Methyl 5-(dimethylamino)-2-methyl-5-oxopentanoate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 March 2013 to 10 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline 416 and GLP compliant

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate no. 2012/96

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

methyl (2R)-4-(dimethylcarbamoyl)-2-methylbutanoate; methyl (2S)-4-(dimethylcarbamoyl)-2-methylbutanoate

EC Number:

700-204-6

Cas Number:

1174627-68-9

Molecular formula:

C9H17NO3

IUPAC Name:

methyl (2R)-4-(dimethylcarbamoyl)-2-methylbutanoate; methyl (2S)-4-(dimethylcarbamoyl)-2-methylbutanoate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Rhodiasolv Polarclean

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy
- Age at study initiation: (P) Males: 6 weeks old; Females: 5 weeks old; (F1) 22 days
- Weight at study initiation: (P) Males: 183 g to 235 g; Females: 103 g to 169 g; (F1) Males: 46 g to 74 g; Females: 43 g to 69 g
- Fasting period before study: no
- Housing: The animals were housed in polycarbonate cages with stainless steel lids (Tecniplast 2154, 940 cm2) containing autoclaved sawdust (SICSA, Alfortville, France):
 individually for the F0 and F1 except during pairing,
 individually during lactation with their litter for the F0 and F1 females with autoclaved wood shawings (SICSA, Alfortville, France) provided as nesting material, a few days before delivery and during the lactation period.
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch Nos. 4898480, 6318584 and 3839086 (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 19 March 2013 To: 28 January 2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosage forms were administered by gavage, using a plastic syringe fitted with a metal gavage tube, once a day, at approximately the same time.
The quantity of the dosage form administered to each animal was adjusted according to the most recently recorded body weight.
A constant dosage-volume of 5 mL/kg/day was used.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL for Low, Mid and High dose group, respectively

Details on mating procedure:

- M/F ratio per cage: One female was placed with one male, in the latter's cage.
- Length of cohabitation: Each female was placed with the same male until mating occurs or 14 days have elapsed.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually and allowed to litter normally and rear their progeny until weaning.
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test item in samples of each control and test item dose formulation prepared for use around weeks 1, 8, 12, 15, 18, 22, 29 and 32 was determined (eight analytical control of the dosage forms) using High Performance Liquid Chromatography with UV.

Duration of treatment / exposure:

F0 generation :
The dosage forms were administered daily according to the following schedule:
 in the males:
- 10 weeks before pairing,
- during the pairing period,
- until sacrifice (after weaning of the pups).
 in the females:
- 10 weeks before pairing,
- during the pairing period,
- during gestation,
- during lactation until the day before sacrifice (i.e. Days 21 to 24 p.p.),
- until the day before sacrifice for females which did not deliver (i.e. Day 24 p.c.).

F1 generation:
 in the males:
- from weaning (Day 22 p.p.) for 10 weeks before pairing,
- during the pairing period (up to 3 weeks),
- until sacrifice (after weaning of the pups).
 in the females:
- from weaning (Day 22 p.p.) for 10 weeks before pairing,
- during the pairing period (up to 3 weeks),
- during pregnancy,
- during lactation until the day before sacrifice (i.e. Days 21 to 22 p.p.),
- until the day before sacrifice for females which did not deliver (i.e. Days 23 to 25 p.c.).

NB: Day 1 corresponds to the first day of the treatment period.

Frequency of treatment:

The dosage forms were administered daily.

Details on study schedule:

-

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

24/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on:
- an OECD 422 study conducted in Wistar rats (Rhodia SAS project No. 41005299) where the No Observed Effect Level (NOEL) for reproductive toxicity was considered to be 1000 mg/kg/day in the absence of test item treatment related findings at this dose-level,
- a 13-week toxicity study by the oral route (gavage) in rats (CiToxLAB France/Study No. 39389 TCR) where the NOEL was considered to be 1000 mg/kg/day in the absence of test item treatment related findings at this dose-level,
- a pre-natal developmental toxicity study by the oral route (gavage) in rats (CiToxLAB France/Study No. 39390 RSR) where the NOEL for maternal parameters was considered to be 1000 mg/kg/day and the NOEL for embryo-fetal development was considered to be 1000 mg/kg/day.

