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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the absence of any effects on mating performance, fertility, gestation duration, litter size and pup viability in a rat OECD 422 study, the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity of Rhodiasolv Polarclean given by the oral route was considered to be 1000 mg/kg/day. These results are consistent with those reported in the rat 2-generation study.  

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same study

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

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

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:

-

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

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.

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

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

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'

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

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.

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

Reproductive effects observed:

not specified

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).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and OECD 416 compliant study ( Klimisch 1). The other study (OECD 422, see Section "Toxicity to reproduction: other studies") is a screening study and was therefore assigned as supporting study.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

For the purpose of notification in China, a two-generation study of reliability 1 according to Klimish criteria was performed and was selected as key study (Spezia, 2014 (OECD 416, oral route)). The aim of this study was provide information on the effects of the test substance 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.

Three groups of 24 male and 24 female Sprague-Dawley F0 rats received the test item, Rhodiasolv Polarclean, 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.

There were no test item-related deaths in F0 and F1 parents. At 1000 mg/kg/day in F0 parents, round back, emaciated appearance and piloerection were considered to be test item treatment-related but not toxicologically significant. Ptyalism, observed in most of animals of F0 and F1 parents, 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 and F1 males and F0 and F1 females), post-mating (F0 and F1 males), pregnancy and lactation (F0 and F females) periods.

There were no effects on mean estrous cycles parameters of F0 and F1 females nor on sperm parameters in F0 and F1 males. There were no effects on mating, fertility data and delivery data for both generations.

There were no toxicologically significant effects on viability index on Day 4 p.p. and on lactation index in F0 and F1 pups. There were no test item-related clinical signs and no effects of the test item on the mean body weight and mean body weight change in F0 and F1 pups during the lactation period. There were no effects of the test item on F0 pup physical and reflex development nor on F1 pup physical development.

In F1 generation, no effects were reported on male and female sexual development landmarks. In addition, there were no effects on mean acoustic startle reflex test results and all animals were positive for the pupil constriction reflex at 4 weeks of age (reflex development). There were no effects on mean number of horizontal movements and rearing (motor activity).

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.

In conclusion, 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). 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).

Short description of key information:
For the purpose of notification in China, a two-generation study (OECD 416, GLP compliant) was performed in order to provide information of the effects of the test substance 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.
In 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).

Justification for selection of Effect on fertility via oral route:
The only one two-generation study available (key study)

Effects on developmental toxicity

Description of key information

The test item, Rhodiasolv Polarclean, was administered by gavage, once daily, from days 6 to 20 post-coitum, inclusive, to mated female Sprague-Dawley rats at dosages of 100, 300 or 1000 mg/kg/day in a GLP-compliant study in accordance with OECD Guideline 414.
On the basis of the results obtained in this study:
-  the No Observed Effect Level (NOEL) for maternal parameters was considered to be 1000 mg/kg/day,
-  the NOEL for embryo-fetal development was considered to be 1000 mg/kg/day.
The test item, Rhodiasolv Polarclean, did not elicit any teratogenic potential.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 Aug 2013 to 24 Jan 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study conducted according to OECD guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy
- Age at study initiation: 10/11 weeks old
- Weight at study initiation: mean body weight of 252 g
- Fasting period before study: no
- Housing: The animals were individually housed in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing autoclaved sawdust (SICSA, Alfortville, France), was placed under each cage. Each cage contained an object for enrichment of the environment for the rats (rat hut).
- Diet: SSNIFF R/M-H pelleted maintenance diet ad libitum
- Water: tap water ad libitum
- Acclimation period: 4 or 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 13 Sep 2012 To: 11 Oct 2012

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. The test item dose formulations were prepared for up to 7 days (10 days is the maximum
authorized: Stability Results from Rhodia SAS project No. 41005298, 14-day Dose Range Finding toxicity study), stored at +4°C "prior-to-use", protected from light and delivered in brown flasks.

ADMINISTRATION OF DOSING SOLUTIONS:
The dose formulations 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 dose formulation 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method applied consisted of sampling 1 mL of dose formulations and diluting it appropriately with diluent to reach the nominal concentration of injection. The diluted samples were analyzed by High Performance Liquid Chromatography with Ultra-Violet detection (HPLC-UV), bracketed by standard solutions and quantified by the mean response factors calculated for the standard solutions.

