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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
February 1996-August 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Only two dose levels instead of three and only 12 females per dose level were used, but the test results provide useful and reliable information.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
, two instead of three dose levels were used, 12 instead of 20 animals per dose level were used
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl (2S)-2-hydroxypropanoate
EC Number:
606-097-1
Cas Number:
186817-80-1
Molecular formula:
C11H22O3
IUPAC Name:
2-ethylhexyl (2S)-2-hydroxypropanoate
Constituent 2
Reference substance name:
2-ethylhexyl-S-lactate
IUPAC Name:
2-ethylhexyl-S-lactate
Details on test material:
Batch no.: EHL 951116-2
Purity: 98.2 %

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 14-15 weeks
- Weight at study initiation: mean weight: 208 g
- Fasting period before study: none
- Housing: mated females were house individually in wire mesh cages (18 × 32 × 18 cm)
- Diet (e.g. ad libitum):the Institute's cereal-based rodent diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%):30-70 %
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark, 12 hours light

IN-LIFE DATES: From: February 7, 1996-March 19 and 20, 1996

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were exposed to the test atmospheres in nose-only inhalation units. Each unit consisted of a cylindrical teflon-coated column, surrounded by a transparent cylinder. The column had a volume of ca. 50 L and consisted of a top assembly, with two rodent tube sections underneath and an exhaust section at the bottom. The rodent tube sections had 40 ports for animal exposure.
- Method of holding animals in test chamber: plastic animal holder (Batelle)
- Source and rate of air: humidified pressurized air
- Method of conditioning air: humidified
- System of generating particulates/aerosols: The low-concentration test atmosphere was generated by passing metered amounts of the test material using a roller pump (Golson, Villiers le Bel, France) to a compressed air driven, all glass nebulizer (Institute's design). The generated mixture of test material and air was subsequently mixed with metered amounts of pressurized air (using a rotameter) and was again mixed at the entrance of the exposure unit with metered amounts of pressurized air. The resulting test atmosphere was directed downward through the mixing chamber towards the animals' noses.
The high-concentration test atmosphere were generated by nebulizing the test material using compressed air driven steel nebulizers (Institute's design). These nebulizers consisted of an atomizer (Lechler Informatic Buro Holland, Oudorp, the Netherlands) and a glass jar containing the test materials. During operation, the test material was drawn through a suction tube to the atomizer and was blown against a baffle which was fitted below the nozzel orifice in such a way that the larger droplets were removed by impaction. The impacted test material drained back into the test material supply at the bottom of the jar. The resulting aerosol was also mixed with humidified pressurized air in the way described above and directed towards the animals.
- Temperature, humidity, pressure in air chamber: The daily mena temperature was 22.7 ± 0.6 °C, 22.6 ± 0.4 °C and 22.6 ± 0.4 °C for the control, low and high concentration level, respectively. The daily relative humidity was 56 ± 6 %, 52 ± 5 % and 52 ± 6 %, respectively. A positive pressure in the central column and a slightly negative pressure in the outer cylinder, which enclosed the entire animal holder, were maintained to prevent dilution of the test atmosphere by air leaking from the animal's thorax to the nose.
- Air flow rate: the mean daily airflows were 26.5, 55.9 and 70.4 L/min for the control, low and high concentration level, respectively
- Air change rate: given the volume of ca 50 L of each inhalation unit, the air change rate was calculated to be 0.53, 1.12 and 1.41 air changes per minute for the control, low, and high concentration level, respectively.
- Method of particle size determination: once per week (except for the control test atmosphere) using an 11-stage cascade impacter (Institute's design) with the largest cut off size of 4.2 µm. The Mass Median Aerodynamic Diameter (MMAD) and the mean geometric standard deviation (gsd) were calculated (Lee, 1972).
- Treatment of exhaust air: The test atmosphere was exchausted at the bottom of the unit.

