Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Referenceopen allclose all

Endpoint:
multi-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable publication with very detailed description of the conducted methods.
Principles of method if other than guideline:
chronic oral administration of the test substance with regard to offspring development.
GLP compliance:
not specified
Species:
rat
Strain:
other: Wistar-Stamm II BR 46
Sex:
male/female
Route of administration:
oral: drinking water
Vehicle:
water
Details on mating procedure:
Preliminary mating occurred during 20 days (daily changing of sexual partners) with 10 males and 10 females from two groups (treated with 0.015 % test substance, and control without test substance)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
100 weeks
Remarks:
Doses / Concentrations:
0.0 % (w/v), 0.0 mg/kg bw (males, females) -> control group
0.015 % (w/v), 3 mg/day, 6.5 mg/kg bw (males), 10.7 mg/kg bw (females);
0.075 % (w/v), 10 mg/day, 20.7 mg/kg bw (males), 35.7 mg/kg bw (females);
0.3 % (w/v), 60 mg/day, 130 mg/kg bw (males), 214.3 mg/kg bw (females);
Basis:
nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Dose descriptor:
NOAEL
Effect level:
>= 130 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: histopathological examination of reproduction organs and fertility
Remarks on result:
other: Generation not specified (migrated information)
Dose descriptor:
NOAEL
Effect level:
>= 214.3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: histopathological examination of reproduction organs and fertility
Remarks on result:
other: Generation not specified (migrated information)
Dose descriptor:
NOAEL
Generation:
other: F3
Effect level:
10.7 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: weights of ovaries were slightly lower in the two higher dosage groups compared to control
Remarks on result:
other:
Remarks:
F3 generation; according to publication, however, no clear treatment-related signs of toxicity or of macroscopic or microscopic effects on the organs
Reproductive effects observed:
not specified

The only diseases related to the reproductive system in the parental animals were vulvar cancers in one female of group III (administration of 0.3 % diethylcarbonate) and one female of the control group (possibly also a tumour of the intestine). Ovarian cysts were not taken into account as these are observed frequently in rats and were therefore not regarded as relevant. Fertility was not affected in treatment groups compared to the controls in any generation.

In group III of the F3 -generation the weights of the ovaries were slightly decreased compared to the control group and group II (25.0 mg/100 g bw in group II, 0.075 %; 20.5 mg/100 g bw in group III, 0.3 %; 26.1 mg/100 g bw in control group); 10 females per group.

According to the publication no clear treatment-related signs of toxicity or of macroscopic or microscopic effects on the organs were found in any of the treated groups.

The authors of the publication summarised that there were no indications of diethyl carbonate having an organotropic effect.

Conclusions:
Rats were administered the test substance diethyl carbonate chronically at concentrations of up to 0.3 % (w/v) in drinking-water and possible effects on the reproduction system analysed. Histopathological analysis of testes and ovaries resulted in vulvar cancers in one treated female and one control female. Ovarian cysts were not taken into account as these would be frequently observed in rats. They were therefore not relevant in the opinion of the authors of the publication.

Another part of the experiment was a fertility-study over 3 generations (parent -> F1 -> F2 -> F3) in which the offsprings were always treated with the corresponding dose of the test substance of their parents. Compared to the controls the fertility of the treated animals was not affected by the test substance as stated in the publication. A lower weight of the ovaries in F3-females was observed in the highest dose group compared to the control group and the next lower treatment group (20.5 mg/100 g bw at 0.3 % versus 25.0 mg/100 g bw at 0.075 % and 26.1 mg/100 g bw in the control group). There was no statement made about the statistical significance of this data nor was it discussed in the results or discussion section. The data was only presented in a table. A concluding statement about the effect of the substance on ovary weight appears therefore not possible for this study. Other effects related to ovaries were not reported. There was no generation F4 bred with generation F3 as parents to investigate further influences on reproduction.

