Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 08 December 2015 to 21 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 08 December 2015 to 21 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Version dated March 1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: MR92143651
- Expiration date of the lot/batch: 01 January 2017
- Purity: 86.4% (No correction was made for purity of the test substance)
- Purity test date: 18 June 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Yes, maximum temperature: 50°C, maximum duration: unknown (until liquefaction).
- Solubility and stability of the test substance in the solvent/vehicle: Stability of the test item in the vehicle for the range of concentration 0.5 to 100 mg/mL: 8 Days when stored at room temperature (+ 15 to + 25°C) or refrigerated (+ 2 to + 8°C) (validation study no. AB21059; data not shown).
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not assessed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The preparation was warmed in a water bath at 40°C ± 5°C (except for the fourth preparation of group 3 and 4, warmed in a water bath at 47.3 °C and 46.9 °C, respectively. However the temperatures of preparations were measured and were below 45°C) under magnetic stirring for at least 10 minutes until a solution is obtained. The test item was prepared as a solution in the vehicle at concentrations of 10, 30 and 100 mg/mL.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Females (if applicable) nulliparous and non-pregnant: N/A
- Age at study initiation: Virgin males: approximately 10 Weeks ; Virgin females: approximately 8 Weeks.
- Weight at study initiation: males: 298 to 339 g; females: 164 to 214 g.
- Fasting period before study: All animals were fasted for approximately 16 hours before sampling for clinical laboratory determinations.
- Housing: Male rats (throughout the study except during mating) and female rats (during the pre-mating period) were housed in groups of 5 of the same sex and dose group in plastic cage meeting European directive 2010/63/EU requirements. During cohabitation with males and during gestation and lactation (together with their litter), females were housed individually in plastic cage meeting European directive 2010/63/EU requirements.
- Diet (e.g. ad libitum): Rat pelleted commercial complete rodent diet ad libitum (Diet reference Safe, A04C-10).
- Water (e.g. ad libitum): Softened and filtered (0.2 μm) mains drinking water was available ad libitum.
- Acclimation period: 7 Days between animal arrival and the start of treatment

DETAILS OF FOOD AND WATER QUALITY:
Rat pelleted commercial complete rodent diet sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
Water is analysed twice a year for chemical and bacterial contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3°C (target range).
- Humidity (%): > 35% (target).
- Air changes (per hr): At least 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light (artificial)/12 hours dark (except when required for technical acts).

IN-LIFE DATES: From: 08 December 2015 To: 26 January 2016
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Preparation:
The test item was prepared as a solution in the vehicle..
Method:
The test item was weighed and then the vehicle was added. The preparation was warmed in a water bath at 40°C ± 5°C under magnetic stirring for at least 10 minutes until a solution is obtained.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was chosen because it allowed to prepare appropriate concentrations of the test item.
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/Day
- Lot/batch no. (if required): 14090129/B
- Purity: 100%
Details on mating procedure:
- M/F ratio per cage: one male and one female
- Length of cohabitation: maximum of 14 Days.
- Proof of pregnancy: sperm in a vaginal smear or a vaginal plug referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: N/A
- After successful mating each pregnant female was caged (how): During gestation and lactation (together with their litter), females were housed individually in plastic cage meeting European directive 2010/63/EU requirements.
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Four samples of 1 g were taken from each formulation, including the vehicle, used on the first Day of treatment and on a suitable Day during each of the third and penultimate Weeks of treatment. The samples were stored at room temperature (+ 15 to + 25°C).
One set of samples was analysed at the Test Facility, within the defined stability period (see above), using a validated method. The transfer and validation of the analytical method was the subject of a separate study plan (study number AB21059).
Duration of treatment / exposure:
Males: 14 Days before mating, throughout the mating period and up to the Day before necropsy.
Females: 14 Days before mating, throughout the mating period, during gestation (the first Day of gestation is designated as G0) and up to to 4 Days after parturition (the first Day of birth is designated as L0).
Mated females that failed to produce a viable litter: until Day 25 or 26 post-coitum.
Unmated female: for 20 Days after the last Day of the mating period.
Frequency of treatment:
Once daily.
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 rats/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: in the absence of severe clinical signs during the DRF phase, the same dose levels were selected for the main study phase
- Rationale for animal assignment (if not random): Performed during the acclimatisation period, using a computer-generated randomisation. The mean body weight of each group at allocation was not statistically significantly different from each other (analysis of variance), each sex being considered separately.
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All adults were observed twice daily at the beginning and at the end of each working Day (including Weekends and public holiDays) to detect any which were moribund. Animals judged to be in a moribund condition were necropsied. A full clinical examination was performed on each weighing Day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed daily for clinical signs During the treatment period, the animals were observed before and at least once after treatment to detect any abnormalities in appearance, behaviour or other signs of reaction to treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights for males were recorded Weekly. Individual body weights for females were recorded:
− Weekly during pre-mating and mating periods (only pre-mating data was reported)
− on Days 0, 7, 14 and 20 of gestation
− on Days 1 and 4 of lactation.