Details on mating procedure:

The females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as day 0 post-coitum (p.c.).

Duration of treatment / exposure:

From day 6 to day 20 p.c., inclusive.

Frequency of treatment:

daily

Duration of test:

see above

No. of animals per sex per dose:

24 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The dose-levels were selected in agreement with the Sponsor, following the results of a previous study OECD 422 study conducted in Wistar rats (Rhodia SAS project No. 41005299). In this OECD 422 study, there were no toxicologically significant effects up to 1000 mg/kg/day. Therefore, 1000 mg/kg/day was selected as the high dose-level for this study. Mid and low dose-levels were selected in order to cover approximately 3-fold intervals

Maternal 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: From arrival, each animal was observed once a day as part of the routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: on days 2, 4, 6, 9, 12, 15, 18 and 21 p.c..

FOOD CONSUMPTION: Yes
- The quantity of food consumed by each female was recorded for the following intervals: days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c..

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: Females were submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
- number and distribution of uterine scars
- number and distribution of dead and live fetuses.
- A gross evaluation of placentas was also undertaken.

Fetal examinations:

- External examinations (included the observation of all visible structures, surfaces and orifices): Yes: [all per litter]
- Soft tissue examinations (included the observation of all the organs and structures of the neck, thorax and abdomen): Yes: [half per litter]
- Skeletal examinations (included the observation of all the bones and cartilage structures of the head, spine, rib cage, pelvis and limbs): Yes: [half per litter]
- Head examinations: Yes: [ half per litter]

Others:
- The body weight of each live fetus was recorded.
- The sex of each fetus was determined at the time of hysterectomy.The sex of fetuses was determined by visual assessment of anogenital distance and was confirmed by examination of sexual organs at detailed dissection of the soft tissues or at evisceration.

Statistics:

Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by Fisher exact probability test.

Indices:

The following parameters will be calculated:

For each pregnant female:
 body weight change for different intervals,

 net body weight (presented as carcass weight):
Body weight on day 21 p.c. - gravid uterine weight

 net body weight change:
Body weight on day 21 p.c. - body weight on day 6 p.c. - gravid uterine weight

For each litter:
 total number of resorptions:
Sum of scars + early resorptions + late resorptions

 total number of dead fetuses:
Sum of dead males + dead females + undefined dead fetuses

 % of dead fetuses per litter:
Total number of dead fetuses
__________________________x 100
Number of implantation sites

 total number of live fetuses:
Sum of live male + live female fetuses

 % of live fetuses per litter:
Total number of live fetuses
__________________________x 100
Number of implantation sites

 % of pre-implantation loss:
Number of corpora lutea - Number of implantations
_______________________________________________x 100
Number of corpora lutea

 % of post-implantation loss:
Number of implantation sites - Number of live fetuses
________________________________________________x 100
Number of implantation sites

 average fetal body weight:
Sum of individual fetal weights
___________________________
Number of fetuses

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

The test item concentrations in the administered dose formulations analyzed in weeks 1 and 3 remained within an acceptable range of variation (+1.7% to +2.5%) when compared to the nominal values.

Pregnancy status:

At termination on day 21 p.c., there were 23, 22, 21 and 21 dams with live fetuses in the vehicle control, 100, 300 and 1000 mg/kg/day groups, respectively.

Mortality:

There were no unscheduled deaths.

Clinical signs:

There were no test item treatment-related findings. The clinical signs recorded (reddish vaginal discharge and cutaneous lesion) are commonly

observed in this species and strain of rat.

Body weight, body weight change and food consumption:

There were no effects on mean body weights, mean body weight changes and mean food consumptions.

Necropsy and hysterectomy data:

There were no test item treatment-related findings at necropsy. There were a few findings commonly observed in this species and strain of rat which concerned:

- in the 100 mg/kg/day group, two pregnant females with a reddish content in vagina or a blackish content in uterus, and one non-pregnant female (A20204) with a bilateral dilatation and serous content of uterine horns,

- in the 1000 mg/kg/day, one pregnant female with a brownish content in vagina.

There were no effects on mean hysterectomy data (pre- and post-implantation losses and early and late resorptions).

Fetal examination:

There were no effects of the treatment with the test item on mean fetal body weights or fetal sex ratios.