Date conclusive but not sufficient for classification.
Endpoint:
toxicity to reproduction
Remarks:
other: subacute: 7 hours at 5 subsequent days for 4 weeks
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No official guideline method but methodology and results well documented; no GLP
Principles of method if other than guideline:
Inhalation studies were carried out according to methods from Farbenfabriken Bayer A.G., Werk Wuppertal-Elberfeld, Germany.
GLP compliance:
no
Species:
rat
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals: male and female Wistar-II-rats (weight 160-200 g), male and female Wistar-II-SPF-rats (weight 130-150 g) from the breeder Winkelmann, Kirchborchen, Germany;
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
other: probably whole body
Vehicle:
air
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Details on methodology see under 8 Analytical methods in this IUCLID dossier:
"DIÄTHYLCARBONAT - KONZENTRATIONSBESTIMMUNG IN DER INHALATIONSLUFT NACH VERDAMPFUNG" (original German title of the study)
Diethyl carbonate - determination of concentration in inhalation air after evaporation (German title translated into English)
Author: Dipl.-Chem. A. Eben, FARBENFABRIKEN BAYER AG, INSTITUT FÜR TOXIKOLOGIE
Report Nr.: 2182
Date: 31.07.1970

Summary air analyses:
Diethyl carbonate was determined in the air being inhaled according to a modified method of H. GARSCHAGEN (Z. analyt. Chem. 241, 32, 1868; Weinberg and Keller 14, 131, 1967). The air being inhaled was conveyed through a vessel cooled down to -30 °C (absorption vessel). Determination by gas chromatography.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
7 hours at 5 subsequent days
Remarks:
Doses / Concentrations:
18.995 mg/L
Basis:
no data
average analytical concentration
No. of animals per sex per dose:
10
Control animals:
yes
Dose descriptor:
NOAEC
Effect level:
>= 18.995 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absolute and relative weights of testes and ovaries not different to control group
Remarks on result:
other:
Remarks:
18.995 mg/l was the average measured concentration. The term "NOAEC" was not used in study report. The NOAEC was spotted out by the author of the IUCLID dossier.
Dose descriptor:
other: -
Generation:
other: no F1 generation
Effect level:
0 mg/kg bw/day (nominal)
Based on:
other:
Remarks:
-
Sex:
male/female
Basis for effect level:
other: -
Remarks on result:
other:
Remarks:
-
Dose descriptor:
other: -
Generation:
other: no F2 generation
Effect level:
0 mg/kg bw/day (nominal)
Based on:
other:
Remarks:
-
Sex:
male/female
Basis for effect level:
other: -
Remarks on result:
other:
Remarks:
-
Reproductive effects observed:
not specified

Rats exposed to the test substance did not show a different behavior to the rats of the control group.

The weight gain of organs was also not significantly different in the rats treated with the test substance.

The hematological examinations did also give no evidence of a difference between rats exposed or not exposed to the substance. The measured values were within the range of normal physiological values.

 

The average activities and measured concentrations of values indicating the function of liver and kidney were also found to be the same in the test group and the control group. The values were all within the normal physiological range.

 

At the autopsy of the animals the organ weights of the treated and untreated group were not significantly different (average absolute organ weights in mg and average relative organ weights in mg/100 g of body weight; comparison of males and females separately; comparison of the different types of organs separately). Examined organs were: thyroid, heart, lung, liver, spleen, both kidneys, adrenal glands, testes, ovaries.

Absolute weight of testes:

Control 2835 mg; treated 2743 mg

Relative weight of testis (per 100 g bw):

Control 1162 mg/100g; treated 1226 mg/100g

Absolute weight of ovaries:

Control 78.1 mg; treated 70.8 mg

Relative weight of ovaries (per 100 g bw):

Control 41.0 mg/100g; treated 38.7 mg/100g

There were no significant differences between the absolute and relative weights of the reproductive systems of the animals (testes and ovaries).

Conclusions:
In respect of the 28d study it was concluded in the study report:
In male and female groups of rats, exposed to diethyl carbonate for 7 h on 5 d for 4 weeks at a concentration of 18.995 mg/l air, no significant differences were found compared to the control group in respect of behaviour, body weight gain, blood status, functionality of liver and kidneys and organ weights. There were no significant differences between the absolute and relative weights of the reproductive systems of the animals (testes and ovaries).
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
214.3 mg/kg bw/day
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
18 995 mg/m³
Additional information

Short description of key information:
Oral exposure:
oral administration of 0.015, 0.075, 0.3 % (w/v) in drinking water to rats (approximately 6.5, 20.7, 130 mg/kg/d for male rats with an average weight of 460 g; 10.7, 35.7, 214.3 mg/kg/d for female rats); no effects on the reproduction and fertility of the animals was seen in any dose group (see endpoint record 7.8.1.001 (key data) for more details)

Inhalation exposure:
Male and female rats were exposed to 18.995 mg/L of diethyl carbonate for 4 weeks (7 h/d). No effects to the reproductive organs of the animals were observed (see endpoint records 7.8.1.003 and 7.5.2.001 for more details, key data each).