TEST ATMOSPHERE
- Brief description of analytical method used: The actual concentration of 2-ethylhexyl lactate in the test atmosphere was detemined at least 3 times per exposure day by means of gravimetric analysis. Preliminary experiments had shown that the 2-ethylhexyl lactate test atmosphere almost exclusively consisted of aerosol and not of vapour and that gravimetrical analysus was a suitable method to measure the actual concentration in air. Measured test atmosphere samples were obtained by passing approximately 50 or 30 liters of test atmosphere for the low- and high-concentration level, respectively, at a mean sampling speed of 5 L/min, through fiber glass filters (Sartorius). Before and immediately after sampling the filters were weighed. The actual concentration was calculated by dividing the amount of test material on the filter by the sample volume.
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle: compressed air was used to generate aerosols
- Composition of vehicle: compressed air
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle:
- Lot/batch no. of vehicle (if required):
- Purity of vehicle:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual concentration of 2-ethylhexyl lactate in the test atmosphere was detemined at least 3 times per exposure day by means of gravimetric analysis. Preliminary experiments had shown that the 2-ethylhexyl lactate test atmosphere almost exclusively consisted of aerosol and not of vapour and that gravimetrical analysus was a suitable method to measure the actual concentration in air. Measured test atmosphere samples were obtained by passing approximately 50 or 30 liters of test atmosphere for the low- and high-concentration level, respectively, at a mean sampling speed of 5 L/min, through fiber glass filters (Sartorius). Before and immediately after sampling the filters were weighed. The actual concentration was calculated by dividing the amount of test material on the filter by the sample volume.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: for mating two females were placed with one male
- Length of cohabitation: every consecutive morgning vaginal examinations were made to ascertain copulation by detection of a vaginal plug or sperm cells in a vaginal smear. Upon evidence of copulation, positive females were housed individually. This procedure was continued until the required number of females showed evidence of copulation.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from gestation day 6 up to and including day 15
Frequency of treatment:
6 hours per day
Duration of test:
from gestation day 0 to day 21
No. of animals per sex per dose:
12 mated females per dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale:
Exposure levels selected were based on similar studies with ethyl lactate (TNO reports V90.322 and V91.416) showing moderate to severe nasal irritating and toxic (local) effects at levels of 600 and 2500 mg/m³, whereas sligh systemic toxicity (reduced food consumption and growth retardation) was observed at the 2500 mg/m³ level only. The No-Observed-Adverse-Effect level (NOAEL) in these studies was 200 mg/m³. In addition, the in vitro hydrolysing acitivity of these compounds by nasal olfactory epithelium was taken into account (TNO-report V92.339) with 2-ethylhexyl lactate showing an approximately 6.5 times higher affinity and a 2.5 times higher velocity than ethyl lactate. The target levels chosen for the present study, therefore, were: 0, 200 and 600 mg/m³.
- Rationale for animal assignment (if not random): The mated females were distributed ove the three experimental groups in such a way that the animals from the same day of pregnacy were, as far as possible, equally distributed over all groups. Females mated by the same male were placed in different groups.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each mated female was observed daily from gestation day 0. Signs of ill health and reaction to treatment as well as mortatily were recorded. On working days, all cages were checked again late in the afternoon for dead or moribund animals to minimize loss of animals from the study. During weekends and holidays only one check per day was carried out.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: if necessary, the females were handled to appraise its physical condition

BODY WEIGHT: Yes
- Time schedule for examinations: body weights of mated females were recorded on gestation days 0, 6, 10, 15 and 21

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
- Organs examined: the uteri (including the fetuses), ovaries and placentas.
The females killed on day 21 were examined for gross abnormalities and the dam that died during the study was also examined macroscopically.

OTHER:
Food consumption of mated females was measured for each animal individually by weighing the feeders on days 0, 6, 10, 15 and 21 of gestation.
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 of live and dead fetuses; sex of fetuses, number of grossly visible malformed fetuses and fetuses with external abnormalities; weight of ovaries; weight of uterus, empty; weight of the fetuses; weight of placentas; gross evaluation of placentas
Fetal examinations:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: No
Statistics:
As a level of significance was considered: P < 0.05
Adult data:
Clinical and necropsy findings were evaluated by Fisher's exact probability test. Body weight, body weight gain, organ weights and food consumption data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
Mating, Caesarian section and litter data:
Fisher's exact probability test was used to evaluate fetal external, visceral and skeletal observations, the number of mated and pregnant females, females with live fetuses and male and female fetuses. Number of corpora lutea, implantations, live and dead fetuses, and early and later resorptions were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U-test. Placental and fetal weight were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
Indices:
The following indices were calculated for each group at the Caesarian section:
Pre-implantation loss = ((number of corpora lutea - total number of implantation sites)/ number of corpora lutea ) × 100
Post-implantation loss = ((total number of implantation sites - number of live fetuses)/ total number of implantation sites) × 100
Fecundity index = (number of females pregnant/ number of males mated) × 100
Gestation index = (number of females with live fetuses/ number of females pregnant) × 100
Live fetus index = (number of live fetuses/ number of implantation sites) × 100
Sex ratio = (number of live male fetuses/ total number of live fetuses) × 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
A visually increased breathing rate was observed in animals exposed to 600 mg/m³ during the entire treatment period and occasionaly in animals exposed to 200 mg/m³.
During the exposure period dams exposed to 2-ethylhexyl lactate tended to consume less food when compared with the controls. In the 600 mg/m³ the difference reached a level of statistical significance. After the exposure period animals of the 200 mg/m³ group consumed slightly more food than the controls (statistically significant), probably to compensate for the slight decrease in food intake during the preceeding exposure period.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
600 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Effect level:
200 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
The slightly retarded ossification observed at the levels of 200 and 600 mg/m³, is most probably not due to systemic toxicity but might be related to stress caused by the combination of maintaining the animals in restraining tubes and the irritative properties of the test material. No teratogenic effects were observed in this study.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
600 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
2-Ethylhexyl lactate in not teratogenic or developmentally toxic.
Executive summary:

In a developmental toxicity study, 2-ethylhexyl lactate was administered to 12 mated female Wistar rats/dose by inhalation, nose only at dose levels of 0, 200 and 600 mg/m³ from gestation day 6 up to and including day 15 during 6-hour sessions.

During exposure the breathing rate was visually increased in animals exposed to 600 mg/m³ during the entire treatment period and occasionally in animals exposed to 200 mg/m³. Further, dams exposed to the test substance consumed less for than the controls, the difference being statistically significant at 600 mg/m³ only. At 200 mg/m³ this decrease was followed by a slight but statistically significant increase. The maternal LOAEC is 200 mg/m³ based on the increase in breathing rate and changes in food consumption; a maternal NOAEC could not be identified.

Slightly retarded ossification was seen in the foetuses of the dams exposed to 200 and 600 mg/m³, but was considered to be a minor developmental effect, most attributable to the stress caused by combination of maintaining the animals in restraining tubes and the irritative properties 2-ethylhexyl lactate. The developmental NOAEC is 600 mg/m³.

No teratogenic effects were observed; the teratogenic NOAEC is 600 mg/m³.