- Rationale for animal assignment (if not random): not applicable

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays. From arrival, each animal was observed at least once a day as part as routine examination. From the start of the treatment period each animal was observed at least once a day, at approximately the same time for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were performed on all animals once a week until sacrifice. Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern).


BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each male was recorded on the first day of treatment (Day 1), then once a week until sacrifice.
The body weight of each female was recorded on the first day of treatment (Day 1), then once a week until mated, on Days 0, 7, 14, 20 p.c. (post-coitum) and Days 1, 4, 7, 14 and 21 p.p..


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. The quantity of food consumed by each male was measured once a week over a 7-day period from the first day of treatment until the start of the pairing period and after the pairing period until sacrifice. The quantity of food consumed by each female was measured once a week over a 7-day period, from the first day of treatment until the start of the pairing period, during gestation (Days 0-7, 7-14 and 14-20 p.c.), and during lactation (Days 1-7, 7-14, and 14-21 p.p.).
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable

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

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning as follows:
 during the last 3 weeks of the pre-pairing period,
 during the pairing period, until the females are mated.

Sperm parameters (parental animals):

Parameters examined in F0(P) and F1 male parental generations:
Full seminology investigations were performed on the first ten surviving F0 and F1 males from the control- and high-dose groups.
Since sperm motility is assessed immediately after sampling, it was also assessed on the first ten surviving F0 and F1 males from the low- and intermediate-dose groups. Investigation consisted in testis weight, epididymis weight, epididymal sperm motility, epididymal sperm morphology and epididymal sperm count; sperm count in testes.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of pups (four/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
- OBSERVATIONS OF THE PROGENY OF THE F0 FEMALES DURING THE LACTATION PERIOD: number and sex of pups, number of live, dead and cannibalized pups, presence of gross anomalies, clinical signs, body weight, physical and reflex development.
- CLINICAL EXAMINATIONS OF THE F1 GENERATION AFTER WEANING: Morbiditiy/Mortality, clinical signs, detailed clinical examination, body weight, food consumption, sexual development.
- NEUROBEHAVIORAL TESTS IN THE F1 GENERATION: Auditory startle reflex (at 4 weeks old), Pupil constriction (at 4 weeks old), Spontaneous locomotor activity (at 8 weeks old).
- OBSERVATIONS OF THE PROGENY OF THE F1 FEMALES DURING THE LACTATION PERIOD: Litter size, clinical signs, body weight, Anogenital distance, Pup physical development.

GROSS EXAMINATION OF DEAD PUPS:
yes: A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all found dead pups. Special attention was paid to the reproductive organs and to whether the pup has fed (e.g. presence of milk in the stomach).

Postmortem examinations (parental animals):

SACRIFICE
On completion of the treatment period, all surviving males and females were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination:
- F0 males: after weaning of the F1 progeny,
- F0 surviving females: at the weaning of their litter (i.e. Days 22 to 25 p.p.),
- F0 females which did not deliver: on Day 25 p.c. (after body weight recording to check for a possible unnoticed delivery),


GROSS NECROPSY
- Gross necropsy consisted of complete macroscopic post-mortem examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
A microscopic examination was performed on:
 all tissues listed in the Tissue Procedure Table 7.8.1/1 for the F0 and F1 animals of the control and high dose groups (groups 1 and 4) sacrificed at the end of the treatment period,
 reproductive organs of all animals that did not mate and of any male with abnormalities at sperm analysis,
 all macroscopic lesions of all the animals of the low- and intermediate-dose groups sacrificed on completion of the treatment period,
 all animals found dead or prematurely sacrificed.

The body weight of each animal sacrificed as scheduled was recorded before sacrifice. The organs specified in the Tissue Procedure Table 7.8.1/1 were weighed wet as soon as possible after dissection.