Justification for selection of Effect on fertility via oral route:
the only data on fertiliy via the oral route

Justification for selection of Effect on fertility via inhalation route:
the only data on fertiliy via the inhalation route

Effects on developmental toxicity

Description of key information
In a study from Bayer (1969), diethyl carbonate was not toxic to mother animals, embryotoxic or teratogenic in the rat when exposed via drinking water ad libitum at a concentration < 1 % (10000 ppm, 956 mg/kg/day) during day 6 to 15 of gestation (see endpoint record 7.8.2.001 for more details (key study)).
However, in a publication from 1966, some effects on the weight development of organs in female rats (thyroid, adrenal glands, ovaries) may have occurred. This was not conclusively discussed in the publication. The data is not deemed to be sufficient for a classification. Conservatively, a LOAEL of 35.7 mg/kg bw was pointed out (7.8.2.002, key study).
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable publication with very detailed description of the conducted methods.
Principles of method if other than guideline:
Method: other: multigeneration toxicity study
GLP compliance:
no
Species:
rat
Strain:
other: Wistar II BR 46
Route of administration:
oral: drinking water
Vehicle:
water
Analytical verification of doses or concentrations:
not specified
Control animals:
yes, concurrent vehicle
Dose descriptor:
NOAEL
Effect level:
>= 214.3 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: other
Remarks on result:
other: -
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Dose descriptor:
LOAEL
Effect level:
35.7 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: weight of organs, F3 generation
Remarks on result:
other: -
Abnormalities:
not specified
Developmental effects observed:
not specified

In summary it is stated in the publication that the administration of the test substance in every treatment group (I-III) and every generation (F1 -F3) did not affect the fetal development nor the development of the newborns compared to the control.

In an additional experiment male and female animals from the F1 -generation of the lowest treatment group and control group (10 weeks old, average weight 230 g) were used to measure the respiration of oxygen. In both groups no differences were observed (respiratory quotient 0.82 for both groups).

In 20 weeks old F3 -animals of the two highest treatment groups (0.075, 0.3 % (w/v)) and the control group analysis of the blood sugar was conducted. The obtained values did not differ between the treatment groups and the control.

In the publication it is summarised that the histological analysis of treatment animals and control animals including weights of endocrine organs did not show any differences between treated and control animals for the generation F1 -F3.

However, for the F3 generation it is in addition referred to a table where organ weights are listed upon treatment.

There is no info about the statistical significance of this table data. This data is also not further discussed. Abnormalities in shape etc. have apparentyl not occurred.

Overview of organ weights for F3 generation:

Dose group Sex Number of animals Pituitary average weight (mg) Pituitary average weight (mg/100 g bw) Thyroid average weight (mg) Thyroid average weight (mg/100 g bw) Adrenal gland average weight (mg) Adrenal gland average weight (mg/100 g bw) Ovaries average weight (mg) Ovaries average weight (mg/100 g bw)
Control m 10 11.3 2.4 20.9 4.5 49.2 10.4 / /
0.075 % m 10 11.1 2.3 20.5 4.3 37.8 7.9 / /
0.3 % m 10 11.3 2.4 21.2 4.5 42.8 9.0 / /
Control f 10 11.8 4.0 14.7 4.9 64.3 21.7 77.1 26.1
0.075 % f 10 12.1 4.2 16.7 5.8 56.7 19.5 72.3 25.0
0.3 % f 10 11.4 3.8 18.1 6.0 53.0 17.6 61.8 20.5
Control m+f 20 11.6 3.2 17.8 4.7 56.8 16.1 / /
0.075 % m+f 20 11.6 3.2 18.6 5.0 47.3 13.7 / /
0.3 % m+f 20 11.4 3.1 19.7 5.2 47.9 13.3 / /

For the author of the IUCLID dossier, it may be disputable if there was some effect of the substance on weight development for the following organs (boldly marked numbers): thyroid, adrenal gland, ovaries.

