Dimethyl carbonate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Apr 2016 - 5 Dec 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In a decision (31 Aug 2015) referring to the REACH-regulation, European Chemicals Agency (ECHA) has prescribed the performance of a Pre-natal developmental toxicity study (test method: EU B.31./OECD 414) in rabbits, oral route.

The objective of the study is detecting adverse effects on pregnant female rabbits and on the development of the conceptuses consequent to exposure of the female with dimethyl carbonate by oral administration from gestation day 6 to 27.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2016

Reference 2

Reference Type:

publication

Title:

Historical control data on prenatal developmental toxicity studies in rabbits.

Author:

Ema, Makato et al.

Year:

2012

Bibliographic source:

Congenit Anom. 52:155-61. doi: 10.1111/j.1741-4520.2012.00365.x.

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

This study was performed on the basis of:
− OECD Guidelines for Testing of Chemicals No. 414, 22 January 2001 (1).
− OECD Guidance No. 43 on Mammalian Reproductive Toxicity Testing and Assessment, 24th July 2008 (2).
− Regulation (EC) No. 440/2008, B.31. Prenatal developmental toxicity study, 30 May 2008 (3).

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl carbonate

EC Number:

210-478-4

EC Name:

Dimethyl carbonate

Cas Number:

616-38-6

Molecular formula:

C3H6O3

IUPAC Name:

dimethyl carbonate

Test material form:

liquid

Specific details on test material used for the study:

Chemical name: dimethyl carbonate
CAS number: 616-38-6
EC number: 210-478-4
Batch no.: PC 12321
Purity: 99.91 % (GC)
Production date: September 2015
Expiry date: September 2018

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Test animals:
Supplier: S & K-LAP Kft., Császár út 135, 2173 Kartal, HUNGARY
Justification of strain: New Zealand White rabbit as a non-rodent is one of the standard strains for embryo-fetal development studies.
Number of animals arrived: 88+4 reserve females
Age: young, healthy and breeding mature rabbits
Body weight range at insemination: 3616-4394 g
Acclimatisation period: 6 days
Hygienic level during the study: Good conventional

Husbandry of test animals:
Animal health: Only animals in an acceptable health condition were used for the test. The breeder/supplier certified the healthy status.
Animal house: 2120 Dunakeszi, Pálya u. 2., E Building, Animal room: 6
Housing: Animals were housed individually in metal cages
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 18 - 22 °C (one occasion 2nd May, 2016, 22 °C)
Relative humidity: 38 - 74 % (one occasion May 02, 2016 100%*)

* = Reason for high humidity and temperature: failure of the air-conditioning system.
The environmental parameters were recorded daily during the study.

Food and Feeding:
Diet: C.HYF separate rabbit diet mixture produced by Cargill Takármany Zrt., 5300 Karcag, Madarasi út 0399, ad libitum
The supplier provided an analytical certificate of the standard diet for the batch used.
Animals received tap water from water bottles ad libitum

Identification of individual animals:
Individual animal identification of the rabbits was performed by numbered ear tag by the breeder. The cages were marked with individual identity cards with information about study number, sex, dose group, date of insemination, date of Caesarian section, cage number and individual animal number.

Identification of litters:
At necropsy the litters were identified with the litter numbers. The flasks used for fixation and all sheets used for recording beared these numbers only up to the end of fetal examinations (blind examination of fetuses).

Identification of fetuses:
In the course of Caesarean section the fetuses were identified individually by the litter number by means of water-proof plastics. The heads from the half of each litter were removed and identified by ear punching.
For skeletal examination the fetuses were identified by means of water-proof plastic ribbon tied around their waist.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Aqua purificata Ph. Hg. VIII.; batch number: 1601-5508 and 1602-5519; expiry dates: July 08, 2016 and August 19, 2016; supplier: Parma Produkt Kft.; storage: at room temperature

Details on exposure:

Rationale for route of administration:
Oral administration of the test item by gavage is an accepted method for treatment of rabbits and is a possible route for human exposure. Furthermore, the choice is also in line with the by ECHA requested information.

Treatment volume:
The treatment volume was 10 ml/kg body weight. The actual volume of administration was adjusted for each animal based on the most recent body weight measured.