Postmortem examinations (offspring):

SACRIFICE
On completion of the treatment period, all surviving males and females were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination:
 F1 surviving males: after weaning of the F2 generation,
 F1 surviving females: at the weaning of their litter (on Days 22 to 23 p.p.),
 F1 females which did not deliver: Days 24 to 26 p.c. (after body weight recording to check for a possible unnoticed delivery),
 F1 females with total litter loss.

Pups were sacrificed by an intraperitoneal injection of sodium pentobarbital:
 pups not selected on Day 4 p.p.: on Day 4 p.p.,
 pups at weaning: on Day 22 p.p. (see § Study plan adherence).

- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY (PROGENY OF THE F0 AND F1 GENERATIONS)
The following pups were carefully examined externally for gross external abnormalities:
 pups found dead,
 pups culled on Day 4 post-partum,
 pups sacrificed on Days 22 to 25 post-partum.

In addition, for the following pups a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed. Special attention was paid to the reproductive organs.
 pups showing external abnormalities or clinical signs (see § Study plan adherence),
 pups found dead,
 one randomly selected F1 and F2 pup/sex/litter sacrificed on Days 22 to 25 post-partum.


HISTOPATHOLOGY / ORGAN WEIGTHS
A microscopic examination was performed on:
 all the tissues listed in Tissue Procedure Table 7.8.1/2 for one randomly selected F1 pup/sex/litter not selected at weaning, and for one randomly selected F2 pup/sex/litter,
 all macroscopic lesions,
 all pups with external abnormalities.

Body weight was recorded for one F1 and F2 pup/sex/litter sacrificed on Days 22 to 25 post partum and randomly selected for a macroscopic post mortem examination.
The organs specified in Tissue Procedure Table 7.8.1/2 were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

Statistics:

Body weights, food consumption and reproductive data:
Data were compared by one-way variance analysis and Dunnett's test, (mean values being considered as normally distributed, variances being considered as homogeneous) or by Fisher exact probability test (proportions).

Organ weights:
PathData software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01) according to the following sequence. Data of non pregnant females are not included in group mean calculations and statistics.

Auditory startle reflex:
Statistical analysis of auditory startle reflex data was performed using the SAS Enterprise Guide software, version 2.05.89 (SAS Release 8.02 TS Level 02M0, SAS Institute Inc).

Reproductive indices:

The following parameters were calculated:

 post-implantation loss (manually calculated):

Number of implantation sites - Number of live pups
_____________________________________________ x 100
Number of implantation sites

 mating index:
Number of mated animals
_____________________ x 100
Number of paired animals

 fertility index:
Number of pregnant female partners
_______________________________ x 100
Number of mated pairs

 gestation index:
Number of females with live born pups
________________________________ x 100
Number of pregnant females

Offspring viability indices:

The following parameters were calculated:

 live birth index:
Number of live born pups
_____________________ x 100
Number of delivered pups

 viability index on Day 4 p.p.:
Number of surviving pups on Day 4 p.p.
_______________________________________ x 100
Number of live born pups