Conclusions:
In this study rats administered the test substance diethyl carbonate via drinking-water (0.015, 0.075, 0.3 % (w/v)) were mated and the resulting offsprings treated as their parents. In total three generations were bred like this and all offsprings received the corresponding parental concentration of the test substance. The authors of the publication summarised that they never found any differences in fetal development, development of the newborns, blood sugar composition, oxygen respiration and histopathological analysis, including weights of endocrine organs, in the treated groups and the control. The authors of the publication concluded that the test substance diethyl carbonate had no embryotoxic or teratogenic effects. They also pointed out that it may, however, not possible to fully exclude the possibility of teratogenicity.

For the author of the IUCLID dossier, it may be disputable if there was some effect of the substance on weight development of the following organs (boldly marked numbers in the table, in results section): thyroid, adrenal gland, ovaries. These data on the F3 generation were presented in a table of the publication but was not further discussed in respect of their significance.

Data conclusive but not sufficient for classification.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study from 1969, no official guideline but information on methodology extensive, no GLP
Principles of method if other than guideline:
Female rats received diethyl carbonate from day 6 to 15 of gestation in drinking water at various concentrations. Inspection of fetuses - inner organs and bone skeleton for abnormalities.

* "LORKE, D. : Zur Methodik der Untersuchungen embryotoxischer
und teratogener Wirkungen an der Ratte.
Arch. exper. Pathol. Pharmakol. 246, 147, 1963."

* Translated into English: LORKE, D.: About the methodology in examinations of embryotoxic and teratogenic effects in the rat. Arch. exper. Pathol. Pharmakol. 246, 147, 1963.

** "LORKE, D. : Embryotoxische Wirkungen an der Ratte.
Arch. exper. Pathol. Pharmakol. 250, 360, 1965."

** Translated into English: LORKE, D.: Embryotoxic effects in the rat. Arch. exper. Pathol. Pharmakol. 250, 360, 1965.

*** "Zur statistischen Signifikanzprüfung wurde der Rangsummentest
nach WILCOXON verwendet (WINNE, D. : Arzneimittelforschung
14, 119, 1964; DOCUMENTA GEIGY: Wissenschaftliche Tabellen,
Basel 1962). Eine Differenz wurde als signifikant bezeichnet,
wenn die Irrtumswahrscheinlichkeit kleiner als 5 % (p< 0, 05) war."

*** Translated into English: For testing the statistical significance the Wilcoxon Rank-Sum test was used (WINNE, D.: Arzneimittelforschung 14, 119, 1964; DOCUMENTA GEIGY: Scientific tables, Basel, 1962). A difference was regarded as significant when the error probability was below 5 % (p < 0.05).
GLP compliance:
no
Species:
rat
Strain:
other: FB 30
Details on test animals or test system and environmental conditions:
females:
age: 2.5 - 3.5 months
weight: 200-250 g

Keeping of animals:
kept in singles, in Makrolon cages Type II at a room temperature of 24-26 °C and an average relative humidity of 70 %.

Feed:
Altromin R-Pressfutter and tap drinking water ad libitum.
Route of administration:
oral: drinking water
Vehicle:
water
Details on mating procedure:
overnight, with males aged 3-6 months, weight 350-500 g
confirmation of insemination: vaginal smear
No. of animals per sex per dose:
10 fertilised rats
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
All mother animals tolerated the treatment well without any negative effects: they were eating well, were putting on weight, appeared to be vital and had smooth furs. All rats appeared to be healthy.
Dose descriptor:
NOAEL
Effect level:
>= 956 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Remarks on result:
other: -
Remarks:
-
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No embryotoxicity, teratogenicity or negative effect on fetal development was found at the highest dose level of 1 % in drinking water.
Dose descriptor:
NOAEL
Effect level:
>= 956 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: embryotoxicity
Remarks on result:
other: -
Remarks:
-
Abnormalities:
not specified
Developmental effects observed:
not specified

Additional remarks to results:

The water with the substance was willingly consumed. During the treatment period, on day 6-15 of the pregnancy, the mother animals of the different treatment groups did not drink statistically different amounts of water.

The percentage of pregnancy was not significantly different for the different treatment groups: control group 10 out of 11, 100 ppm group 10 out of 11, 100 ppm and 1000 ppm groups 10 out of 10.