Treatment period:
The inseminated females were treated daily from the 6th up to and including the 27th day of gestation. Dosing was completed in the morning hours, at approximately the same time each day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the dosing solutions in the main study, the concentrations have been verified using GC-FID.
The concentrations were in the range 92 - 99 % of the nominal concentrations.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Information from the Validation Study (Study No. 864-100-1282, Toxi-Coop ZRT., September 01, 2016) where the analytical methodolgy was validated:
Specificity: No interfering substance was detected at the retention time of the test item and the internal standard.
Repeatability (7 replicates): CV% ≤ 4.0 %
Linear range: 0.1 – 5 mg/mL
Limit of Quantification: 0.1 mg/mL
Recovery from water: 101% at 1 mg/mL concentration level, 100% at 100 mg/mL concentration level
Stability in water: at least 8 days at room temperature, at least 8 days in refrigerator
Stock solution stability: at least 8 days at 5 +/- 3 °C
Stability in the autosampler: at least 23 hours

Details on mating procedure:

Insemination procedure:
Day of insemination was regarded as day 0 of gestation.
Synchronization of the cycle was completed 48 hours prior to insemination by administering PMSG (gonadotropin) hormone subcutaneously into the neck region. The insemination procedure was performed by the breeder. Each female was given diluted sperm containing at least 2 million spermatozoa and 1 ml Receptal hormone preparation with intramuscular injection into the femur region to provoke ovulation. The sperm originated from New Zealand White male rabbits from the same source as the females.

Duration of treatment / exposure:

The inseminated females were treated daily from the 6th up to and including the 27th day of gestation. Dosing was completed in the morning hours, at approximately the same time each day.

Frequency of treatment:

The inseminated females were treated daily from the 6th up to and including the 27th day of gestation.

Duration of test:

The inseminated females were treated daily from the 6th up to and including the 27th day of gestation
Before expected delivery, on the gestation day 28, a Caesarean section was performed on each doe. Euthanasia of the animals was executed by lethal injection of Release® administrated intravenously.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control, water (vehicle) only; number of inseminated females: 23

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low, administered at 10 mg/mL; number of inseminated females: 22

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Mid, administered at 30 mg/mL; number of inseminated females: 22

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High, administered at 100 mg/mL; number of inseminated females: 23

Control animals:

yes

Details on study design:

Randomisation:
Females were randomly assigned to dose groups on the basis of their body weight on the day of insemination in such a way that the group averages of the body weight were as similar as possible on the first day (day 0) of gestation.

Rationale for dose level selection:
The dose levels were selected by the Sponsor with the highest dose level at 1000 mg/kg b.w./day (limit dose according to OECD TG 414) based on the results of the Dose Range Finding Prenatal Developmental Toxicity Study of Dimethyl Carbonate in Rabbits by Oral Administration (Study number 864-410-1280; Toxi-Coop Zrt.).

Examinations

Maternal examinations:

During the study animals were checked for mortality and clinical signs.
Body weight and food consumption of the does were also recorded.

The day of insemination was regarded as day 0 of gestation. A Caesarean section and gross pathology were performed on gestational day 28.
Euthanasia by Release® administrated intravenously. Organs of the does were examined macroscopically incl. viscera.

Organs with pathological changes which could not be diagnosed macroscopically were fixed in 4 % neutral formaldehyde solution. Corresponding organs from control animals were kept for comparison. No microscopic examination of the retained tissues was performed in the absence of any treatment-related effects.

Ovaries and uterine content:

The ovaries and uterus were removed and the uterus of the pregnant females was weighed. Uterus of each female was examined for early, late embryonic and fetal death and for the number of live fetuses. The number of corpora lutea per ovaries were counted. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded.

Fetal examinations:

Euthanasia of the viable fetuses was made by subcutaneous injection of Release®. Live fetuses and their placentas were weighed individually (litter mean was calculated). The crown-rump length of fetuses was measured (accuracy 1 mm) (litter mean was calculated). Fetuses and their placentas were examined externally.

Visceral examination:
Fresh visceral examination of each fetus was performed. Head of about 50 % of each litter was removed and fixed in modified Sanomiya solution and washed in 90 % isopropanol for Wilson-sections. Examination of the heads was done by Wilson's free-hand razor blade method.
Viscera of all fetuses were examined (including gender determination) freshly by a microscope. The head of ca. 50% of the fetuses were examined by Wilson's free-hand razor blade method after fixation. After cartilage-bone staining all skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

Each fetus was weighed and examined for external abnormalities. The placentas were weighed and examined externally.

Statistics:

Data evaluation:Data were individually recorded on data sheets, transferred, and compiled by computer or compiled manually.
The parameters to be evaluated are listed below. Means and standard deviations and/or percentages were calculated. Statistical analysis was performed with SPSS PC+ software. The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance was carried out. If the obtained result is positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.