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period (Day 14).
- Anaesthetic used for blood collection: Blood was withdrawn from the retro-orbital sinus under isoflurane anaesthesia (AErrane®, Baxter SA)
- Animals fasted: Yes ( for at least 16 hours)
- How many animals: Five randomly selected animals/sex/group.
- Parameters checked in table no. 7.5.1.1 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes.
- Time schedule for examinations: Males: at the end of the treatment period, the Day before necropsy (Day 31). Females: during lactation, shortly before necropsy (Day 4 of lactation).
- Dose groups that were examined:
- Battery of functions tested: The following tests were performed: auditory reflex, pupillary reflex, righting reflex, fore- and hind-limb grip strength and locomotor activity in an open field test.
Estrous cyclicity (parental animals):
Not evaluated
Sperm parameters (parental animals):
Not evaluated
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
For each litter, the following data were recorded:
− number of pups born (live and dead)
− external abnormalities of the pups
− number, weight and sex of pups alive on postnatal Days 1 and 4.

GROSS EXAMINATION OF DEAD PUPS:
Pups that died were also necropsied; their stomach was examined for the presence of milk, if possible. Defects or cause of death were evaluated, if possible.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
Moribund adults and females showing signs of parturition difficulties or total litter death were killed by carbon dioxide inhalation and exsanguination then necropsied.

Surviving adult animals were killed by carbon dioxide inhalation followed by exsanguination then necropsied according to the following schedule:
− males: after completion of the mating period (31 Days of oral administration)
− females: on Day 4 of lactation (mated females that failed to produce a viable litter by Day 26 or 27 post-coitum were killed and necropsied)
− apparently unmated female: 21 Days after the last Day of the mating period (Day 50).

GROSS NECROPSY / HISTOPATHOLOGY / ORGAN WEIGHTS
The list of the organs/tissues examined is indicated in table 7.8.1.1
Postmortem examinations (offspring):
Pups that died were necropsied; their stomach was examined for the presence of milk, if possible. Defects or cause of death were evaluated, if possible.
Statistics:
The following parameters were analysed statistically on each occasion for males and females separately, by the Provantis data acquisition system:
− body weights and body weight gains, food consumption, gripping test, open field test, haematology, coagulation and serum clinical chemistry parameters, pre-coital interval,terminal body weights, absolute and relative organ weights.
The best transformation for the data (none, log or rank) was determined depending upon the kurtosis of the data, the probability of the Bartlett's test for homogeneity of the variances and the similarity of the group sizes. Non- or log-transformed data were analysed by parametric methods. Rank transformed data were analysed using non-parametric methods. The homogeneity of means was assessed by analysis of variance (ANOVA).
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or Shirley's test for non-parametric data.
If no trend was found and the means were not homogeneous, the data were analysed by a stepwise parametric or non-parametric Dunnett's test to look for significant differences from the control group.
Open field test and pre-coital interval data were analysed using a SAS software package. Levene’s test will be used to test the equality of variance across groups and Shapiro-Wilk's test will be used to assess the normality of the data distribution in each group. Data with homogeneous variances and normal distribution in all groups will be analysed using ANOVA followed by Dunnett’s test. Data showing non homogeneous variances or a non-normal distribution in at least one group will be analysed using Kruskal-Wallis test followed by Wilcoxon's rank sum test.
Reproductive indices:
- Pre-coital interval (in Days)
- Pre-Implantation Loss
- Pre-Birth Loss
- Male and female fertility index (in %)
- Gestation index (in %)
-
Offspring viability indices:
- Live birth index (in %)
- Viability index (in %)
- Sex ratio (proportion of male pups in %)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Two females given 1000 mg/kg/Day had decreased activity and/or piloerection noted after the first administration only. There were no test item-related clinical signs for females given 100 and 300 mg/kg/Day and for males at all dose levels.
Vehicle-related clinical signs included hypersalivation, abnormal foraging and pedalling and were observed in all groups.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female in each of the control (no. 115) and 1000 mg/kg/Day (no. 172) groups were sacrificed in moribund condition on G 24 and 23, respectively. Female no. 115 was sacrificed moribund after giving birth to 10 stillborn pups and female no. 172 was unable to deliver. Before sacrifice, both females presented decreased activity, pallor, marked piloerection, subdued and/or rapid breathing. At necropsy, the cause of the poor health of both females could not be determined.
In addition, one female in each of the 100 and 1000 mg/kg/Day groups (nos. 137 and 176, respectively) were euthanized following total litter death on L 1 or L4, respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
There was a lower mean body weight gain from Day 1 to Day 15 for males given 1000 mg/kg/Day (- 66%) when compared with the control. Thereafter, body weight gain improved but the overall mean body weight gain (Day 1 to Day 29) remained lower (- 40%), with an absolute mean body weight at the end of the treatment period statistically significantly lower compared with the control group (- 7%).
A lower mean body weight gain from Day 8 to Day 15 was also observed for males given 300 mg/kg/Day compared with the control but at a lesser intensity (- 27%). However, terminal mean body weight remained comparable with the control group.
There was no obvious effect of treatment on mean body weight gain or absolute body weight for males at 100 mg/kg/Day.

