea in the high-dose group was also lower compared to historical controls (range 11.0-14.5) but higher than lower numbers of corpora lutea observed incidentally in intermediate dose groups of other studies conducted at the testing laboratory (lowest value observed: 8.7). Therefore, the lower number of corpora lutea in the present high-dose group is of doubtful toxicololgical significance and probably due to chance.

Key result

Dose descriptor:

NOAEL

Remarks:

(General toxicity)

Effect level:

750 mg/kg diet

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Dietary equivalent of 37 mg/kg bw/day based on dietary concentration corrected for measured decrease upon storage in animal room (-27% after 24 hr)

Key result

Dose descriptor:

NOAEL

Remarks:

(Reproductive toxicity)

Effect level:

>= 2 500 mg/kg diet

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Dietary equivalent of 129 mg/kg bw/day based on dietary concentration corrected for measured decrease upon storage in animal room (-22% after 24 hr)

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Pup weight on lactation days 1 and 4 and weight gain between days 1-4 were not affected by treatment.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Viability was not affected by treatment. The live birth index and the number (stillborn pups) and percentage (pup mortality day 1) of pups found dead on day 1 were comparable between the groups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex ratio on days 1 and 4 was not affected by treatment.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of pups delivered in the high-dose group was statistically significantly lower than in the control group (8.4 at the high-dose versus 11.2 in controls). This was due to the lower number of corpora lutea (probably due to chance) in the high-dose group.

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

The number of pups lost between days 1-4 in the high-dose group was lower than that in the control and intermediate-dose groups. The higher pup loss in the latter groups was mainly due to the total loss of 2 litters in each of these groups and considered unrelated to treatment.

External malformations:

no effects observed

Description (incidence and severity):

Macroscopic examination of stillborn pups and pups that died during the study revealed no treatment-related abnormalities.

Description (incidence and severity):

CLINICAL SIGNS:
No treatment-related findings. In the high-dose group on day 1, the number of cold pups and pups with no milk in the stomach was statistically significantly lower than in the control group. This was due to a relatively high incidence of this finding in controls.

Key result

Dose descriptor:

NOAEL

Remarks:

(Developmental toxicity)

Effect level:

>= 2 500 mg/kg diet

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Dietary equivalent of 129 mg/kg bw/day based on dietary concentration corrected for measured decrease upon storage in animal room (-22% after 24 hr)

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In an oral (diet) developmental toxicity screening test in rats (OECD TG 422), a NOAEL for maternal toxicity of 750 mg/kg diet (ca. 37 mg/kg body weight/day) was established based on decreases in body weight gain and food consumption. No adverse effects on development of the offspring were noted in any group (NOAEL 2500 mg/kg diet, 129 mg/kg bw/day). The NOAELs expressed as mg/kg bw/day were based on nominal dietary test material concentrations corrected for loss on storage in the animal room.

Executive summary:

The developmental toxicity upon repeated oral exposure was examined in a study performed according to OECD TG 422 and in compliance with GLP. Twelve female Wistar rats per group received the test material via their diet, at concentrations of 0, 200, 750 and 2500 mg/diet during a 10-week premating period and during mating (one week), gestation and lactation until postnatal day 4. The total exposure duration of the dams was up to 103 days. Homogeneity and correct concentration of the test material in the diet were confirmed by analysis. The test substance was not stable in the diet upon storage in the animal room. The decrease in concentration during a 1-day storage period was 18%, 27% and 22% at the low-, mid- and high-dose, respectively. To minimize loss of test material under experimental conditions as much as possible, the feed in the feed hoppers was replaced daily by a fresh portion from the freezer (stability in the freezer was confirmed by analysis). Moreover, the fresh feed was provided in the afternoon instead of in the morning because rats eat most of their feed during the dark period. The following endpoints were evaluated to assess general toxicity: daily clinical observations, neurobehavioural examination (grip strength; functional observational battery including sensory reactivity to different stimuli; spontaneous motor activity), body weight, food consumption, routine haematology and clinical chemistry (in week 10 of the premating period), organ weights, macroscopic examination and histopathological examination of a wide range of tissues and organs (except for the kidneys which were examined in all groups, only high-dose animals and controls were subjected to histopathology). Endpoints to assess reproductive / developmental toxicity included parental fertility and reproductive performance (weight and morphology of sex organs, mating index, pre-coital time, fertility indices, female fecundity index, gestation index and length, pre- and post-implantation loss) and litter data (numbers of stillborn, live and dead pups, external abnormalities, pup weight on lactation days 1 and 4, necropsy of stillborn pups and pups that died during the study).