- External examination: there were no external variations or malformations.

- Soft tissues examination: there were no soft tissue variations considered to be related to treatment with the test item; there were no soft tissue malformations. In the 300 mg/kg/day group, one litter had a fetus with a hemorrhagic eye. This variation was isolated and recorded in one fetus only.

- Skeletal examination: there were no cartilage abnormalities, skeletal variations or malformations considered to be related to treatment with the test item. In the 1000 mg/kg/day group, 1/21 litter had a fetus with bipartite cartilage of thoracic vertebra(e). This finding was isolated and considered to be of no toxicological significance in the absence of associated variations or malformations. In the 1000 mg/kg/day group, two litters had a fetus with absent lumbar vertebra(e). This malformation was isolated and recorded in the Historical Control Data (two fetus from 2/141 litters) and therefore considered not to be related to the treatment with the test item.

Conclusions:

The test item, Rhodiasolv Polarclean, was administered by gavage, once daily, from days 6 to 20 post-coitum, inclusive, to mated female Sprague-Dawley rats at dosages of 100, 300 or 1000 mg/kg/day in a GLP-compliant study in accordance with OECD Guideline 414.
On the basis of the results obtained in this study:
- the No Observed Effect Level (NOEL) for maternal parameters was considered to be 1000 mg/kg/day,
- the NOEL for embryo-fetal development was considered to be 1000 mg/kg/day.
The test item, Rhodiasolv Polarclean, did not elicit any teratogenic potential.

Based on the results of this study, the test substance Rhodiasolv Polarclean is not classified for developmental toxicity / teratogenicity according to the classification criteria of the Regulation (EC) 1272/2008 (CLP) and of the Directive 67/548/EEC.

Executive summary:

The potential toxic effects of the test item, Rhodiasolv Polarclean, on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (day 6 to day 20 post-coitum inclusive) was investigated in a GLP-compliant study in accordance with OECD Guideline 414.

In this study, three groups of 24 mated Sprague-Dawley rats were administered the test item, Rhodiasolv Polarclean (batch No. CY12018030), once daily from day 6 to day 20 post-coitum, by gavage, at dosages of 100, 300 or 1000 mg/kg/day. An additional group of 24 mated females received the vehicle, drinking water obtained by reverse osmosis, under the same experimental conditions and acted as the control group. A dose volume of 5 mL/kg/day was used. The animals were checked daily for mortality and/or clinical signs. Body weight and food consumption were recorded at designated intervals. On day 21 post-coitum (p.c.), females were sacrificed and submitted to macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities.

The test item concentrations in the administered dose formulations analyzed in weeks 1 and 3 remained within an acceptable range of variation (+1.7% to +2.5%) when compared to the nominal values.

At termination on day 21 p.c., there were 23, 22, 21 and 21 dams with live fetuses in the vehicle control, 100, 300 and 1000 mg/kg/day groups, respectively.

There were no unscheduled deaths and no test item treatment-related clinical signs.

There were no effects on mean body weights, mean body weight changes and mean food consumptions.

There were no test item treatment-related findings at necropsy.

There were no effects on mean hysterectomy data (pre- and post-implantation losses and early and late resorptions).

There were no effects of the treatment with the test item on mean fetal body weights or fetal sex ratios were noted at fetal examination: There were no external variations or malformations. There were no soft tissue variations considered to be related to treatment with the test item; there were no soft tissue malformations. There were no cartilage abnormalities, skeletal variations or malformations considered to be related to treatment with the test item.

In conclusion, on the basis of these results:

- the No Observed Effect Level (NOEL) for maternal parameters was considered to be 1000 mg/kg/day,

- the NOEL for embryo-fetal development was considered to be 1000 mg/kg/day.

The test item, Rhodiasolv Polarclean, did not elicit any teratogenic potential.

Based on the results of this study, the test substance Rhodiasolv Polarclean is not classified for developmental toxicity / teratogenicity according to the classification criteria of the Regulation (EC) 1272/2008 (CLP) and of the Directive 67/548/EEC.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and OECD 414 compliant study ( Klimisch 1)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

One developmental toxicity study of reliability 1 according to Klimisch cotation criteria is available and was selected as key study (Spezia, 2013 (OECD 414, oral route)).