Females:
There was a lower mean body weight gain from Day 7 to 20 of gestation for females given 1000 mg/kg/Day (-20 %) when compared with the control.
There was no obvious test item-related effect on mean body weight or body weight gain during the pre-mating or lactation periods at any dose level, or during the gestation period at 100 and 300 mg/kg/Day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
There was a slightly lower mean food consumption between Day 1 to Day 8 for males given 1000 mg/kg/Day (- 11%) when compared with the control.
There was no obvious effect on mean food consumption for males treated at lower doses.

Females:
There was a dose-related lower mean food consumption between Day 1 to Day 4 of lactation for females given 300 and 1000 mg/kg/Day (- 17% and - 21%, respectively) when compared with the control.
There was no obvious effect on mean food consumption for females during the pre-mating or gestation period at any dose level and during the lactation period at 100 mg/kg/Day.

In addition, when compared with the historical control data (see Addendum 6), there was a lower mean food consumption for both sexes during the pre-mating, gestation and/or lactation periods in all groups receiving the vehicle with or without the test item. This finding was therefore considered to be associated with the vehicle (i.e. corn oil).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The liver weight was increased for both sexes at 1000 mg/kg/Day and for males given 300 mg/kg/Day. The liver weight increase for females at 300 mg/kg/Day was only marginal.
There was increased weight of thyroids which was observed at all dose levels, except for females given 300 mg/kg/Day.

Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The liver was enlarged at necropsy for males given 1000 mg/kg/Day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The increase in liver weight correlated with minimal test item-related centrilobular hepatocellular hypertrophy, which was noted for both sexes at 300 and 1000 mg/kg/Day.
In the thyroid glands, there was a minimal diffuse follicular cell hypertrophy at all dose levels for both sexes.
Histopathological findings: neoplastic:
not examined
Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There was no obvious test item-related influence on mating performance of the males and females in any group. All animals mated with the exception of 1 pair in the 1000 mg/kg/Day group. This isolated finding was considered incidental since all other females mated within the first 4 Days of the start of the mating period. The majority of females in all group showed evidence of insemination within the first 4 Days of pairing (approximate duration of a normal oestrous cycle). The mean pre-coital interval was consequently comparable in all groups.
There was no effect of treatment on fertility of either sex in any group. There was a single mated female in each of the control, 100, 300 and 1000 mg/kg/Day groups (nos. 112, 139, 157 and 174, respectively) that did not become pregnant. These isolated cases in each group, including the control, were considered incidental.
Key result
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to the highest dose tested.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive performance
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect of treatment on mating performance or fertility.
Remarks on result:
not determinable due to absence of adverse toxic effects
Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The live birth index (number of pups born alive/number of pups born) was slightly lower in the 100 and 300 mg/kg/Day (93.5 and 91.3%, respectively) compared with the concurrent control (100%) and the mean historical control data (99.5%). However, without any dose relationship and any similar finding at the high dose of 1000 mg/kg/Day, this was considered incidental.
There was a lower viability index (number of pups alive on PND 4/number of pups born alive) in the 100 and 1000 mg/kg/Day groups (87.1% and 77.3%, respectively) compared with concurrent control (100%) and the historical control data range (94.1% to 100%). At 100 mg/kg/Day, this finding was disproportionally influenced by one atypical female (no. 137) with total litter death on PND 1. At 1000 mg/kg/Day, three out of six females (nos. 175, 176 and 178) had approximately half or total litter death by PND 4 which is indicative of a test item-related effect.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a test item-related lower mean body weight for both sexes at 100, 300 and 1000 mg/kg/Day on PND 1 (6.4 g, 6.1 g and 5.4 g, respectively) compared with the control (6.8 g). This difference from the control group was accentuated on PND 4 (10.3 g, 9.3 g, 8.9 g and 7.2 g in groups 1 to 4, respectively) with lower mean body weight gain between PND 1 and 4 (3.4/3.6 g, 3.0/3.0 g, 2.9/2.9 g, and 1.5/1.7 g for males/females in groups 1 to 4, respectively). However, the effect was considered not adverse at 100 mg/kg/Day only since mean body weight of pups remained within or close to the historical control data range (minimum of 9.7 g for males and 9.6 g for females on PND 4).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There was no pattern in the incidences of the pup observations noted, such as a haematoma, thin appearance, pale and tail constriction that suggested any association with maternal treatment.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Delivery and Litter Data:

Parturition and Duration of Gestation:

There were 7, 9, 9 and 6 females that completed delivery in the control, 100, 300 and 1000 mg/kg/Day groups, respectively.

There was no test item-related effect on the mean duration of gestation. However, the mean duration of gestation for all groups, including the control, (22.4 to 22.7 Days) was slightly higher than the mean historical control data (22.1 Days). A potential effect of the vehicle could not be excluded.