 lactation index:
Number of surviving pups on Day 21 p.p.
________________________________________ x 100
Number of surviving pups on Day 4 p.p.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, test item treatment-related effects but of no toxicological significance (see details below).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 300 and 1000 mg/kg/day, variations in body weight gain during premating and post-mating periods for F0 generation but of no toxicological significance (see details below).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 300 and 1000 mg/kg/day, variations in body weight gain during premating and post-mating periods for F0 generation but of no toxicological significance (see details below).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
- F0 GENERATION: There was no treatment-related death. At 1000 mg/kg/day, round back, emaciated appearance and piloerection were considered to be test item treatment-related but of no toxicological significance taking into account the low number of animals concerned and their limited duration (1 to 3 days). Ptyalism, observed in most of animals, was also considered to be test item treatment-related but of minor toxicological importance.
- F1 GENERATION: There was no treatment-related death. Ptyalism was observed in most of animals given 1000 mg/kg/day. This finding was considered to be related to the treatment with the test item and of minor toxicological importance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- F0 GENERATION: There were no effects on mean body weight both during premating (F0 males and F0 females) and post mating (F0 males) periods.
In the 300 and 1000 mg/kg/day groups, statistically significant episodes of higher or lower mean body weight gain were recorded during premating (F0 males and F0 females) and post-mating (F0 males) periods. Taking into account the amplitude of the changes and the absence of effect on mean body weight and on mean food consumption, these findings were considered to be not toxicologically significant.
There were no effects on mean body weight and mean body weight changes in females during the pregnancy and lactation periods.
There were no treatment-related effects on mean food consumption.
- F1 GENERATION: There were no toxicologically significant effects on mean body weight, mean body weight changes and mean food consumption during the premating (males and females), post-mating (males), pregnacy (females) and lactation (females) periods.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): not applicable.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no effects on mean estrous cycles parameters in F0 and F1 females.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no effects on sperm parameters in F0 and F1 males.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- There were no effects on mating and fertility data in F0 and F1 generation.
- There were no effects on delivery data in F0 and F1 generation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
F0 GENERATION: When compared with controls, there were slight to moderate, statistically significant increases in mean absolute and relative-to-body liver weights in high-dose males and females (up to +14%, p<0.05 or 0.01). This change was considered to be related to test item treatment, although the livers were not examined microscopically.
There were also minimal, statistically significant increases in mean relative-to-body kidney weights in high dose males. The relationship to test item administration was considered to be unlikely, in view of the very low magnitude of this difference and of the opposite trend in females.
The statistically significant changes in organ weights for seminal vesicles and thyroids were considered not to be test item-related since these differences were minimal and not dose-related.
F1 GENERATION: When compared with controls, there were slight, statistically significant increases in mean absolute and relative-to-body liver weights in high-dose males (up to +16%, p<0.01). This change was considered to be related to test item treatment, although the livers were not examined microscopically. Statistically significant changes in organ weights for uterus were seen at 100 and 300 mg/kg/day and were considered not to be test item-related since these differences were of low grade and not dose-related.


GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no macroscopic findings attributed to the test item administration in F0 and F1 parents.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no microscopic test item-related changes in the genital tract in F0 and F1 parents.
When compared with controls, there was a slight increase of the mean numbers of corpora lutea and primordial follicles in high-dose females. In view of the absence of organ weight correlation (no increased ovarian weights) and of the low magnitude of these observations, these variations were not considered to be toxicologically significant.
The few macroscopic findings noted at the end of the treatment period in other organs were of those commonly recorded in the species and none were considered to be related to the test item administration in parents. For F0 generation, the livers were not observed, except one liver sampled from one mid-dose F0 female. This female had a macroscopic finding (red discoloration) correlating with moderate focal angiectasia. This microscopic isolated finding was not related to test item administration in view of the incidence. For F1 generation, the livers were not examined, except the liver sampled from one mid-dose and one high-dose males because of accentuated lobular pattern at gross examination. This correlated with slight or moderate increased glycogen content. In view of the low incidence of this finding and the minor toxicological importance of this lesion, this finding was unrelated to test item administration.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The NOAEL for parental toxicity was considered to be 1000 mg/kg/day (based on absence of adverse effects at this dose level in the F0 generation).

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING = F0 and F1 pups):
There were no toxicologically significant effects on viability index on Day 4 p.p. and on lactation index in F0 and F1 pups.

CLINICAL SIGNS (OFFSPRING = F0 and F1 pups):
There were no test item-related clinical signs in F0 and 1 pups during the lactation period.

BODY WEIGHT (OFFSPRING = F0 and F1 pups):
There were no effects on mean body weight and mean body weight change in F0 and F1 pups during the lactation period.

SEXUAL MATURATION (OFFSPRING = F1 GENERATION):
There were no effects on male and female sexual development landmarks in F1 generation.

