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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:
1995-12-08 to 1999-12-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl lactate
EC Number:
228-503-2
EC Name:
2-ethylhexyl lactate
Cas Number:
6283-86-9
Molecular formula:
C11H22O3
IUPAC Name:
2-ethylhexyl 2-hydroxypropanoate
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-Ethylhexyl lactate
- Molecular formula (if other than submission substance): C11H22O3
- Molecular weight (if other than submission substance): 202.3 g/mol
- Physical state: Clear, colourless liquid
- Analytical purity: 98.2 %
- Lot/batch No.: EHL 951116-2
- Thermostability: Stable at ambient temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING . The generated mixture of test material and air was subsequently mixed with meterd amounts of pressurized air and was again mixed at the entrance of the exposure unit with metered amounts of humidified pressurized air.

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: 10-11 weeks
- Weight at study initiation: No data
- Fasting period before study: No
- Housing: during acclimatization period: In groups of four in suspended stainless group cages; shortly before mating, males were housed individually in smaller suspended wire mesh cages with wire mesh floor and front; mated females were housed individually in wire mesh cages before and after exposure; during the daily 6-hour exposure period (day 6-15 of gestation) the female rats were individually placed in inhalation tubes
- Diet (e.g. ad libitum): TNO rodent diet batch 2020, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 6 days quarantine, afterwards 13 days acclimation

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

IN-LIFE DATES: 1996-02-07 to 1996-03-20

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
> 1.7 - < 2.7 µm
Geometric standard deviation (GSD):
1.55
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only inhalation units consisting of a cylindrical Teflon-coated steel column surrounded by a transparent cylinder. The column had a volume of 50 L and consisted of a top assembly, with two rodent tube sections underneath and an exhaust section at the bottom. The rodent tube section had 40 ports for animal exposure
- Method of holding animals in test chamber: the animals were secured in plastic animal holders, positioned radially through the outer cylinder around the central column
- Source and rate of air: see below
- System of generating particulates/aerosols: The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. Each test atmosphere was generated separately. The low-concentration test atmosphere was generated by passing metered amounts of the test material using a roller pump (Gilson, 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 humidified pressurized air (using a second rotameter). The resulting test atmosphere was directed downwards through the mixing chamber towards the animal's noses. The test atmosphere was exhausted at the bottom of the unit. The high-concentration test atmosphere was generated by nebulizing the test material using compressed air driven steel nebulizers (Institute's design). These nebulizers consisted of an atomizer (Lechler Informatie Buro Holland, Oudorp, the Netherlands) and a glass jar containing the test material. 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 nozzle 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 also directed towards the animals.
- Temperature, humidity, pressure in air chamber: several empty ports of the rodent tube section were used for temperature and relative humidity measurements
- Air flow rate: Before the start of the first exposure, the rate of airflow through the nebulizers was established at the pressures used. The pressure settings were recorded at regular intervals (approximately hourly). In this way, the total exposure airflows were monitored indirectly through the aerosol generation system.
- Method of particle size determination: Particle size distribution measurement was carried out in each test atmosphere (except for the control test atmosphere) once per week using an 11-stage cascade impactor with a largest cut off size of 4.2 µm. The Mass Median Aerodynamic Diameter (MMAD) and the mean geometry standard deviation (gsd) were calculated.

TEST ATMOSPHERE
- Brief description of analytical method used: The actual concentration of 2-ethylhexyl lactate in the test atmosphere was determined at least 3 times per exposure day by means of gravimetric analysis. Measured test atmosphere samples were obtained by passing approximately 50 or 30 liters test atmosphere for the low- and high-concentration level, respectively, at a mean sampling speed of ca 5 L/min, through fibre glass filters.
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.
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 determined at least three times per exposure day by means of gravimetric analysis.
Details on mating procedure:
- Impregnation procedure: Co-housed
- M/F ratio per cage: 1/2
- Length of cohabitation: Upon evidence of copulation, positive females were housed individually
- Proof of pregnancy: Vaginal plug/sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From day 6 up to day 15 of gestation daily six hours exposure sessions.
Frequency of treatment:
Daily
Duration of test:
After termination of the exposure period, the rats were observed up to gestation day 21.
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/m³ air (nominal)
Remarks:
chosen on the basis of similar studies with ethyl lactate (NOAEL)
Dose / conc.:
230 mg/m³ air (analytical)
Dose / conc.:
600 mg/m³ air (nominal)
Remarks:
chosen on the basis of similar studies with ethyl lactate (highest dose without adverse systemic effects)
Dose / conc.:
594 mg/m³ air (analytical)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were based on similar studies with ethyl lactate showing moderate lo severe nasal irritating and toxic (local) effects at levels of 600 and 2500 mg/m³, whereas slight 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.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: On the weekend once a day, on working days twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each mated female was observed daily from gestation day 0 and, if necessary, handled to appraise its physical condition. Signs of ill health and reaction to treatment as well as mortality were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of the mated female rats were recorded on gestation days 0, 6, 10, 15 and 21.