Pre-Implantation Loss:

There was a lower mean number of corpora lutea in the 300 and 1000 mg/kg/Day groups (11.7 and 10.6, respectively) compared with the concurrent control (12.5) and the 100 mg/kg/Day group (12.9). However, since the majority of individual values fall within the control range, this finding was considered of no biological significance.

There was no obvious test item-related effect on the mean percentage pre-implantation loss in any group. However, as a consequence of the lower number of copora lutea, there was a lower mean number of implantation sites in the 300 and 1000 mg/kg/Day groups compared with the concurrent control and historical control data range.

Pre-Birth Loss:

There was no test item-related effect on the mean percentage pre-birth loss. However, the mean percentage pre-birth loss for the control, 300 and 1000 mg/kg/Day groups (16.5 to 20.7%) was higher than the mean historical control data (8.1%). A potential effect of the vehicle could not be excluded.

As a consequence of the lower number of corpora lutea (see section 10.11.2), the mean number of pups born was lower in the 300 and 1000 mg/kg/Day (8.9 and 7.3, respectively) compared with the control (10.6) and the mean historical control data (10.9).

Conclusions:
Oral (gavage) administration of 1-Phenyldecane-1,3-dione at doses of 100, 300 and 1000 mg/kg/Day was associated with a transient reduction in body weight gain and/or food consumption for males and females at 300 and 1000 mg/kg/Day. In addition, adaptative non-adverse test item-related changes in the liver at 300 and 1000 mg/kg/Day and in the thyroid glands at all dose levels were observed. The No Observed Adverse Effect Level (NOAEL) for parental toxicity is therefore 1000 mg/kg/Day.
There was no effect of treatment on mating performance or fertility. In addition, there were no test item-related pathological changes in the male or female genital system. The No Observed Adverse Effect Level (NOAEL) for reproductive performance including gonadal function, mating behaviour, conception and parturition is therefore 1000 mg/kg/Day for both sexes.
Developmental changes included a lower pup viability at 1000 mg/kg/Day and a lower pup weight at all dose levels (considered not adverse at 100 mg/kg/Day) on PND 1 and through to termination. The No Observed Adverse Effect Level (NOAEL) for postnatal development is therefore 100 mg/kg/Day.
Executive summary:

The objectives of this study were to evaluate the potential toxic effects of the test item, 1-Phenyldecane-1, 3-dione, when exposed for a minimum of 28 Days and the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. Parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No-Observed-Adverse-Effect-Levels (NOAELs) were evaluated. This GLP study was carried out according to OECD test guideline No. 422 (March 1996).

Three groups of 10 male and 10 female Wistar Han rats were given the test item, 1-Phenyldecane-1,3-dione, by daily oral (gavage) administration at dose levels of 100, 300 and 1000 mg/kg/Day from 14 Days before mating (Day 1) until Day 31 for males or until Day 4 of lactation for females. A control group of 10 male and 10 female Wistar Han rats was given 10 mL/kg/Day of the vehicle (corn oil).

Clinical condition, body weight and food consumption of the animals were monitored throughout the study. Clinical laboratory determinations were performed at the end of the pre-mating period (Day 14). After two Weeks of treatment, one male and one female of the same group were paired for a maximum of 14 Days. The females were allowed to give birth and litter parameters, including the number of pups born, pup survival, sex and pup weights were recorded up to postnatal Day 4. Functional tests were performed at the end of the treatment period for 5 selected males in each group and on Day 4 of lactation for 5 selected females in each group.

The males were necropsied after the pairing period. The dams and pups were necropsied on Day 4 of lactation. All animals were submitted to a macroscopic examination and selected organs were weighed. Selected organ/tissue samples were fixed and preserved at necropsy for half of the animals/group and reproductive organs samples were fixed and preserved at necropsy from all animals. Histopathological examinations were performed on selected organs/tissues from animal killed moribund, from half of the group 1 and 4 animals killed at the end of the treatment period, from all males suspected to be infertile or that failed to sire, and from all females that failed to deliver healthy pups.

There was no obvious test item-related mortality in any group. However, one female in each of the control and 1000 mg/kg/Day groups were sacrificed in a moribund condition following dystocia.

Two females given 1000 mg/kg/Day had decreased activity and/or piloerection noted after the first administration only. There were no test item-related clinical signs for females given 100 and 300 mg/kg/Day and for males at all dose levels.

There was a reduced body weight gain for males given 300 (from Day 8 to Day 15) and 1000 mg/kg/Day (from Day 1 to Day 15), and for females given 1000 mg/kg/Day (from Days 7 to 20 of gestation).

There was a reduce food consumption for males given 1000 mg/kg/Day (from Day 1 to Day 8) and for females given 300 and 1000 mg/kg/Day (from Day 1 to Day 4 of lactation).

On Day 14, there was no obvious effect of treatment on haematology, coagulation and serum clinical chemistry parameters from males or females at any dose level.

There was no obvious effect of treatment on functional tests (i.e. auditory reflex, pupillary test, righting reflex, gripping test or open field test) for either sex at any dose level.