ORGAN WEIGHTS (OFFSPRING = F0 and F1 offsprings):
There were no changes in body or organ weights in treated groups when compared with controls.

GROSS PATHOLOGY (OFFSPRING = F0 and F1 offsprings):
There were no macroscopic findings attributed to the test item administration.

HISTOPATHOLOGY (OFFSPRING = F0 and F1 offsprings):
There were no test item-related microscopic findings.

OTHER PARAMETERS (OFFSPRING):
PHYSICAL AND REFLEX DEVELOPMENT:
There were no effects of the test item on F0 and F1 pups physical development. There were no effects of the test item on F0 pup reflex development.

NEUROBEHAVIORAL DEVELOPMENT OF THE F1 GENERATION:
- Acoustic startle test: There were no effects on mean acoustic startle reflex test results.
- Pupil constriction test: All animals were positive for the pupil constriction reflex at 4 weeks of age.
- Motor activity: There were no effects on mean number of horizontal movements and rearing.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

neuropathology

other:

Remarks on result:

other: based on the absence of effect at this dose-level

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The test item, Rhodiasolv Polarclean, was administered by gavage to F0 and F1 male and female Sprague-Dawley rats at 100, 300 or 1000 mg/kg/day, daily for 10 weeks prior to pairing, through mating and gestation, and until the end of the lactation period of the F2 generation [Day 21 post partum (p.p.)].

Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day (based on absence of adverse effects at this dose level in the F0 generation).

The NOEL for mating behavior, fertility and gestation of F0 generation and for development, growth and survival of the F1 progeny up to weaning of the F2 generation was considered to be 1000 mg/kg/day (based on the absence of effect at this dose-level).

Executive summary:

The objective of this study, performed according to OECD 416 guideline and in compliance with GLP, was to provide general information concerning the effects of the test item, Rhodiasolv Polarclean, on the integrity and performance of the male and female reproductive systems, including gonadal function, the estrous cycle, mating behavior, conception, gestation, parturition, lactation and weaning, and on the growth and development of the offspring.

Methods

F0 and F1 generations:

Three groups of 24 male and 24 female Sprague-Dawley F0 rats received the test item, Rhodiasolv Polarclean (batch No. CY 12331065), daily for 10 weeks prior to pairing, during pairing, through gestation and lactation until weaning of the F1 pups [Day 21 post-partum (p.p.)]. On Day 22 p.p., one or two F1 males and one or two F1 females per litter (from as many litters as possible) were selected to obtain 24 animals/sex/group and therefore constitute the F1 generation. The F1 rats received the test item, daily for 10 weeks prior to pairing, during pairing, through gestation and lactation until weaning of the F2 pups [Day 21 post-partum (p.p.)].

The test item was administered by oral administration (gavage, 5 mL/kg) at 100, 300 or 1000 mg/kg/day. Control groups of 24 males and 24 females received the vehicle alone (drinking water treated by reverse osmosis), under the same experimental conditions, and acted as a reference F0 or F1 control groups.

Examination of F0, F1 and F2 generations:

Clinical signs and mortality were checked daily. Food consumption and body weight were recorded at designated intervals. Males and females were paired until mating was obtained or 14 days have elapsed. Pairs with no evidence of mating after 14 days were separated and the female was placed for a further 7-day period with a different male from the same dose-level group who has already mated. The F0 and F1 females were allowed to deliver normally, and rear their progeny. Pregnancy and litter parameters were recorded. The F0 and F1 parents were sacrificed after weaning of their progeny. During lactation, the F1 and F2 pups were observed daily for survival and clinical signs. Body weight was measured at designated intervals and the sex-ratio was recorded. On Day 4 post-partum, the size of each litter was adjusted to obtain eight pups per litter. Physical and/or reflex development was assessed at designated end-points.