The results showed no treatment-related changes at 200 and 750 mg/kg diet. At 2500 mg/kg diet, body weight (gain) and food consumption were statistically significantly decreased during the entire or substantial parts of the study. No female reproductive toxicity parameters were affected except for a slight statistically significant decrease in corpora lutea compared to the historical controls. The observed decrease in corpora lutea is of doubtful toxicological relevance and probably due to chance. Pre- and post-implantation loss were not affected by treatment. The viability of pups was not affected by treatment. The mean number of pups delivered in the high-dose group was statistically significantly lower than in the control group. This was due to the lower number of corpora lutea (probably due to chance) in the high-dose group. No treatment-related clinical findings or body weight changes were observed. Macroscopic examination of stillborn pups and pups that died during the study revealed no treatment-related abnormalities.

Based on these results the NOAEL for maternal toxicity was 750 mg/kg diet. This dietary level provided ca. 37 mg/kg bw/day in female rats (based on the overall mean substance intake during the gestation period). The intake of the test material per kg body weight was calculated from the nominal dietary concentration corrected for the loss measured after storage in the animal room for one day. The NOAEL for developmental toxicity was 2500 mg/kg diet based on the absence of effects up to the highest dose tested. This dietary level provided ca. 129 mg/kg bw/day in female parental rats.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

with 10-week premating period

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (E)-3,4,5,6,6-pentamethylhept-3-en-2-one and 3,5,6,6-tetramethyl-4-methyleneheptan-2-one

EC Number:

946-245-5

Molecular formula:

C12H22O

IUPAC Name:

Reaction mass of (E)-3,4,5,6,6-pentamethylhept-3-en-2-one and 3,5,6,6-tetramethyl-4-methyleneheptan-2-one

Test material form:

liquid

Details on test material:

According to ECHA communication with reference to Annotation number: SUB-C-2114629169-42-01/F, the substance ID for Koavone has been changed with EC# from 939-627-8 to 946-245-5, and the IUPAC name from Reaction mass of (3R,5R)-3,5,6,6-tetramethyl-4-methylideneheptan-2-one and (3R,5S)-3,5,6,6-tetramethyl-4-methylideneheptan-2-one and (E)-3,4,5,6,6-pentamethylhept-3-en-2-one to Reaction mass of (E)-3,4,5,6,6-pentamethylhept-3-en-2-one and 3,5,6,6-tetramethyl-4-methyleneheptan-2-one.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Wistar strain: Crl:WI(Han)
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at initiation of treatment: 5 (males) - 6 (females) weeks in the main study; about 6 weeks in the range-finding study.
- Weight at initiation of treatment: about 100 g (males) - 130 g (females) in the main study; about 155 g (males) - 125 g (females) in de range-finding study.
- Housing: in macrolon cages with wood shavings (Lignocel, type 3/4) as bedding and shreds of paper (Enviro-dri) as environmental enrichment. Range-finding study: 2 rats of the same sex/cage; Main study: Premating period: 4 rats of the same sex/cage; mating period: one male and one female per cage; after mating, females were housed individually during gestation and with their litter during lactation.
- Diet: powdered Rat and Mouse no. 3 breeding diet, RM3 (SDS Special Diets Services, Witham, England), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days
- Allocation: by computer randomization proportionately to body weight

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2 in the main study, 18±3 in the range-finding study
- Humidity (%):45-65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES:
Range-finding study: in-life dates not reported.
Main study: From 11 May 2011 to 1-2 August (males) / 15-22 August (females) 2011

Administration / exposure

Route of administration:

oral: feed

Details on oral exposure:

The test material was mixed into the diet using a mechanical blender.
Range-finding study: Nominal concentrations in diet: 0, 230, 1150, 3850 and 7700 mg/kg diet.
The experimental diets were prepared just before the start of the study and stored in a refrigerator. The feed in the feeders was refreshed twice per week.

Main study: Nominal concentrations in diet: 0, 200, 750 and 2500 mg/kg diet.
Fresh batches of the experimental diets were prepared about once every 4-5 weeks, divided into daily amounts, packed in plastic bags, and stored in a freezer (<-18°C). Once daily in the afternoon (generally between 3-4 PM on working days), a fresh portion from the freezer was given to the animals. This frequency and timing of feed refreshing was applied because stability analyses showed that the concentration of the test substance in the feed decreased during storage at room temperature (by 18-27% after one day).