There was no obvious test item-related effect on the mating performance or fertility of either sex in any group. There were 7, 9, 9 and 6 females that completed delivery in the control, 100, 300 and 1000 mg/kg/Day groups, respectively.

There was no obvious test item-related effect on number of corpora lutea.

There was no obvious test item-related effect on the pre-birth loss, pup sex ratio or on the live birth index in any group.

There was a test item-related reduce viability index in the 1000 mg/kg/Day group compared with concurrent control. There was no obvious effect of test item on viability index at lower dose levels.

There was no obvious maternal treatment-related clinical observation for any pup in any group.

There was a dose-related lower pup weight for both sexes at all dose levels on PND 1 and through to termination. However, the effect was considered not adverse at 100 mg/kg/Day since values remained within or close to the historical control data range.

Adaptative non-adverse test item-related changes were observed in the liver (i.e. centrilobular hepatocellular hypertrophy) for both sexes at 1000 mg/kg/Day and for males given 300 mg/kg/Day and the thyroid glands (i.e. diffuse follicular cell hypertrophy) at all dose levels for both sexes. There were no test item-related pathological changes in the male or female genital system.

It should be noted that there was some vehicle-related effects including clinical signs (i.e. hypersalivation, abnormal foraging, pedalling and straub tail), reduce food consumption (consistent with the oily nature of the vehicle), a suspected prolongation of gestation and higher pre-birth loss.

In conclusion, oral (gavage) administration of 1-Phenyldecane-1,3-dione at doses of 100, 300 and 1000 mg/kg/Day was associated with a transient reduction in body weight gain and/or food consumption for males and females at 300 and 1000 mg/kg/Day. In addition, adaptative non-adverse test item-related changes in the liver at 300 and 1000 mg/kg/Day and in the thyroid glands at all dose levels were observed. The No Observed Adverse Effect Level (NOAEL) for parental toxicity is therefore 1000 mg/kg/Day.

There was no effect of treatment on mating performance or fertility. In addition, there were no test item-related pathological changes in the male or female genital system. The No Observed Adverse Effect Level (NOAEL) for reproductive performance including gonadal function, mating behaviour, conception and parturition is therefore 1000 mg/kg/Day for both sexes.

Developmental changes included a lower pup viability at 1000 mg/kg/Day and a lower pup weight at all dose levels (considered not adverse at 100 mg/kg/Day) on PND 1 and through to termination. The No Observed Adverse Effect Level (NOAEL) for postnatal development is therefore 100 mg/kg/Day.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Reddish solid below 25°C / liquid above 25°C
Details on test material:
- State of aggregation: N/A
- Particle size distribution: N/A
- Mass median aerodynamic diameter (MMAD): N/A
- Geometric standard deviation (GSD): N/A
- Shape of particles: N/A
- Surface area of particles: N/A
- Crystal structure: N/A
- Coating: N/A
- Surface properties: N/A
- Density: 1.06 g/cm3
- Moisture content: N/A
- Residual solvent: N/A
- Activation: N/A
- Stabilisation: N/A
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: MR92143651
- Expiration date of the lot/batch: 01 January 2017
- Purity: 86.4% (No correction was made for purity of the test substance)
- Purity test date: 18 June 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Yes, maximum temperature: 50°C, maximum duration: unknown (until liquefaction).
- Solubility and stability of the test substance in the solvent/vehicle: Stability of the test item in the vehicle for the range of concentration 0.5 to 100 mg/mL: 8 Days when stored at room temperature (+ 15 to + 25°C) or refrigerated (+ 2 to + 8°C) (validation study no. AB21059; data not shown).
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not assessed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The preparation was warmed in a water bath at 40°C ± 5°C (except for the fourth preparation of group 3 and 4, warmed in a water bath at 47.3 °C and 46.9 °C, respectively. However the temperatures of preparations were measured and were below 45°C) under magnetic stirring for at least 10 minutes until a solution is obtained. The test item was prepared as a solution in the vehicle at concentrations of 10, 30 and 100 mg/mL.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Females (if applicable) nulliparous and non-pregnant: N/A
- Age at study initiation: Virgin males: approximately 10 Weeks ; Virgin females: approximately 8 Weeks.
- Weight at study initiation: males: 298 to 339 g; females: 164 to 214 g.
- Fasting period before study: All animals were fasted for approximately 16 hours before sampling for clinical laboratory determinations.
- Housing: Male rats (throughout the study except during mating) and female rats (during the pre-mating period) were housed in groups of 5 of the same sex and dose group in plastic cage meeting European directive 2010/63/EU requirements. During cohabitation with males and during gestation and lactation (together with their litter), females were housed individually in plastic cage meeting European directive 2010/63/EU requirements.
- Diet (e.g. ad libitum): Rat pelleted commercial complete rodent diet ad libitum (Diet reference Safe, A04C-10).
- Water (e.g. ad libitum): Softened and filtered (0.2 μm) mains drinking water was available ad libitum.
- Acclimation period: 7 Days between animal arrival and the start of treatment

DETAILS OF FOOD AND WATER QUALITY:
Rat pelleted commercial complete rodent diet sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
Water is analysed twice a year for chemical and bacterial contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3°C (target range).
- Humidity (%): > 35% (target).
- Air changes (per hr): At least 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light (artificial)/12 hours dark (except when required for technical acts).

