Registration Dossier

Diss Factsheets

Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
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
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

Administration / exposure

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

Examinations

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

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

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.