Where significant heterogeneity is found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there is a positive result, the inter-group comparisons are performed using the Mann-Whitney U-test. Chi2 test was performed if feasible.

Does or litters were excluded from the data evaluation in cases of:

- A death of the doe unrelated to the treatment (total exclusion)
- Non pregnant females i.e. females with no implantation and no corpora lutea (total exclusion)
- Body weight, body weight gain and food consumption of does with complete post implantation loss or of does with less than 2 implantations independent of their viability (partial exclusion)
- Body weight, body weight gain, food consumption, clinical signs and necropsy findings of females with no implantation but corpora lutea (partial exclusion)
- Circumstances unrelated to the test item which are considered to be reason for exclusion, at the discretion of the study director

Study report contains all data of these animals although not used for data evaluation.
Fetus was considered retarded in body weight/crown-rump length, when weight/crown-rump length was below average minus two-fold standard deviation of all control fetuses (24.23 g and 8.36 cm).

Historical control data:

The malformations found at external, visceral and skeletal examination were judged to be incidental based on the historical control data of Toxi-Coop Zrt. (Appendix XXV to study report) and on historical control data of New Zealand White rabbits and Japanese White rabbits (a strain with similar control data) of other laboratories published in scientific literature (Ema, Makato et al. (2012): Historical control data on prenatal developmental toxicity studies in rabbits. Congenit Anom. 52:155-61. doi: 10.1111/j.1741-4520.2012.00365.x.).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related mortality, clinical signs or necropsy findings during the in-life phase.
Clinical signs such as dispnoea and whitish secretum at the snout area in one control animal (necropsy revealed brownish hard swelling in two lung lobes) and snuffle breathing in one female in the 100 mg/kg bw/day group from gestation day 22 and 24 respectively up to the end of the in-life phase up to (whitish yellowish formations in the lungs were recorded at necropsy). Also whitish and/or yellowish formations with pin-prick or pin-head sized haemorrhages in the lungs were found in the lungs in another female in the 100 and in one in the 300 mg/kg bw/day group. There were no macroscopic changes found in the high dose group at necropsy.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no treatment-related mortality, clinical signs or necropsy findings during the in-life phase.
One female died in the control group on gestational day 27. Bloody snout and forelimb, secretum around the nose were observed on the day before. Dark red lungs and whitish secretum on the lung surface was recorded at necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Summary body weight, food consumption:
The food consumption and body weight of the maternal animals was not influenced by the treatment.

There were no statistically significant differences indicated in the body weight or corrected body weight values except the body weight gain was statistically significantly higher in the 1000 mg/kg bw/day dose group between gestation days 24 and 27. This was considered unrelated from the treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption of the does was similar in all groups in the different periods of the study.

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One female aborted in the 100 (No: 4710) and one in the 1000 mg/kg bw/day group (No.: 5229).
This occurred between gestations days 18 and 20. Both had vaginal orifice and clotted blood pieces or blood smear was found in the cages. There were no macroscopic changes observed in these females.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of corpora lutea, implantations, viable fetuses, pre- and postimplantation loss was similar in all groups.
There was a statistically significant difference indicated in the sex ratio of viable fetuses (both sexes (p<0.05)) in the 1000 mg/kg bw/day dose group. This was judged to be incidental, considering that the treatment started from implantation and the mean mortality rate of the conceptuses was similar in the experimental groups.

Does with total post implantation loss (total intrauterine mortality) (partial exclusion, body weight, body weight gain and food consumption excluded) of conceptuses:

Control group: No.: 4009
100 mg/kg bw/day dose group: none
300 mg/kg bw/day dose group: none
1000 mg/kg bw/day dose group: none

Doe died unrelated to the treatment (total exclusion):
Control group: No.: 4715

One female aborted in the 100 (No: 4710) and one in the 1000 mg/kg bw/day group (No.: 5229).

Hence, the number of evaluated litters was 66 (17 each in the control, 100, 300 and 15 in the 1000 mg/kg bw/day dose groups respectively).

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

The number of inseminated females was 90 in the study. The number of females with implantation was 19, 18, 17 and 16 in the control, 100, 300 and 1000 mg/kg bw/day respectively.