IN-LIFE DATES: From: 08 December 2015 To: 26 January 2016

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Gavage using a plastic cannula.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Preparation:
The test item was prepared as a solution in the vehicle..
Method:
The test item was weighed and then the vehicle was added. The preparation was warmed in a water bath at 40°C ± 5°C under magnetic stirring for at least 10 minutes until a solution is obtained.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was chosen because it allowed to prepare appropriate concentrations of the test item.
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/Day
- Lot/batch no. (if required): 14090129/B
- Purity: 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of preparations::
Four samples of 1 g were taken from each formulation, including the vehicle, used on the first Day of treatment and on a suitable Day during each of the third and penultimate Weeks of treatment. The samples were stored at room temperature (+ 15 to + 25°C).
One set of samples was analysed at the Test Facility, within the defined stability period (see above), using a validated method. The transfer and validation of the analytical method was the subject of a separate study (study number AB21059).
Duration of treatment / exposure:
Males: 14 Days before mating, throughout the mating period and up to the Day before necropsy.
Females: 14 Days before mating, throughout the mating period, during gestation (the first Day of gestation is designated as G0) and up to to 4 Days after parturition (the first Day of birth is designated as L0). Mated females that failed to produce a viable litter: until Day 25 or 26 post-coitum. Unmated female: for 20 Days after the last Day of the mating period.
Frequency of treatment:
Once daily.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 rats/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In the absence of severe clinical signs during the DRF phase, the same dose levels were selected for the main study phase/
- Rationale for animal assignment (if not random): Performed during the acclimatisation period, using a computer-generated randomisation. The mean body weight of each group at allocation was not statistically significantly different from each other (analysis of variance), each sex being considered separately.
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes / No / Not specified
- Time schedule: All adults were observed twice daily at the beginning and at the end of each working Day (including Weekends and public holiDays) to detect any which were moribund. Animals judged to be in a moribund condition were necropsied. A full clinical examination was performed on each weighing Day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed daily for clinical signs During the treatment period, the animals were observed before and at least once after treatment to detect any abnormalities in appearance, behaviour or other signs of reaction to treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights for males were recorded Weekly. Individual body weights for females were recorded:
− Weekly during pre-mating and mating periods (only pre-mating data was reported)
− on Days 0, 7, 14 and 20 of gestation
− on Days 1 and 4 of lactation.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY: N/A

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period (Day 14).
- Anaesthetic used for blood collection: Blood was withdrawn from the retro-orbital sinus under isoflurane anaesthesia (AErrane®, Baxter SA)
- Animals fasted: Yes ( for at least 16 hours)
- How many animals: Five randomly selected animals/sex/group.
- Parameters checked in table no. 7.5.1.1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes.
- Time schedule for examinations: Males: at the end of the treatment period, the Day before necropsy (Day 31). Females: during lactation, shortly before necropsy (Day 4 of lactation).
- Dose groups that were examined:
- Battery of functions tested: The following tests were performed: auditory reflex, pupillary reflex, righting reflex, fore- and hind-limb grip strength and locomotor activity in an open field test.
Activity was monitored by a video image analysis system (Videotrack supplied by Viewpoint, Lyon, France). The arena was divided into nine equal invisible regions; the time spent by the animal in each type of region (i.e. corner, centre or lateral) was recorded. Motor activity was divided into three categories: ambulatory activity (in which the centre of the image moved at more than 7 cm/sec), small movements (including grooming etc.) and inactivity. The proportion of time spent engaged in each type of activity and the total distance travelled by the rat was calculated.

IMMUNOLOGY: No

OTHER: No
Sacrifice and pathology:
Terminal Examinations
Moribund adults and females showing signs of parturition difficulties or total litter death were killed by carbon dioxide inhalation and exsanguination then necropsied.

Surviving adult animals were killed by carbon dioxide inhalation followed by exsanguination then necropsied according to the following schedule:
− males: after completion of the mating period (31 Days of oral administration)
− females: on Day 4 of lactation (mated females that failed to produce a viable litter by Day 26 or 27 post-coitum were killed and necropsied)
− apparently unmated female: 21 Days after the last Day of the mating period (Day 50).