In this study, three groups of 24 mated Sprague-Dawley rats were administered the test item, Rhodiasolv Polarclean, once daily from day 6 to day 20 post-coitum, by gavage, at dosages of 100, 300 or 1000 mg/kg/day. An additional group of 24 mated females received the vehicle, drinking water obtained by reverse osmosis, under the same experimental conditions and acted as the control group.

At termination on day 21 p.c., there were 23, 22, 21 and 21 dams with live fetuses in the vehicle control, 100, 300 and 1000 mg/kg/day groups, respectively.

There were no unscheduled deaths and no test item treatment-related clinical signs.There were no effects on mean body weights, mean body weight changes and mean food consumptions.There were no test item treatment-related findings at necropsy.

There were no effects on mean hysterectomy data (pre- and post-implantation losses and early and late resorptions).

There were no effects of the treatment with the test item on mean fetal body weights or fetal sex ratios were noted at fetal examination: There were no external variations or malformations. There were no soft tissue variations considered to be related to treatment with the test item; there were no soft tissue malformations. There were no cartilage abnormalities, skeletal variations or malformations considered to be related to treatment with the test item.

In conclusion, on the basis of these results:

- the No Observed Effect Level (NOEL) for maternal parameters was considered to be 1000 mg/kg/day,

- the NOEL for embryo-fetal development was considered to be 1000 mg/kg/day.

The test item, Rhodiasolv Polarclean, did not elicit any teratogenic potential.

Justification for selection of Effect on developmental toxicity: via oral route:
Only one developmental toxicity study available on the substance (key study)

Toxicity to reproduction: other studies

Additional information

One repeated dose toxicity study of reliability 1 according to Klimisch cotation criteria was performed prior to the two-generation study and was selected as supporting study (McRae LA and Watson P., 2012 (OECD 422, oral route)).

In this study,Rhodiasolv Polarclean was administered once daily orally (by gavage) tothree groups, each of ten male and ten female Wistar Han rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 30, 300 and 1000 mg/kg bw/day. An additional group of control animals received the vehicle alone (purified water).

There were no deaths during the study. Clinical findings were confined to transient episodes of increased salivation, in one 300 mg/kg bw/day and among animals of either sex at 1000 mg/kg bw/day. There were no treatment-related changes in the behavioural parameters measured, functional performance or sensory reactivity.Weight development in all test animals was comparable to that of the controls throughout the treatment period. No adverse effects on food consumption, food efficiency or water consumption were detected in test animals in comparison with controls. There was no evidence of treatment-related changes in the haematological parameters measured and no convincing treatment-related adverse effects identified for the biochemistry profile. There were no macroscopic abnormalities detected. An increase in absolute and relative liver weight was identified in 1000 mg/kg bw/day males. Females at this dose level showed an increase in absolute and relative kidney weight in comparison with controls. Individual values all fell the historical ranges for rats of the age and strain used. Treatment related microscopic findings were confined to minimal to slight centrilobular hepatocyte hypertrophy in four out of five males receiving 1000 mg/kg bw/day. None of these changes were considered to be toxicologically significant, therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg/day. No treatment-related systemic toxicity was detected in either sex at 30 and 300 mg/kg bw/day, therefore the ‘No Observed Effect Level’ (NOEL) for systemic toxicity was considered to be 300 mg/kg/day.

There were no treatment-related effects on mating, conception rate or gestational length in animals of either sex treated with 30, 300 or 1000 mg/kg bw/day. Of the litters born, litter size at birth and subsequently on Day 1 and Day 4post partumas well as offspring body weight gain and litter weights were comparable to controls. No adverse effects were detected in surface righting reflex on Day 1. No effect on the reproductive organs of parental animals was detected during post mortem or histopathological assessments. There was no evidence of any developmental effects observed in offspring from treated litters. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was therefore considered to be 1000 mg/kg/day.

Justification for classification or non-classification

Based on the absence of any effectson 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 offspringreported in a rat OECD 416 study and the absence of any effect on embryo-fetal development in a rat OECD 414 study (NOAEL = 1000 mg/kg/day in both studies), the test substance Rhodiasolv Polarclean is not classified for reproductive toxicity according to the classification criteria of the Regulation (EC) 1272/2008 (CLP) and of the Directive 67/548/EEC.