DIET PREPARATION
- Rate of preparation of diet: every 4-5 weeks in the main study; once in the range-finding study
- Storage temperature of food: <-18°C in the main study; refrigerator in the range-finding study

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose confirmation analyses were conducted using a HPLC method with UV detection (at 247 nm) after extraction with acetonitrile.
Range-finding study: Stability was checked by analysis of samples (from diets containing 230 or 7700 mg substance/kg feed) taken just after diet preparation and after storage in the animal room for up to four days or in a refrigerator at 2-10°C for four days (samples were stored frozen pending analysis). Compared with the concentration measured at the start of the storage period (which was close to the expected value), the concentration was decreased by about 10% after four days storage in the refrigerator, by about 20% after one day storage in the animal room and by about 50% after four days storage in the animal room.

Main study: Stability under simulated experimental conditions was checked by analysis of samples immediately after diet preparation and after storage in the animal room (open container) for one day or in a freezer (<-18°C) for at least 5 weeks. The test substance was stable in the diet when stored in a freezer. The concentrations measured after one day storage in the animal room were 18% (low dose), 27% (mid dose) and 22% (high dose) lower than those measured at the start of the storage period.
Homogeneity and achieved concentration were confirmed by analysis of samples from diets prepared shortly before initiation of treatment. For homogeneity, 5 samples per concentration (low, mid, high dose) were analysed. The measured concentrations were generally within 90-110% of the nominal concentrations.

Duration of treatment / exposure:

Range-finding study: 14 days
Main study males: up to 83 days (70 days premating, 7 days mating, 5-6 days until sacrifice)
Main study females: up to 103 days (70 days premating, 1-4 days mating, 21/22 days gestation, 4-6 days lactation)

Frequency of treatment:

Continuous administration via the diet (fixed dietary concentrations)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg diet

Remarks:

Low dose group; Dietary equivalent to 16, 15, 14, 14 mg/kg bw/day for premating males, premating females, gestation females and lactation females, respectively. Calculated from nominal dietary concentrations; due to instability actual concentrations were lower (loss is maximally 30% ).

Doses / concentrations 2

Dose / conc.:

750 mg/kg diet

Remarks:

Mid dose group; Dietary equivalent to 58, 56, 51, 45 mg/kg bw/day for premating males, premating females, gestation females and lactation females, respectively. Calculated from nominal dietary concentrations; due to instability actual concentrations were lower (loss is maximally 30% ).

Doses / concentrations 3

Dose / conc.:

2 500 mg/kg diet

Remarks:

High dose group; Dietary equivalent to 202, 171, 165, 195 mg/kg bw/day for premating males, premating females, gestation females and lactation females, respectively. Calculated from nominal dietary concentrations; due to instability actual concentrations were lower (loss is maximally 30% ).

No. of animals per sex per dose:

Range-finding study: 4 rats/sex/dose
Main study: 12 rats/sex/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale main study: selection based on 2-week range-finding study.
- Rationale for animal assignment: computerised randomisation proportionately to body weight. Animals were allocated one day before initiation of treatment.

Examinations

Observations and examinations performed and frequency:

RANGE-FINDING STUDY:
CAGE SIDE OBSERVATIONS:
- Time schedule: at least once daily.
- Additional checks: daily afternoon check for dead or moribund animals.

BODY WEIGHT AND FOOD CONSUMPTION:
- Twice weekly.

MAIN STUDY:
CAGE SIDE OBSERVATIONS:
- Time schedule: at least once daily.
- Additional checks: daily afternoon check for dead or moribund animals.

DETAILED CLINICAL OBSERVATIONS (in standard arena): Not conducted.

BODY WEIGHT:
- Males: on day of allocation, at initiation of treatment, weekly thereafter, and on the day of scheduled sacrifice.
- Females: on day of allocation, at initiation of treatment, weekly during the premating and mating period, on days 0, 7, 14 and 21 during presumed gestation (mated females), and on days 1 and 4 of lactation. Non-mated females were weighed one per week after the mating period.

FOOD CONSUMPTION:
- Except during the mating period, food consumption was measured from day 0 onwards on the same days as body weight was measured (except during the last week of the pre-mating period body weight, food was given on day 64 instead of day 63 because some of the animals were fasted for 1 night for haematology and clinical chemistry). Results were expressed as g feed/animal/day and g feed/kg body weight/day.