Result table:

(values of the individual animals in the test groups are averaged)

amount in drinking water (ppm) weight gain (g) number of implantations number of fetuses number of resorptions average weight fetuses (g) average weight placenta (g) fetuses with minor changes in bone formation fetuses with abnormalities underdeveloped fetuses < 1 g
control 130 12.9 12.0 0.9 3.83 0.509 4.6 0 0
100 136.2 13.0 12.1 0.9 3.86 0.539 5.4 0 0
1000 134.7 11.8 10.5 1.3 4.11 0.565 4.9 0 0
10000 134.4 13.1 12.3 0.8 4.03 0.515 5.3 0 0
Conclusions:
According to this study diethyl carbonate is not toxic to mother animals, embryotoxic or teratogenic in the rat when exposed via drinking water ad libitum at a concentration < 1 % (10000 ppm, 956 mg/kg/day) during day 6 to 15 of gestation.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Publication of acceptable quality standard.
Principles of method if other than guideline:
Teratogenic effects of a single dose of the test substance.
GLP compliance:
no
Species:
hamster, Syrian
Strain:
not specified
Details on test animals or test system and environmental conditions:
Animal weight: 100-120 g;
Age of the animals: 8-10 weeks
obtained from: NIH breeding facilities
Route of administration:
intraperitoneal
Vehicle:
other: saline
Details on exposure:
The substance was injected once intraperitoneally on Day 8 of gestation.
Analytical verification of doses or concentrations:
not specified
Frequency of treatment:
single dose (once)
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes
Abnormalities:
not specified
Developmental effects observed:
not specified

Results on the mother animals:

No maternal deaths occurred during gestation whether the animals belonged to the control or the treatment groups. Also there were no differences between the number of implantations from control and treated animals (average number of implantations in control group: 252/19 = 13.3; average number of implantations in treatment groups: 4.2 µmol/g, 39/3 = 13; 8.5 µmol/g, 42/3 =14).

Results on offspring:

The total number of dead or resorbed fetuses in the control group was 28 from the total number of implantations of 252 (11.1 %). Compared to that 5.1 and 21.4 % of dead or resorbed fetuses were observed in the low dose group (4.2 µmol/g, 496 mg/kg), and the high dose group (8.5 µmol/g, 1004 mg/kg), respectively. The percentage of malformed living fetuses increased in the treated groups compared to the control group (7.6 % for the low dose, 16.6 % for the high dose compared to 0 % in the control group). The observed malformations in the living fetuses of hamsters treated with diethyl carbonate were: Exencephaly, encephalocele, microcephaly, cleft palate, cleft lip, anophthalmia, microphthalmia, myelocele, spina bifida, omphalocele, extremities, growth retardation.

Conclusions:
Female Syrian hamsters were injected the test substance diethyl carbonate on Day 8 of gestation intraperitoneally. No maternal deaths occurred (treatment or control). The percentage of malformation in the offsprings increased with the injected concentration (0 % control, 7.6 % low dose, 16.6 % high dose). The conclusion of the authors was that the test substance diethyl carbonate is teratogenic.

Remark of the author of this IUCLID dossier: It is difficult to conclude a teratogenic effect on the test substance diethyl carbonate since the number of animals used in this study was very low (3 per dose) and even the lowest injected dose was rather high (496 mg/kg). Additionally intraperitoneal injection to pregnant animals is not the typical way of administration in order to evaluate teratogenicity. In the oppinion of the author of this IUCLID dossier the significance of these study results are limited. Therefore the purpose flag of this study has been set as "disregarded study".
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
yes
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Animals
Species / Strain: Rat, Han: of Wistar origin
Source: TOXI-COOP ZRT. 1103 Budapest, Cserkesz u. 90.
Hygienic level: SPF at arrival and kept in good conventional environment during the study
Animal health: Only healthy animals were used. Healthy status was certified by the breeder
Age of females at arrival: 6.4-7.1 weeks
Age of animals at mating: Females: Young adult and nulliparous females, 7.4-8.1 at start of the mating period
Males: 13-14 weeks at start of the mating period
Body weight of females at mating: The group averages of the body weight of the females were as similar as possible on the first day of gestation
Number of animals
used for mating: 70 females
40 males
Number of animals involved
in the study: animals needed to achieve at least 5 sperm positive females/dose group (70 females for mating, 32 sperm positive females included to the study)
Acclimatization time: 7 days
4.4.1 Reason for Selection of Species
The rat is commonly used species for toxicological studies in accordance with international recommendations. The Wistar strain is a well-known laboratory model with sufficient historical data. Rats are recommended as rodents for prenatal developmental toxicity studies.
4.4.2 Identification of Animals
The individual identification of the animals was performed in the pre-experimental phase by numbering written on the tail with a permanent marker pen.
Males were identified from 1 to 40 and females were marked from 101 to 170.
After randomization of the sperm positive females, the animals’ cages were identified at least by identity cards, with information at least about study number, sex, dose group, cage number, individual animal numbers, the date of mating and scheduled necropsy.
Litters:
At Caesarean section, the litters were identified with the litter numbers. All sheets used for recording data of the fetuses were marked only with these numbers and not with the dose groups up to the end of fetal examinations (to avoid bias).
Fetuses:
In the course of the Caesarean section, after removal from the uterus the fetuses were randomly allocated and identified thereafter.
Identification numbering at data evaluation:
aabbccc/d
a= evaluation number (starting from 01 in each dose group, issued after completion of the in-life phase, used at maternal and fetal data evaluation)
b= litter number (starting from 01, issued at Caesarean section continously for only litters with live fetuses independent from the dose group)
c= identification number of the dam
d= fetus number