The following females were excluded from the data evaluation:
Circumstances unrelated to treatment which were considered to be reason for exclusion, at the discretion of the study director:
- Aggressive behave: Control group: 4023
- Swollen leg: 1000 mg/kg bw/day dose group: 5254

Non pregnant females i.e. females with no implantation and no corpora lutea (total exclusion):
No corpora lutea and no implantation:
Control group: No.: 5249
100 mg/kg bw/day dose group: No.: 4732, 5275, 4741
300 mg/kg bw/day dose group: No.: 4719, 5257, 4699, 5248, 4697
1000 mg/kg bw/day dose group: No.: 5241, 4001, 5239, 4017, 5259

Females with no implantation but corpora lutea (partial exclusion; body weight, body weight gain, food consumption, clinical signs and necropsy findings excluded):
Control group: No.: 5266, 4016
100 mg/kg bw/day dose group: 4010
300 mg/kg bw/day dose group: No.: none
1000 mg/kg bw/day dose group: No.: 4706

Other effects:

no effects observed

Description (incidence and severity):

The mean body weight, crown rump length of the fetuses as well as the weight and relative weight of the placentas was similar in the groups.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: findings in general

Remarks on result:

other: Substance caused no mortality, clinical signs and macroscopic alterations revealed at necropsy, no effects on the food consumption and body weight of the maternal animals. Substance was judged not to increase the pre- and post-implantation mortality.

Results (fetuses)

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The mean body weight, crown rump length of the fetuses as well as the weight and relative weight of the placentas was similar in the groups.

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically significant difference indicated in the sex ratio of viable fetuses (both sexes (p<0.05)) in the 1000 mg/kg bw/day dose group. This was judged to be incidental, considering that the treatment started from implantation and the mean mortality rate of the conceptuses was similar in the experimental groups.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Summary Malformations:
There was no dose response indicated in the number of litters with malformed fetuses as well as no significant increase in the fetal incidence of malformations.
The malformations found at external, visceral and skeletal examination were judged to be incidental based on the historical control data of Toxi-Coop Zrt. and on historical control data of New Zealand White rabbits and Japanese White rabbits (a strain with similar control data) of other laboratories published in scientific literature (Ema, Makato et al. (2012): Historical control data on prenatal developmental toxicity studies in rabbits. Congenit Anom. 52:155-61. doi: 10.1111/j.1741-4520.2012.00365.x.).

The incidence of litters with malformed fetuses was five in the control, six both in the 100 and 1000 mg/kg bw/day and 3 in the 300 mg/kg bw/day dose group.

Summary Variations: There was no increase indicated in the incidence of external and visceral variations. The incidence of skeletal abnormalities increased statistically significantly (p<0.05) in the 1000 mg/kg bw/day dose group due to the skeletal variations. However considering that the increases of these variations (bipartite sternebra, unossified metacarpal of pollex and less than 7/7 ossified proximal and medial phalanges) were slight and in most of the cases just partially significant statistically (either per fetal basis or on litter basis) and the values were around or within the historical control level this change was considered as non-adverse.

Details External Examination:
Malformations:
One fetus was observed with encephalocele, open eye (bilateral) and hiperflexion of the forelimb (not confirmed by skeletal examination) in the 1000 mg/kg bw/day dose group. These malformations occur sporadically with low incidence unrelated to the treatment according to the experience with this species in this laboratory. Malrotated limb is included in the historical control data of Toxi-Coop Zrt. (Appendix XXV). Similar head malformations like exencephaly or chranioschisis as well as open eye occur sporadically with low incidences in New Zealand White rabbits as well as in Japanese White rabbits another strain with similar background data according to scientific literature (Ema et al., 2012) (10). Moreover considering that this was a single fetus found with external malformations it was judged to have no relationship with the test item.

Variations:
There were no dose-related differences in the incidence of growth retarded fetuses (crown rump length below 8.4 cm and body weight under 24.2g). Slightly shorter maxilla and slightly protruding tongue of one fetus in the 1000 mg/kg bw/day dose group was categorized as a variation. Considering the low incidence this was considered to have no relationship to the treatment.

Placental abnormalities:
One placenta was found with an absent lobe in the 100 mg/kg bw/day dose group without a relationship to the treatment.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Summary Malformations
There was no dose response indicated in the number of litters with malformed fetuses as well as no significant increase in the fetal incidence of malformations.

The malformations found at external, visceral and skeletal examination were judged to be incidental based on the historical control data of Toxi-Coop Zrt. and on historical control data of New Zealand White rabbits and Japanese White rabbits (a strain with similar control data) of other laboratories published in scientific literature (Ema, Makato et al. (2012): Historical control data on prenatal developmental toxicity studies in rabbits. Congenit Anom. 52:155-61. doi: 10.1111/j.1741-4520.2012.00365.x.).