GROSS PATHOLOGY: Yes (see list of organs examined in table 7.8.1.2)

HISTOPATHOLOGY: Yes (see list of organs/tissues examined table 7.8.1.2)
Other examinations:
Mating; pregnancy and parturition, and Litter data. Data described in section 7.8.1 (Toxicity to reproduction)
Statistics:
The following parameters were analysed statistically on each occasion for males and females separately, by the Provantis data acquisition system:
− body weights and body weight gains, food consumption, gripping test, open field test, haematology, coagulation and serum clinical chemistry parameters, pre-coital interval,terminal body weights, absolute and relative organ weights.
The best transformation for the data (none, log or rank) was determined depending upon the kurtosis of the data, the probability of the Bartlett's test for homogeneity of the variances and the similarity of the group sizes. Non- or log-transformed data were analysed by parametric methods. Rank transformed data were analysed using non-parametric methods. The homogeneity of means was assessed by analysis of variance (ANOVA).
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or Shirley's test for non-parametric data.
If no trend was found and the means were not homogeneous, the data were analysed by a stepwise parametric or non-parametric Dunnett's test to look for significant differences from the control group.
Open field test and pre-coital interval data were analysed using a SAS software package. Levene’s test will be used to test the equality of variance across groups and Shapiro-Wilk's test will be used to assess the normality of the data distribution in each group. Data with homogeneous variances and normal distribution in all groups will be analysed using ANOVA followed by Dunnett’s test. Data showing non homogeneous variances or a non-normal distribution in at least one group will be analysed using Kruskal-Wallis test followed by Wilcoxon's rank sum test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Two females given 1000 mg/kg/Day had decreased activity and/or piloerection noted after the first administration only. There were no test item-related clinical signs for females given 100 and 300 mg/kg/Day and for males at all dose levels.
Vehicle-related clinical signs included hypersalivation, abnormal foraging and pedalling and were observed in all groups.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female in each of the control (no. 115) and 1000 mg/kg/Day (no. 172) groups were sacrificed in moribund condition on G 24 and 23, respectively. Female no. 115 was sacrificed moribund after giving birth to 10 stillborn pups and female no. 172 was unable to deliver. Before sacrifice, both females presented decreased activity, pallor, marked piloerection, subdued and/or rapid breathing. At necropsy, the cause of the poor health of both females could not be determined.
In addition, one female in each of the 100 and 1000 mg/kg/Day groups (nos. 137 and 176, respectively) were euthanized following total litter death on L 1 or L4, respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
There was a lower mean body weight gain from Day 1 to Day 15 for males given 1000 mg/kg/Day (- 66%) when compared with the control. Thereafter, body weight gain improved but the overall mean body weight gain (Day 1 to Day 29) remained lower (- 40%), with an absolute mean body weight at the end of the treatment period statistically significantly lower compared with the control group (- 7%).
A lower mean body weight gain from Day 8 to Day 15 was also observed for males given 300 mg/kg/Day compared with the control but at a lesser intensity (- 27%). However, terminal mean body weight remained comparable with the control group.
There was no obvious effect of treatment on mean body weight gain or absolute body weight for males at 100 mg/kg/Day.

Females:
There was a lower mean body weight gain from Day 7 to 20 of gestation for females given 1000 mg/kg/Day (-20 %) when compared with the control.
There was no obvious test item-related effect on mean body weight or body weight gain during the pre-mating or lactation periods at any dose level, or during the gestation period at 100 and 300 mg/kg/Day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
There was a slightly lower mean food consumption between Day 1 to Day 8 for males given 1000 mg/kg/Day (- 11%) when compared with the control.
There was no obvious effect on mean food consumption for males treated at lower doses.

Females:
There was a dose-related lower mean food consumption between Day 1 to Day 4 of lactation for females given 300 and 1000 mg/kg/Day (- 17% and - 21%, respectively) when compared with the control.
There was no obvious effect on mean food consumption for females during the pre-mating or gestation period at any dose level and during the lactation period at 100 mg/kg/Day.
In addition, when compared with the historical control data (see Addendum 6), there was a lower mean food consumption for both sexes during the pre-mating, gestation and/or lactation periods in all groups receiving the vehicle with or without the test item. This finding was therefore considered to be associated with the vehicle (i.e. corn oil).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
On Day 14, there was no obvious effect of treatment on haematology or coagulation parameters for males or females at any dose level.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
On Day 14, there was no obvious effect of treatment on serum clinical chemistry parameters for males or females at any dose level.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There was no obvious effect of treatment on the auditory, pupillary and, righting reflexes, or on the gripping and open field tests for the males or females at any dose level.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The liver weight was increased for both sexes at 1000 mg/kg/Day and for males given 300 mg/kg/Day. The liver weight increase for females at 300 mg/kg/Day was only marginal.
There was increased weight of thyroids which was observed at all dose levels, except for females given 300 mg/kg/Day.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The liver was enlarged at necropsy for males given 1000 mg/kg/Day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The increase in liver weight correlated with minimal test item-related centrilobular hepatocellular hypertrophy, which was noted for both sexes at 300 and 1000 mg/kg/Day.
In the thyroid glands, there was a minimal diffuse follicular cell hypertrophy at all dose levels for both sexes.
Histopathological findings: neoplastic:
not examined
Details on results:
The changes in the liver at 300 and 1000 mg/kg/Day and in the thyroid glands at all dose levels.are considered adaptive non-adverse reactions likely the consequence of hepatic enzyme induction.
In view of the changes in the thyroid glands, no NOEL could be established in this study.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to the highest dose tested
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Formulation Analysis:

No test item was detected in the vehicle sample.