FOOD CONSUMPTION: Yes
- 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.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21; gross abnormalities were examined
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
Fetal examinations:
Half of the foetuses of each litter were fixed in Boum's fixative, examined for soft tissue anomalies according to a method modified after Barrow and Taylor (1969) and then discarded. The other half of the fetuses were fixed in 70 % alcohol, subsequently partly eviscerated, and then cleared in potassium hydroxide and stained with Alizarin Red S modified after Dawson (1926). They were examined for skeletal abnormalities and then retained.
The foeto-pathological examinations were initially restricted to all foetuses of the control group and the high-concentration group. As some alterations were found in the ossification of the skeletons in the latter group the skeletal examinations were extended to the intermediate dose group, after consultation with the Sponsor.
Statistics:
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
be 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 foetal external, visceral and skeletal observations, the number of mated and pregnant females, females with
live foetuses and male and female foetuses. Number of corpora lutea, implantations, live and dead foetuses and early and late resorptions were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann-Whitney U-test. Placental and foetal 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, post-implantation loss, fecundity index, gestation index, live foetus index, sex ratio.
Historical control data:
N.A.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Abnormalilics seen during exposure were confined to visually increased breathing rate in animals exposed lo 600 mg/m³ during Ihe entire treatment period and occasionally in rats exposed lo 200 mg/m3. One animal of the control group (A 101) lost part of its tail due to a mechanical trauma. Daily clinical observations did not reveal any noticeable differences in the animals' appearance, general condition or behaviour amongst the various groups.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One animal of the low dose group died on day 11 of gestation because it turned around in the inhalation tube and subsequently suffocated. All other females survived until scheduled Caesarian section.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no significant differences in body weight or body weight change between the control group and the groups exposed to the test substance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption of the high dose group was statistically significantly decreased when compared to the control group throughout the exposure period. The food consumption of the 200 mg/m³ was slightly decreased during the exposure period, but increased thereafter.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross examination at autopsy did not reveal any significant differences of the maternal organs and tissues among the various groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Uterus and ovary weights:
Mean reproductive organ weights and net maternal body weight change during gestation were evaluated. No statistically significant differences in gravid and empty uterus weight, ovary weight, carcass weight and the net weight change (body weight gain from day 0 to 21 of gestation minus gravid uterine weight) were observed between the control group and he groups treated to 2-ethylhexyl lactate.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between the control group and the groups exposed to the test substance in the numbers of corpora lutea, implantations, live and dead foetuses and early and late resorptions nor in pre- and post-implantation loss or in the sex ratio of the foetuses.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between the control group and the groups exposed to the test substance in the numbers of corpora lutea, implantations, live and dead foetuses and early and late resorptions nor in pre- and post-implantation loss or in the sex ratio of the foetuses.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between the control group and the groups exposed to the test substance in the numbers of corpora lutea, implantations, live and dead foetuses and early and late resorptions nor in pre- and post-implantation loss or in the sex ratio of the foetuses.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between the control group and the groups exposed to the test substance in the numbers of corpora lutea, implantations, live and dead foetuses and early and late resorptions nor in pre- and post-implantation loss or in the sex ratio of the foetuses.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
From the 12 mated female rats per group, 11 of each group were pregnant.
Other effects:
not examined
Details on maternal toxic effects:
Details on maternal toxic effects:
Clinical signs and mortality:
One animal of the low dose group died on day 11 of gestation because it turned around in the inhalation tube and subsequnetly suffocated. Daily clinical observations did not reveal any differences between dose and control group animals.

Maternal body weight and body weight change:
There were no significant differences in body weight or body weight change between the control group and the groups exposed to the test substance.

Food consumption:
The food consumption of the high dose group was statistically significantly decreased when compared to the control group throughout the exposure period. The food consumption of the 200 mg/m³ was slightly decreased during the exposure period, but increased thereafter.

Parental necropsy observations:
Gross examination at autopsy did not reveal any significant differences of the maternal organs and tissues among the various groups.

