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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
See attached document with the justification for the category/read-across approach.

Data source

Reference Type:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
Limit test:

Test material

Constituent 1
Reference substance name:
Reaction mass of dimethyl esters of adipic, glutaric and succinic acids
Reaction mass of dimethyl esters of adipic, glutaric and succinic acids
Details on test material:
see information in the field 'Confidential details on test material'

Test animals

other: Crl:CD BR
Details on test animals or test system and environmental conditions:
- Source: Charles River Breeding Laboratories Inc., Kingston, NY
- Age at study initiation: females: 63 days, males: 84 days
- Weight at study initiation: females: 146-198g; males: 303-396g
- Housing: individually in stainless steel wiremesh cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

- Temperature (°C): 21-25°C
- Humidity (%): 40-60%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
Type of inhalation exposure (if applicable):
whole body
unchanged (no vehicle)
Details on exposure:
- Exposure apparatus: 1.4 m3 stainless steel and glass NYU-style inhalation chamber
- Method of holding animals in test chamber: in cages
- System of generating particulates/aerosols: 0.16 and 0.4 mg/L: liquid DBE was metered into a furnace maintained at 50-300°C; for the 1.0 mg/L concentration, mixed aerosol/vapour athmospheres were generated using a Spraying Systems Nebulizer
- Temperature, humidity, pressure in air chamber:
- Air flow rate: about 300 L/min

- Brief description of analytical method used: GC-FID
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Samples were analyzed using a Hewlett-Packard Model 5710 Gas Chromatograph (GC) equipped with a flame ionization detector. DBE was chromatographed isothermally at 150°C on a 3' x 2 mm ID glass column packed with 10% SP-1000 on Chromosorb W-AW 1001120 mesh.
Details on mating procedure:
The female rats were cohabited overnight with mature males (1:1). Mating was verified each morning by detection ol a copulation plug in the vagina or on the cage hoard. The day a plug was found was designated Day 1 of gestation (Day 1G). Mated females were stratified by body wcight and assigned to groups by random sampling within each stratum (24 per group).
Duration of treatment / exposure:
In the definitive study, groups of 24 pregnant rats were targeted for exposure to 0 (control), 0.16, 0.4, and 1.0 mg DBE /L. Rats were exposed to whole-body inhalation for 6 hr per day from Days 7 through 16 (10 exposures). The control dams were exposed to air only.
Frequency of treatment:
6h per day
Duration of test:
Day 7 to 16 (10 exposures)
Doses / concentrationsopen allclose all
Doses / Concentrations: 0.0, 0.16, 0.4, 1.0 mg/L
Basis: nominal conc.
Doses / Concentrations: 0.0, 0.15, 0.38, 0.99 mg/L
Basis: analytical conc.
No. of animals per sex per dose:
24 pregnant rats per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results of a pilot study