4.4.3 Environmental Conditions
Room number: 403
Housing: before mating: 1-3 females per cage 1-2 males per cage
during mating: 1 male and 1-3 females / cage
during gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage
Cage type: Type II polypropylene/polycarbonate
Bedding: LignocelHygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (D-73494 Rosenberg, Holzmühle 1, Germany). Details of bedding quality is reported.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.5-22.3 °C
Relative humidity: 31 - 63 %
Ventilation /
Room sanitation: above 10 air exchanges/hour by central air-condition system at the end of each working day floors were swept and then mopped with an acceptable disinfectant. Water bottles were cleaned on a rota basis as required during the course of the study
The temperature and relative humidity were checked and recorded once daily during the study.

Route of administration:
oral: gavage
Vehicle:
vegetable oil
Details on exposure:
4.4.4 Food and Water Supply
Animals received ssniff®SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum, and tap water from municipal supply, as for human consumption from 500 mL bottle ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
For contents of the standard diet, see Appendix XVII/A. The supplier provided an analytical certificate (Appendix XVII/B) of the diet for the batch used.
The drinking water was periodically analyzed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Copies of the relevant certificates of analysis are maintained in Toxi-Coop Zrt.’s archive.
Details of food are reported.
4.5 Administration
4.5.1 Route of Administration and Reason for the Selection
The test item was administered orally (by gavage) from gestational day 5 to 19, daily. The route of application was selected in compliance with international guidelines. The oral route is a potential route for human exposure of the test item.
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
The females were paired to males in the mornings for two to four hours (one male: one to three females) until the number of sperm positive females per group achieves at least twenty two. Vaginal smears were prepared from each female, stained with 1 % aqueous methylene blue solution and examined for presence of sperm and for estrus cycle.
The day of mating is regarded as day 0 of pregnancy (vaginal plug and/or sperm in the vaginal smear). Sperm positive females were separated and caged if possible in groups of 2 animals. Caging of the females individually was avoided if possible.
Duration of test:
In-life time during test: 20 gestational days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
The total number of pregnant females and evaluated litters was 18 in the control, 20 in the 100, 19 in the 300 and 20 in the 1000 mg/kg bw/day dose group).
Control animals:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of systemic toxicity.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
None of the pregnant females died before scheduled termination. There were no clinical signs
of systemic toxicity.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weights of the dams were similar in the experimental groups in the different time
periods. There was no statistical difference indicated, except slightly lower body weight gain
values at 100 and 300 mg/kg bw/day between GD 11 and 14. There was no statistically
significant difference seen for this interval in the 1000 mg/kg bw/day dose group, hence
these minor differences were not attributed to the treatment of the dams with the test item.
The corrected body weight and body weight gain were very similar in the groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption of the dams was very homogenous among the dose groups. There was
no effect of the test item indicated. A statistically significantly higher mean food consumption
in the 1000 mg/kg bw/day dose group was not test item related as it occurred before
treatment (from gestation day 0 to 3).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Thyroid Hormone (FT3 (free T3), T4 (free T4)) and TSH
There were no significant differences observed in the means of hormone level values in the
test item treated groups and control. The levels of TSH were below the limit of detection.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Thyroid Weight
There were no significant differences revealed at statistical evaluation in the mean values of the
0, 100, 300 and 1000 mg/kg bw/day groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The findings revealed at gross pathology such as dilated renal pelvis in three dams in the
100 mg/kg bw/day group as well as in one dam in the 300 mg/kg bw/day group were not
attributed to the treatment of the test item considering the lack of dose response (no finding at all
in the 1000 mg/kg bw/day group) and one control dam with a dilated renal pelvis. In addition,