Achieved concentration of formulation of 1-Phenyl decane-1.3-dione at 10, 30 and 100 mg/mL in corn oil as vehicle used for the first Day of treatment and on the third and penultimate Week of treatment, were in agreement with acceptance criteria with a deviation from nominal concentration ranging from - 9.9% to - 1.7%.

Applicant's summary and conclusion

Conclusions:
Oral (gavage) administration of 1-Phenyldecane-1,3-dione at doses of 100, 300 and 1000 mg/kg/Day was associated with a transient reduction in body weight gain and/or food consumption for males and females at 300 and 1000 mg/kg/Day. In addition, adaptative non-adverse test item-related changes in the liver at 300 and 1000 mg/kg/Day and in the thyroid glands at all dose levels were observed. The No Observed Adverse Effect Level (NOAEL) for parental toxicity is therefore 1000 mg/kg/Day.
Executive summary:

The objectives of this study were to evaluate the potential toxic effects of the test item, 1-Phenyldecane-1, 3-dione, when exposed for a minimum of 28 Days and the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. Parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No-Observed-Adverse-Effect-Levels (NOAELs) were evaluated. This GLP study was carried out according to OECD test guideline No. 422 (March 1996).

Three groups of 10 male and 10 female Wistar Han rats were given the test item, 1-Phenyldecane-1,3-dione, by daily oral (gavage) administration at dose levels of 100, 300 and 1000 mg/kg/Day from 14 Days before mating (Day 1) until Day 31 for males or until Day 4 of lactation for females. A control group of 10 male and 10 female Wistar Han rats was given 10 mL/kg/Day of the vehicle (corn oil).

Clinical condition, body weight and food consumption of the animals were monitored throughout the study. Clinical laboratory determinations were performed at the end of the pre-mating period (Day 14). After two Weeks of treatment, one male and one female of the same group were paired for a maximum of 14 Days. The females were allowed to give birth and litter parameters, including the number of pups born, pup survival, sex and pup weights were recorded up to postnatal Day 4. Functional tests were performed at the end of the treatment period for 5 selected males in each group and on Day 4 of lactation for 5 selected females in each group.

The males were necropsied after the pairing period. The dams and pups were necropsied on Day 4 of lactation. All animals were submitted to a macroscopic examination and selected organs were weighed. Selected organ/tissue samples were fixed and preserved at necropsy for half of the animals/group and reproductive organs samples were fixed and preserved at necropsy from all animals. Histopathological examinations were performed on selected organs/tissues from animal killed moribund, from half of the group 1 and 4 animals killed at the end of the treatment period, from all males suspected to be infertile or that failed to sire, and from all females that failed to deliver healthy pups.

There was no obvious test item-related mortality in any group. However, one female in each of the control and 1000 mg/kg/Day groups were sacrificed in a moribund condition following dystocia.

Two females given 1000 mg/kg/Day had decreased activity and/or piloerection noted after the first administration only. There were no test item-related clinical signs for females given 100 and 300 mg/kg/Day and for males at all dose levels.

There was a reduced body weight gain for males given 300 (from Day 8 to Day 15) and 1000 mg/kg/Day (from Day 1 to Day 15), and for females given 1000 mg/kg/Day (from Days 7 to 20 of gestation).

There was a reduce food consumption for males given 1000 mg/kg/Day (from Day 1 to Day 8) and for females given 300 and 1000 mg/kg/Day (from Day 1 to Day 4 of lactation).

On Day 14, there was no obvious effect of treatment on haematology, coagulation and serum clinical chemistry parameters from males or females at any dose level.

There was no obvious effect of treatment on functional tests (i.e. auditory reflex, pupillary test, righting reflex, gripping test or open field test) for either sex at any dose level.

Adaptative non-adverse test item-related changes were observed in the liver (i.e. centrilobular hepatocellular hypertrophy) for both sexes at 1000 mg/kg/Day and for males given 300 mg/kg/Day and the thyroid glands (i.e. diffuse follicular cell hypertrophy) at all dose levels for both sexes. There were no test item-related pathological changes in the male or female genital system.

It should be noted that there was some vehicle-related effects including clinical signs (i.e. hypersalivation, abnormal foraging, pedalling and straub tail), reduce food consumption (consistent with the oily nature of the vehicle), a suspected prolongation of gestation and higher pre-birth loss.

In conclusion, oral (gavage) administration of 1-Phenyldecane-1,3-dione at doses of 100, 300 and 1000 mg/kg/Day was associated with a transient reduction in body weight gain and/or food consumption for males and females at 300 and 1000 mg/kg/Day. In addition, adaptative non-adverse test item-related changes in the liver at 300 and 1000 mg/kg/Day and in the thyroid glands at all dose levels were observed. The No Observed Adverse Effect Level (NOAEL) for parental toxicity is therefore 1000 mg/kg/Day.