The incidence of litters with malformed fetuses was five in the control, six both in the 100 and 1000 mg/kg bw/day and 3 in the 300 mg/kg bw/day dose group.

Summary Variations: There was no increase indicated in the incidence of external and visceral variations. The incidence of skeletal abnormalities increased statistically significantly (p<0.05) in the 1000 mg/kg bw/day dose group due to the skeletal variations. However considering that the increases of these variations (bipartite sternebra, unossified metacarpal of pollex and less than 7/7 ossified proximal and medial phalanges) were slight and in most of the cases just partially significant statistically (either per fetal basis or on litter basis) and the values were around or within the historical control level this change was considered as non-adverse.

Details Skeletal Malformations:
Malformations:
Skeletal malformations such as misshapen, split, fused and/or misaligned sternebra with or without misaligned costal cartilage, split xiphoid cartilage, fused ribs with or without common origin, misshapen 13th rib anlage, bifurcate rib with enlarged transverse process of the vertebra, dumb-bell shaped or bipartite thoracic centrum were found with low incidence, sporadically distributed among the experimental groups and partially also in the control group. There was one fetus found with multiple malformed skeleton in the 300 mg/kg bw/day dose group (Vertebrae: atlas misshapen, dorsal; C V asymmetric bipartite; C VI hemicentric; Th III asymmetric; Th IV asymmetric bipartite; Th III- VII misaligned; 'Th V asymmetric, hemicentric;Th III- VIII misshapen; Th VI and Th VIII hemicentric; Th X misshapen; Sternebrae: 5; 5th -0; 3rd and 4th misaligned ossification and fused; Costae: 12; 3rd and 4th common origin, bilateral; from 6th to 8th common origin, right; from 5th to 7th common origin, left; 7th and 8th fused).

One fetus was found with multiple malformed vertebrae in the 100 mg/kg bw/day dose group (Th VI- IX misaligned, slight; Th VII bipartite, asymmetric; Th VIII hemicentric, left centrum absent or common with Th VII left; Th IX asymmetric dumb-bell shaped (including cartilage)) and one in the high dose group (Th IX displaced, dumb-bell shaped, cartilage misshapen; Th X hemicentric, small (including cartilage), fused with Th XI). The same type or similar malformations occur spontaneously in New Zealand White rabbits according to the Background data of Toxi-Coop Zrt. and to scientific literature (10). Moreover rib (bifurcate, multiple malformed) and vertebral malformations (hemicentric, misshapen) occurred also in the control group in the Prenatal Developmental Toxicity Study of Dimethyl Carbonate in Rabbits by Oral Administration (864-410-1280). Hence, the occurrence of these abnormalities was attributed as no effect of the test item.

Variations:
Incomplete and irregular ossification of the skull bones, slightly enlarged anterior fontanel, bipartite, misaligned (with or withour slightly misaligned costal cartilage), dumb-bell shaped ossification of sternebra, extra ossification centrum, incomplete ossification of the sternum (if less than 5 sternebra ossified), short or/and interrupted 13th rib, bipartite, dumb-bell shaped or asymmetric thoracic vertebral centra, asymmetric pelvic articulation (lumbar/sacral), less than 13 ossified caudal vertebra, incompletely or not ossified pubic bone, talus, small calcaneus, less than 4 ossified metacarpal, unossified pollex metacarpal, less than 7/7 proximal and middle phalanges ossified, incomplete or asymmetric ossification were observed as variations during the skeletal examination.
The incidence of skeletal abnormalities was significantly (p<0.05) increased in the 1000 mg/kg bw/day dose group due to the skeletal variations. Most of the different type of these variations were found with low incidences or were at the actual control level.
The incidence of litters with fetuses with bipartite sternebra (p<0.05) was statistically significantly increased in the 1000 mg/kg bw/day dose group. However this slight increase was not significant if the fetal incidence was evaluated and was around the historical control level (Appendix XXV). There was also a statistical significant increase in the unossified pollex metacarpal ((p<0.05) in the 300 and (p<0.01) in the 1000 mg/kg bw/day group) if evaluated on a fetal basis and not significant in case of the 300 as well as p<0.05 in the 1000 mg/kg bw/day group if evaluated on litter basis. The incidence was within the historical control level (Appendix XXV). There was a statistical significance (p<0.05) indicated in the litter however not the fetal incidence of slightly increased less than 7/7 proximal- and middle phalanges. Considering that the increases of these minor variations were slight and the values were around or within the historical control level this change was considered as non-adverse.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Summary Malformations
There was no dose response indicated in the number of litters with malformed fetuses as well as no significant increase in the fetal incidence of malformations.
The malformations found at external, visceral and skeletal examination were judged to be incidental based on the historical control data of Toxi-Coop Zrt. and on historical control data of New Zealand White rabbits and Japanese White rabbits (a strain with similar control data) of other laboratories published in scientific literature (Ema, Makato et al. (2012): Historical control data on prenatal developmental toxicity studies in rabbits. Congenit Anom. 52:155-61. doi: 10.1111/j.1741-4520.2012.00365.x.).