HAEMATOLOGY:
- Time schedule for collection of blood: at end of premating period (week 10)
- Anaesthetic used for blood collection: Yes (carbon dioxide/oxygen)
- Animals fasted: yes (overnight)
- How many animals: 5/sex/group (from different cages)
- Parameters examined:
. Red blood: haemoglobin, red blood cells, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocytes
. White blood: total white blood cells, differential white blood cells (absolute and relative)
. Coagulation: thrombocytes, prothrombin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: at end of pre-mating period (week 10)
- Anaesthetic used for blood collection: Yes (carbon dioxide/oxygen)
- Animals fasted: Yes (overnight)
- How many animals: 5/sex/group (from different cages)
- Parameters examined: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamuyl transferase, total bilirubin, total protein, phospholipids, albumin, albumin/globulin ratio, glucose, total cholesterol, triglycerides, creatinine, urea, electrolytes (calcium, chloride, potassium, sodium, inorganic phosphate).

NEUROBEHAVIOURAL EXAMINATION:
Time schedule for examinations: towards the end of the pre-mating period (in week 9)
Battery of functions tested:
- Grip strength (forelimb and hindlimb)
- Motor activity
- Observational screen including sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive). Screen components:
Autonomic: lacrimation (R), salivation(R), pupil response to light(Q), palpebral closure(R), piloerection(Q), defaecation(C), urination(C);
Neuromuscular: gait(D,R), mobility(R), forelimb and hindlimb gripstrength(I), landing foot splay(I), righting reflex(R);
Sensorimotor: response(R) to tail pinch, click, touch and approach of a visual object;
Convulsive: clonic and tonic movements(D);
Excitability: ease of removal(R), handling reactivity(R), arousal(R), vocalizations(Q);
Activity: rearing(C), posture(D);
Physiological: body temperature(I)
(C=count data; D=descriptive rank data; I=interval or continuous data; Q=quantal data; R=rank order data).

Sacrifice and pathology:

RANGE-FINDING STUDY:
All animals: gross pathology and organ weights (testes, kidneys, liver, spleen)

MAIN STUDY:
GROSS PATHOLOGY: Yes (all male and female parent animals)
Animals were sacrificed by exsanguination from the abdominal aorta under carbon dioxide/oxygen anaesthesia.
Males were sacrificed about one week after the mating period. Females were sacrificed at or shortly after day 4 of lactation.

ORGAN WEIGHTS:
All male animals: testes, epididymides
5 Animals/sex/group: adrenals, brain, heart, kidneys, liver, spleen, thymus
Paired organs were weighed together.
Organ weights relative to terminal body weight were calculated.

TISSUES PRESERVED:
In neutral aqueous phosphate-buffered 4% formaldehyde solution or Bouin's fixative (testes)
All parental animals:
ovaries (after counting of corpora lutea)
uterus (after counting of the implanation sites)
testes
epididymides
seminal vesicles
prostate
all gross lesions

5 Parental animals/sex/group:
adrenals
bone marrow (femur)
brain (3 levels: cerebrum, cerebellum, medulla/pons)
heart
small and large intestine (including Peyer's patches)
kidneys
liver
lung
lymph nodes (mesenterial and axilary)
peripheral nerve (tibial)
spinal cord (3 levels: cervical, mid-thoracic, lumbar)
spleen
stomach
thymus
thyroid
trachea
urinary bladder

HISTOPATHOLOGY:
Control and high-dose: all preserved organs and tissues.
Low- and mid-dose: kidneys (males only) and gross lesions.
Paraffin sections were stained with haematoxylin-eosin or PAS haematoxylin (testes).
Additional kidney sections of the male control and high-dose animals were processed for immunocytochemical staining of alpha 2-microglobuline using a mouse-anti-rat alpha 2-microglobuline antibody (SSI, Copenhagen, Denmark).

Other examinations:

Examinations to detect reproduction/developmental toxicity are presented in the endpoint study record under section 7.8.1 Toxicity to reproduction.

Statistics:

RANGE-FINDING STUDY:
- Clinical signs and gross pathology findings: Fisher's exact probability test.
- Body weight (gain), food consumption, organ weights: one-way analysis of variance followed by Dunnett's multiple comparison test.