Maternal examinations:
Body weights and clinical signs were recorded on the day after arrival and before mating; observations for morbidity and mortality were made daily. Females selected for the study were weighed on Days 1, 7, 9, 11, 13, 15, 17, and 21G. Feed was weighed on Days 1, 3, 9, 11, 13, 15, 17, 19 and 21G and each afternoon on Days 7-16G (the exposure period). Just before euthanasia on Day 21G. each dam was coded, so personnel involved did not know the exposure group to which any dam or fetus belonged. Dams were euthanized by cervical dislocation, a gross pathologic examination was made, and the liver was weighed. The ovaries were removed and the number of corpora lutea were counted at a magnification of 2.5 x (Ednalite).
Ovaries and uterine content:
The uterus was opened and the number and position of all live, dead, and resorbed conceptuses were recorded. Both gravid and empty uteri were weighed. The uterus of each apparently nonpregnant dam was stained with ammonium sulfate solution to determine whether any very early resorption could be identified.
Fetal examinations:
All fetuses were weighed and examined for external alterations at a magnification of 2.5x. About one-half of the live fetuses in each litter (plus all stunted and malformed fetuses) were examined for visceral alterations. For each litter, the maximum stunted weight (MSW) was calculated by subtracting the lightest weight from the total weight dividing by the remaining number of fetuses, and multiplying by 0.666. A fetus weighing the same or less than the MSW was considered stunted, and its weight was omitted after the mean litter weight was calculated. The heads of these fetuses were fixed in Bouin’s solution and examined. All fetuses were sexed internally and then they were fixed in 70% ethanol, eviscerated, macerated in 1% aqueous KOH, and stained with alizarin red S to permit examination of the skeletons for alterations.
The litter was considered the experimental unit for the purpose of statistical evaluation. The level of significance selected was 0.05.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
All female rats survived the testing period. The number of pregnant rats/group was not altered by exposure to DBE, with the numbers being 24, 21, 20, and 24, control to high concentration groups, respectively. Body weight changes in maternal rats exposed to either 0.4 or 1.0 mg/L, were reduced, whereas those from the 0.16 mg/L were not. Feed consumption was reduced in the 0.4 and 1.0 mg/L group rats during the first 6 days of exposures (means of 23.3, 20.5, and 20.1 g/day, 0, 0.4, and 1.0 mg/L groups, respectively). Clinical observations of perinasal staining (15 rats) and wet fur (20 rats) were seen in the 1.0 mg/L group; while these findings were not seen in the controls. Perinasal staining was seen in 1 rat from the 0.16 mg/L group and in 4 rats from the 0.4 mg/L group; wet fur was also seen in 1 rat from the 0.4 mg/L group. Other clinical observations including alopecia, sores, periocular and facial staining, and swollen extremities were seen but infrequently and with no suggestion of a relationship to DBE exposure. No significant differences in absolute or relative liver weight was found but a significant trend in absolute weight was evident; a trend not seen when related to the body weight where values for all test groups were lower (but not significantly) from that of the controls.

Effect levels (maternal animals)

Key result
Dose descriptor:
Effect level:
0.16 mg/L air (nominal)
Based on:
test mat.
Basis for effect level:
other: perinasal staining in all dose groups
Remarks on result:
not determinable
no NOAEC identified

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
No adverse reproductive effects related to DBE exposure were detected. There was no effect on fetal weights at any exposure level. There was no significant difference between the control and experimental groups in the incidence of external, visceral, or skeletal malformations. Developmental variations among fetuses derived from DBE-exposed female were not significantly different from those of the controls.

Effect levels (fetuses)

Key result
Dose descriptor:
Effect level:
> 1 mg/L air
Based on:
test mat.
Basis for effect level:
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:

Applicant's summary and conclusion

DBE exerts no adverse effect on prenatal development in the rat following inhalation exposures as high as 1.0 mg/L DBE during the period of organogenesis.
Executive summary:

Dibasic Esters (DBE) has been tested in a developmental toxicity study on Crl:CD(SD)BR rats after inhalation exposure using a protocol similar to OECD guideline no 414 in compliance with Good Laboratory Practice.

Pregnant Crl:CD BR rats (7-8 per group) were exposed to either 0.16, 0.4, or 1.0 mg/L DBE by inhalation for 6 hr/day from Days 7 through 16 of gestation (day in which copulation plug was detected was designated Day 1G) in whole-body inhalation chambers. A control group of pregnant rats was exposed simultaneously to air only. Parental examinations included clinical and cage side observations, body weight determination, and determination of feed consumption. Gross pathologic examination was performed on day 21G and included counting of the corpora lutea and determination of number and position of all live, dead, and resorbed conceptuses in the uterus. Liver, gravid and empty uteri were weighed. All fetuses were weighed and examined or external alterations. About one-half of the live fetuses were examined for visceral alterations. All fetuses were sexed and examined for alterations of the skeleton after maceration.


A suppression of both food consumption and the rate of body weight gain was seen in the 0.4 and 1.0 mg/L groups during the first 6 exposure days. Staining on the fur and perineal area was seen in rats exposed to 1.0 mg/L and liver weight decreases, although not statistically significant, occurred in the 2 high exposure groups. None of the reproductive parameters were altered in any of the groups and no fetal effects were detected.


DBE exerts no adverse effect on prenatal development in the rat following inhalation exposures as high as 1.0 mg DBEL during the period of organogenesis.

Based on the given data, Dibasic Esters (DBE) is not classified as reproductive toxicant according to the criteria of Directive 67/548/EC and EU Regulation No. 1272/2008 (CLP).