The incidence of litters with malformed fetuses was five in the control, six both in the 100 and 1000 mg/kg bw/day and 3 in the 300 mg/kg bw/day dose group.

Summary Variations: There was no increase indicated in the incidence of external and visceral variations. The incidence of skeletal abnormalities increased statistically significantly (p<0.05) in the 1000 mg/kg bw/day dose group due to the skeletal variations. However considering that the increases of these variations (bipartite sternebra, unossified metacarpal of pollex and less than 7/7 ossified proximal and medial phalanges) were slight and in most of the cases just partially significant statistically (either per fetal basis or on litter basis) and the values were around or within the historical control level this change was considered as non-adverse.


Details on Visceral Examination:
Malformations:
Open eye and deficient vertex was recorded for the fetus found as malformed also at external examination in the 1000 mg/kg bw/day group (discussed in section 11.7.1). In the same litter one fetus was found with abnormalities of the great arteries such as absent aortic arch and arising of left subclavian artery from the pulmonary artery, and continuation of truncus pulmonalis as aorta descendens. In the 300 mg/kg bw/day group one fetus was found with markedly dilated aortic arch and aorta descendens; markedly thinned, short pulmonary trunk and ductus Botalli as well as dilated left ventricle (pseudo truncus arteriosus).

According to the scientific literature the same type of malformations (including truncus arteriosus persistent and absence of aortic arch, hypoplasia of pulmonrary opening/artery) may occur spontaneously in New Zealand White rabbit as well as in Japanese White rabbits another strain with similar background data according to scientific literature (Ema et al., 2012) (10). Considering this information and the low incidence this malformation was not attributed to the treatment of the does.

Enlarged space between the cerebral hemisphere and thalamus including misshapen thalamus was found in one high dose fetus. Considering that also three control fetuses had this abnormality it was judged to have no relationship to the treatment.

Dilated third brain ventricle was observed in one fetus each in the 100, 300 and 1000 mg/kg bw/day dose group and one fetus had also dilated lateral ventricles besides in the 1000 mg/kg bw/day group. Considering the balanced distribution among the dose groups this malformation was attributed as no effect of the test item. According to the Background data of Toxi-Coop Zrt. (Appendix XXV) the same or similar abnormalities (dilated or slightly dilated third or lateral brain ventricle, external hydrocephaly, dilated aquaductus cerebri) occur in animals unrelated to the treatment with low incidence. This is in line with scientific literature by which dilated cerebral ventricles may occur spontaneously in New Zealand White rabbit as well as in Japanese White rabbits another strain with similar background data (Ema et al., 2012).

Variations:
Brain variations such as slightly enlarged space between the cerebral hemisphere and thalamus, dilated space between the frontal lobes, slightly dilated third brain ventricle, hydro- or convoluted ureter were recorded. There were no treatment-related significant differences observed.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: findings in general

Remarks on result:

other: no increase pre- and post-implantation mortality. no effect on intrauterine development of the embryos/fetuses acc. to fetal weight, crown-rump length, external, visceral and skeletal examinations

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based upon these data, treatment of pregnant New Zealand White rabbits from gestational day 6 to 27 by oral administration of Dimethyl Carbonate, caused no mortality, clinical signs and macroscopic alterations revealed at necropsy, no effects on the food consumption and body weight of the maternal animals. Dimethyl Carbonate was judged not to increase the pre- and post-implantation mortality. Treatment of the does was considered not to influence adversely the intrauterine development of the embryos and fetuses according to the fetal weight and crown-rump length measurements as well as external, visceral and skeletal examinations.

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL maternal toxicity: 1000 mg/kg bw/day (highest administered dose)
NOAEL developmental toxicity: 1000 mg/kg bw/day (highest administered dose)