there was one control dam with a diaphragmatic hernia.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No lesion of the thyroid tissue in any dam was detected at histological examination.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No neoplastoc effects were observed.
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
There were no adverse effects indicated in the number of corpora lutea, percentage of pre- and
post- implantation loss, number of implantations, percentages of early- and late embryonic death
and dead fetuses, percentage of total intrauterine mortality and number of viable fetuses when
comparing dose group mean values to the control group mean values. The late embryonic death
was statistically significantly higher (p<0.05) in the 100 mg/kg bw/day dose group, however
there was no dose response indicated, hence this was not attributed to the test item. According to
the chi squared analysis there was a statistical significant difference (p<0.05) seen in the number
of male and female fetuses in the 100 and 1000 mg/kg bw/day dose groups without a biological
or toxicological relevance, since there was no statistical significance indicated in the percentage
values of male and female fetuses (sex distribution) and the sex distribution in the control group
was at the upper end of the historical control range.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no treatment-related effects were observed in the highest dose group
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no statistical significance indicated in the fetal weight, ano-genital distance (absolute
and normalized) as well as placental weight (absolute and relative).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no dose response indicated in the number of litters with malformed fetuses (two
each in the control, 100 and 300 mg/kg bw/day groups and zero in the 1000 mg/kg bw/day
dose group).
The number of examined fetuses was 229, 225, 230 and 243 in the control, 100, 300 and
1000 mg/kg bw/day groups, respectively.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Body weight retardation (limit: below 2.23 g for males and below 2.13 g for females) was
evaluated as an external variation. There were no treatment related differences in the fetal-
and litter incidences.
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Two fetuses were found with malformation at external examination, one fetus with agnathia
in the 300 mg/kg bw/day group (No.: 1469228/1) and one with anophthalmia in the
100 mg/kg bw/day group (No.: 1558225/11).
According to the experience with this species in this laboratory (Appendix 18.1), agnathia
and anophthalmia occurs sporadically without a relationship to the treatment. Moreover,
these findings were not observed in the high dose group. Thus, these malformations in the
100 and 300 mg/kg bw/day dose group in this study were judged to be incidental.
One placenta had clotted bloody margin in the 1000 mg/kg bw/day group. Considering that
this was a single finding, it was not attributed to the treatment.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of examined fetuses was 114, 112, 112 and 120 in the control, 100, 300 and
1000 mg/kg bw/day groups, respectively.

Malformations:
There were three fetuses found with malformations, one in the control group and two at
300 mg/kg bw/day.
The agnathia of the fetus (No.: 1469228/1) in the 300 mg/kg bw/day group observed at
external examination (see 10.12) was confirmed at skeletal examination as hypoplastic
mandible (absent Meckel’s cartilage was also recorded). In the same group malformation of
the vertebrae (misshapen cartilage of thoracic centrum, and slightly inclined vertebra as well
as the variation bipartite ossification) was observed in another fetus (No.: 1977223/3). One
fetus was found with a hemicentric vertebra in the control group (No. 1544180/5). Considering
the low incidences and the different type of malformations or also the presence in the control
group, these skeletal malformations were not attributed to the treatment.

Variations:
Skeletal findings such as retarded ossification of the skull, slightly larger anterior and
posterior fontanelle, incomplete ossification of the skull bones, bipartite or not ossified supra
occipital, unossified hyoid bone, bipartite supra occipital, less than 2 ossified or bipartite
and/or asymmetric sternebrae, wavy or interrupted ribs, interrupted 14th rib, dumb-bell
shaped or bipartite or/and asymmetric vertebrae, unossified or incompletely ossified
thoracic, lumbar or sacral arches or centra, unossified pubis or ischii, slightly bent humerus,
radius and ulna as well as less than 2.5/3 ossified metacarpal/metatarsal were classified as
variations.
There were no statistically indicated differences in the incidence different type variations.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of examined fetuses was 115, 113, 118 and 123 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.
There were two fetuses found with malformations in the test item treated groups and one in the control group.