MAIN STUDY:
- Body weight (gain), food consumption, haematology, clinical chemistry, organ weights, continuous behavioural data, total distance moved in motor activity test: one-way analysis of variance followed by Dunnett's multiple comparison test.
- Habituation of motor activity: repeated measures analysis of variance on time blocks.
- Behaviour - rank data: Kruskal-Wallis non-parametric one-way analysis of variance followed by Dunnett's multiple comparison test.
- Behaviour - categorical data: Pearson's chi-square analysis.
- General clinical signs and histopathology: Fisher's exact probability test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The death of one low-dose female and one high-dose female on day 4 of lactation was unrelated to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight and body weight gain of male and female rats of the high-dose group were statistically significantly decreased during the entire or substantial parts of the study. Mean terminal body weights of high-dose rats were 11% (males) or 3% (females) lower compared to controls; at the end of the premating period, high-dose females were about 6% lighter than controls. During gestation, mean body weights of high-dose females remained statistically significantly lower compared with controls but weight gain was not significantly affected. During lactation, no statistically significant intergroup differences in body weight or weight gain occurred.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of male and female rats of the high-dose group were statistically significantly decreased during the entire or substantial parts of the study.
Substance intake in successive weeks was calculated was calculated from the nominal dietary concentrations. As the test material was not stable over a 1-day storage period in the animal room, the actual test material intake was lower than indicated below (compared to the concentration measured at the start of the storage period, the concentration measured after storage was decreased by 18%, 27% and 22% at the low-, mid- and high-dose, respectively).
The mean (range) intakes in mg substance/kg bw/day were:
- Premating males: 16 (11-26) at the low-dose, 58 (41-86) at the mid-dose, 202 (144-287) at the high-dose.
- Premating females: 15 (12-20) at the low-dose, 56 (45-73) at the mid-dose, 171 (139-214) at the high-dose.
- Gestation females: 14 (13-16) at the low-dose, 51 (46-59) at the mid-dose, 165 (161-190) at the high-dose.
- Lactation females: 14 at the low-dose, 45 at the mid-dose, 195 at the high-dose.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Red blood cell and coagulation parameters were not affected by treatment.
Total white blood cell count and absolute numbers of lymphocytes, neutrophils, eosinophils, basophils and monocytes were statistically significantly decreased in high-dose males. To a lesser extent, total and differential white blood cell values in males of the low- and mid-dose groups were also decreased compared to concurrent controls. As the mean and individual values in the low- and mid-dose groups were within the historical control range, the lower values in the intermediate dose groups were not ascribed to treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes in clinical chemistry values were limited to higher chloride concentrations in males at all dose levels (statistically significant at the low- and high-dose) and a lower potassium concentration in high-dose males. These changes might be related to the effect on the kidneys observed in males.
A few other statistically significant differences from controls occurred (lower aspartate aminotransferase in females at all dose levels, lower alkaline phosphatase in mid-dose females and lower albumin in low-dose females) but were not ascribed to treatment (reason: lack of dose dependence).

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observational screen parameters, fore- and hindlimb grip strength, and spontaneous motor activity (total distance moved and habituation of activity) were not affected by treatment.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Relative kidney weight was statistically significantly increased in high-dose males. This finding was likely to be related to the alpha 2-microglobulin nephropathy observed in males.
Other statistically significant findings were not ascribed to treatment (slightly lower absolute brain weight in high-dose males, higher absolute and relative adrenal weight in mid-dose females and lower testicular parenchyma weight in the high dose males).

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination revealed no treatment-related changes.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A dose-dependent increase of hyalin droplet nephropathy was found in the male animals, characterised by an abundant presence of eosinophilic hyalin droplets in the proximal tubular cells, and several dilated tubuli filled with eosinophilic debris in the corticomedullary area. This change was accompanied by a dose-dependent increase in basophilic tubuli. Compared with concurrent controls, the high-dose males showed increased alpha 2-microglobulin staining of the cortical tubular epithelial cells and of the corticomedullary tubular cell debris.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

750 mg/kg diet

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

haematology

Remarks on result:

other: Dietary equivalent to 42 mg/kg bw/day based on nominal dietary concentration corrected for measured decrease upon storage in animal room (-27% after 24 hr).

2

Key result

Dose descriptor:

NOAEL

Effect level:

750 mg/kg diet

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Dietary equivalent to 41 mg/kg bw/day based on nominal dietary concentration corrected for measured decrease upon storage in animal room (-27% after 24 hr).

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

2 500 mg/kg diet

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

RANGE-FINDING STUDY:

CLINICAL SIGNS AND MORTALITY

Piloerection in two females fed 7700 mg/kg diet (on days 1-2 only). No mortality.