Terminal examination of F0 and F1 animals:

After weaning of their progeny, F0 and F1 parents were sacrificed. Designated organs were weighed in parents and offspring. Epididymal and testicular sperm parameters were evaluated in F0 and F1 males. A macroscopic post-mortem examination was performed on all F0 and F1 parents and on one F1 and F2 pup per sex and per litter sacrificed at weaning. Any pups which died or were killed prematurely during the lactation period were also submitted for macroscopic post-mortem examination. Macroscopic lesions, reproductive organs, adrenals, brain, kidneys, liver, spleen, thyroids and pituitary glands were sampled in all parent animals. A microscopic examination was performed on macroscopic lesions, reproductive organs of all F0 and F1 parents of the control- and high-dose groups. A detailed histopathological examination was performed on the ovaries and the testes.

Results

F0 Generation:

There were no test item-related deaths or premature euthanasia. At 1000 mg/kg/day, round back, emaciated appearance and piloerection were considered to be test item treatment-related but not toxicologically significant. Ptyalism, observed in most of animals, was also considered to be test item treatment-related but of minor toxicological importance.

There were no toxicologically significant effects on mean body weight, mean body weight changes and mean food consumption during the premating (F0 males and F0 females) and post-mating (F0 males) periods. There were no effects on mean body weight, mean body weight changes and mean food consumption in F0 females during the pregnancy and lactation periods. There were no effects on mean estrous cycles parameters of F0 females. There were no effects on sperm parameters in F0 males. There were no effects on mating and fertility data. There were no effects on delivery data.

F0 Generation offspring:

Several animals were found dead or prematurely sacrificed in all groups, with similar incidence in both control and treated groups and with no macroscopic findings. There were no effects on viability index on Day 4 p.p. and on lactation index. There were no test item-related clinical signs in F0 pups during the lactation period. There were no effects on mean body weight and mean body weight change in F0 pups. There were no effects of the test item on F0 pup physical and reflex development.

F1 Generation:

At 100 mg/kg/day, one male was found dead on Day 111 and at 300 mg/kg/day one male was found dead on Day 122. The causes of these deaths were undetermined but a relationship to the treatment was considered unlikely in absence of deaths at higher doses.

At 1000 mg/kg/day, ptyalism was observed in most of animals; this finding was considered to be related to the treatment with the test item and of minor toxicological importance.

There were no toxicologically significant effects on mean body weight, mean body weight changes and mean food consumption during the premating (F1 males and F1 females) and post-mating (F1 males) periods. there were no effects on mean body weight, mean body weight changes and mean food consumption during the pregnancy and lactation periods in F1 females.

There were no effects on male and female sexual development landmarks.There were no effects on mean acoustic startle reflex test results. All animals were positive for the pupil constriction reflex at 4 weeks of age. There were no effects on mean number of horizontal movements and rearing. There were no effects on mean estrous cycles parameters of F1 females. There were no effects on sperm parameters in F1 males. There were no effects on mating and fertility data. There were no effects on delivery data.

F1 Generation offspring:

During lactation, several animals were found dead or prematurely sacrificed in all groups, with noteworthy higher incidence in control versus treated groups at 100, 300 or 1000 mg/kg/day and with no macroscopic findings. There were no toxicologically significant effects on viability and lactation indexes. There were no test item-related clinical signs in F1 pups. There were no effects on mean body weight and mean body weight changes in F1 pups during the lactation period. There were no effects on F1 pup physical development.

Pathology:

In F0 parents, there were increases in absolute and relative-to-body liver weights in males and females treated at 1000 mg/kg/day. In F1 parents, similar increase in absolute and relative-to-body liver weights was observed in males treated at 1000 mg/kg/day.

There were no macroscopic or microscopic test item-related changes in either F0 generation, F0 generation offspring, F1 generation or F1 generation offspring, including in any genital organs.

Conclusion

Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day (based on absence of adverse effects at this dose-level in the F0 generation).

The NOEL for mating behavior, fertility and gestation of F0 generation and for development, growth and survival of the F1 progeny up to weaning of the F2 generation was considered to be 1000 mg/kg/day (based on the absence of effect at this dose-level).