There were two fetuses found with malformations in the test item treated groups and one in
the control group. Anophthalmia was confirmed for the fetus (No.: 1558225/11) which was
found with this malformation at external examination in the 100 mg/kg bw/day dose group.
Also in the low dose truncus arteriosus persistent was recorded in one single fetus in which
also microphthalmia was observed (No.: 1145159/14). Anophthalmia and microphthalmia
may occur sporadically without a relationship to the treatment (see discussion above).
Truncus arteriosus persistent as a single finding and not in the high dose was not attributed
to the treatment. Hydronephrosis was found in one control fetus (No.: 1443215/14).

The distribution of variations such as slightly or moderately dilated lateral ventricle (one in the
control group), bilateral hydroureter or marked hydroureter (one in the control group) as well as
hydro- or dilated ureter with dilated renal pelvis was equal in the experimental groups or
occurred only in the control group.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects were observed in the highest dose group
Key result
Developmental effects observed:
no
Treatment related:
no
Conclusions:
Treatment of 18 to 20 pregnant Han: WIST Rats per group from gestation day 5 to 19
by oral gavage administration of the test item Diethyl carbonate formulated in the vehicle sunflower oil at dose levels of 0 (vehicle only), 100, 300 or 1000 mg/kg bw/day caused no mortality and no clinical signs or effects on the body weight, food consumption, FT3 (free T3), FT4 (free T4) and TSH hormone levels as well as thyroid weight of the dams or histopathological detectable lesions on the thyroid glands.
The treatment of the dams with Diethyl carbonate at the dose levels of 100, 300 and 1000 mg/kg bw/day under the circumstances as described in this study, caused no effects on the intrauterine parameters (pre-and post-implantation loss, number of implantations and viable fetuses, or the distribution of the sexes) as well as development of the fetuses observed by external, visceral and skeletal examinations.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL maternal toxicity: 1000 mg/kg bw/day
NOAEL developmental toxicity: 1000 mg/kg bw/day
Executive summary:

Summary

Diethyl carbonate was examined for its possible prenatal developmental toxicity and to determine the dose levels for the main study of prenatal developmental toxicity. Groups of 6, 6, 7 and 7 spermium-positive female Han: of Wistar origin rats were treated with Diethyl carbonate by oral administration daily at four dose levels of 100, 200, 500 and 1000 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of six spermium positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 4 mL/kg bw.

During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when spermium was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities, the ano-genital distance was measured. The placentas were weighed and examined externally.

Results

Mortality, clinical signs

There was no mortality in the experimental groups before scheduled necropsy. There were also no clinical signs recorded for the maternal animals.

Food consumption, body weight of dams

There was no indication of a treatment related effect on the food consumption and body weight.

Evaluated litters

On gestation day 20, a total of 5, 5, 6, 6 and 7 litters in the control, 100, 300, 500 and 1000 mg/kg bw/day groups, respectively, were evaluated.

Necropsy findings of dams

There were no treatment related necropsy findings observed.

Intrauterine mortality

The intrauterine parameters such as pre- and post-implantation loss, mean number of viable foetuses and their sex distribution were not influenced by the treatment of the dams with the test item.

Fetal- and placental weight

There were no significant differences observed in the fetal- and placental weight.

External examination

Malformations

There were no malformations found.

Variations

A moderate incidence of body weight retarded fetuses occurred only in the 500 and 1000 mg/kg bw/day dose groups which was not attributed to the treatment.

Conclusion

Under the conditions of the present study treatment with Dimethyl Carbonate at the dose levels of 1000, 500, 200 and 100 mg/kg bw/day revealed no maternal effects and no adverse effects on the pre-and post-implantation loss, the mean fetal/placental weight as well as the ano-genital distance of fetuses.

Based on these observations in this dose range finding study the suggested dose levels for the main study are suggested as follows:

High dose: 1000 mg/kg bw/day

Mid dose: 300 mg/kg bw/day

Low dose: 100 mg/kg bw/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
35.7 mg/kg bw/day
Additional information

Justification for selection of Effect on developmental toxicity: via oral route:
See also discussion under "Short description of key information". The data under 7.8.2.001 (study from Bayer (1969)) is regarded as more profound than this data source. However, in a conservative approach this data delivers a lower effect level, LOAEL of 35.7 mg/kg bw/d.

Justification for classification or non-classification

The data is regarded as conclusive but not sufficient for a classification as reproductive toxicant.

Additional information