 

BODY WEIGHT (GAIN) AND FOOD CONSUMPTION

Body weight, body weight gain and food consumption were decreased, dose-dependently, in male and female rats fed 3850 or 7700 mg/kg diet. The decrease in food consumption was most marked during the first three days of the study, suggesting that this effect was due to reduced palatability of diets containing high concentrations of the test substance.

COMPOUND INTAKE

Mean substance intake over days 0 -14 was 23, 110, 340 and 620 mg/kg bw/day in male rats and 23, 100, 330 and 560 mg/kg bw/day in female rats fed 230, 1150, 3850 or 7700 mg/kg diet, respectively. These values are based on the nominal dietary test substance concentrations. Due to instability of the test substance in the diet the actual substance intake was lower (the concentration of test substance in the diet upon storage in the animal room for 1 or 4 days decreased by 20% or 50%, respectively; diets were refreshed every 3 or 4 days).

 

GROSS PATHOLOGY:

No remarkable findings.

 

ORGAN WEIGHTS:

Absolute spleen weight was decreased in male and female rats fed 7700 mg/kg diet.

Relative liver weight was increased, dose-dependently, in male and female rats fed 3850 or 7700 mg/kg diet.

Relative kidney weight was increased in male rats fed 7700 mg/kg diet.

These organ weight changes were statistically significant.

Applicant's summary and conclusion

Conclusions:

In an oral (diet) Combined Repeated Dose Toxicity Study with Reproduction / Developmental Toxicity Screening Test in rats (10-week premating period), performed according to OECD guideline 422 and in compliance with GLP, the mid-dose level of 750 mg/kg diet was a NOAEL for systemic toxicity. This dietary concentration provided ca. 42 mg/kg bw/day in male rats and ca. 41 mg/kg bw/day in female rats (based on nominal dietary test material concentration corrected for 27% loss on storage in animal room for one day; see remark under discussion).

Executive summary:

The toxicity upon repeated oral exposure was examined in a study performed according to OECD guideline 422 and in compliance with GLP. Groups of 12 male and 12 female Wistar rats received the test material via their diet, at concentrations of 0, 200, 750 and 2500 mg/diet during a 10-week premating period and during mating (one week), gestation and lactation until postnatal day 4. The total exposure duration was up to 83 days in male rats and up to 103 days in female rats. Homogeneity and correct concentration of the test material in the diet were confirmed by analysis. The test substance was not stable in the diet upon storage in the animal room. The decrease in concentration during a 1-day storage period was 18%, 27% and 22% at the low-, mid- and high-dose, respectively. To minimize loss of test material under experimental conditions as much as possible, the feed in the feed hoppers was replaced daily by a fresh portion from the freezer (stability in the freezer was confirmed by analysis). Moreover, the fresh feed was provided in the afternoon instead of in the morning because rats eat most of their feed during the dark period. These doses resulted in final mg/kg bw of circa 10, 40 and 120 mg/kg bw. The following endpoints were evaluated to assess general toxicity: daily clinical observations, neurobehavioural examination (grip strength; functional observational battery including sensory reactivity to different stimuli; spontaneous motor activity), body weight, food consumption, routine haematology and clinical chemistry (in week 10 of the premating period), organ weights, macroscopic examination and histopathological examination of a wide range of tissues and organs (except for the kidneys which were examined in all groups, only high-dose animals and controls were subjected to histopathology). Endpoints to assess reproductive / developmental toxicity included parental fertility and reproductive performance (including sperm analysis) and litter data are discussed in Section on Toxicity to reproduction.

The results showed no treatment-related changes at circa 10 and 40 mg/kg bw. At circa 120 mg/kg bw, body weight (gain) and food consumption were statistically significantly decreased during the entire or substantial parts of the study in rats of both sexes, and total and differential white blood cell counts were decreased in male rats (40%). In addition, male rats showed renal changes consisting of an increased relative kidney weight (at the high-dose level) and a dose-dependent increase of alpha 2-microglobulin nephropathy. This type of nephropathy in male rats is generally regarded as of no relevance for humans. The changes in clinical chemistry values in male rats (higher chloride at all dose levels and lower potassium at the high-dose) might be related to the nephropathy.

Based on these results the NOAEL for general toxicity was 750 mg/kg diet. This dietary level provided ca. 42 mg/kg bw/day in male rats and ca. 41 mg/kg bw/day in female rats (based on the overall mean substance intake during the pre-mating period). The intake of the test material per kg body weight was calculated from the nominal dietary concentration corrected for the loss measured after storage in the animal room for one day.