Uterus and ovary weights:
Mean reproductive organ weights and net maternal body weight change during gestation were evaluated. No statistically significant differences in gravid and empty uterus weight, ovary weight, carcass weight and the net weight change (body weight gain from day 0 to 21 of gestation minus gravid uterine weight) were observed between the control group and the groups treated to 2-ethylhexyl lactate.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEC
Effect level:
600 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
other: No differences in clinical signs, maternal body weight or body weight change and necropsy seen in treated animals in comparison to control animals.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
See box “Details on embryotoxic / teratogenic effects” below.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
See box “Details on embryotoxic / teratogenic effects” below.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
See box “Details on embryotoxic / teratogenic effects” below.
Skeletal malformations:
no effects observed
Description (incidence and severity):
See box “Details on embryotoxic / teratogenic effects” below.
Visceral malformations:
no effects observed
Description (incidence and severity):
See box “Details on embryotoxic / teratogenic effects” below.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Reproduction and litter data at Caesarian section:
From the 12 mated female rats per group, 11 of each group were pregnant.
Reproduction and litter data revealed no treatment-related changes either as evidenced by the absence of statistically significant differences between the
control group and the groups exposed to the test substance in the numbers of corpora lutea, implantations, live and dead foetuses and early and late resorptions nor in pre- and
post-implantation loss or in the sex ratio of the foetuses.

Foetal external observations:
No statistically significant differences were observed for the individual findings. When compared with the control group, the total number of fetal external
observations was slightly albeit statistically significantly increased in the high concentration group. This difference was mainly due to the low number of foetal
observations in the control group: Only one foetus with a small haemorrhage on the head in the control group versus 4 dysmature foetuses from 4 litters (i.e. foetus weight < 75 % of the mean foetal body weight in the control group) and three large foetuses (i.e. foetus weight > 125 % of the mean foetal body weight) plus one foetus with a flexed limb from one litter of the 600 mg/m³ group. Considering the nature of the findings and the low number in the control group, this difference is not considered treatment related.

Findings of the placenta:
Findings of the placenta were limited to two fused placenta in four fetuses of one female control group animal.

Foetal weight and placental weight:
No significant differences in mean foetal body weights were observed between the control group and the groups exposed to the test substance. Mean placental
weight of the 200 mg/m³ group was increased (statistically significantly for both sexes combined). Mean placental weights in of the 600 mg/m³ were comparable to these in the control group.

Visceral examination:
Examination of foetal soft tissues was limited to the control group and the high concentration group.
Visceral malformations:
No visceral malformations were seen in the control group and the high concentration group.
Visceral anomalies:
No visceral variations were observed in the control and the high-concentration group.

Skeletal examinations:
Skeletal examinations were conducted in all groups.
Skeletal malformations:
None of the fetuses showed skeletal malformations
Skeletal anomalies:
Skeletal anomalies were limited to wavy ribs in 3 foetuses out of 2 litters in the high-concentration groups. The incidence in the high-concentration group did not differ significantly from that in the control group.
Skeletal variations:
No statistically significant differences were observed for the individual findings.
Variations in the ossification of the skeletons
When compared with the control group, the 200 and 600 mg/m³ groups showed the following differences: Increase in the number of foetuses and litters with an incompletely ossified frontalis and unossified metatarsals, which was significant in the 200 mg/m³ group. Furthermore, a delay in the ossification of the hind limb phalanges was observed in the 200 and 600 mg/m³ group. The slightly retarded ossification as observed at 200 and 600 mg/m3, was considered to be a minor developmental effect, most attributable to the stress conditions. No teratogenic effects were observed in this study.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
600 mg/m³ air (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Gravimetric analysis:

The mean actual concentrations of 2-ethylhexyl lactate in the test atmospheres (and their standard deviations) were 230 (± 16) and 594 (± 48) mg/m³.

Nominal concentration:

The daily mean airflow through the exposure units were 26.5, 55.9, and 70.4 L/min for the control, low, and high concentration level, respectively. The nominal concentrations were 378 and 751 mg/m³, indicating generation efficiencies of 61 and 79 % for the low and high concentration level, respectively.

Particle size measurement:

Particle size measurement showed that almost all particles in the animals' breathing zone were respirable, viz. they were smaller than or equal to 4.2 µm. The mean mass median aerodynamic siameter (MMAD) was 2.7 and 1.7 µm for the low and high exposure level, respectively. The mean geometric standard deviation was 1.5 for the low concentration level and 1.6 for the high concentration level.

Temperature and relative humidity:

The daily mean 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 relalive humidity was 56 ± 6%, 52 ± 5% and 52 ± 6 %, respectively.

Applicant's summary and conclusion

Conclusions:
In conclusion, no treatment-related effects in developmental parameters or maternal parameters were detected in a developmental toxicity study (OECD 414) after inhalation of 2-ethylhexyl lactate, except slightly retarded ossification. This is considered to be a minor developmental effect, most attributable to the stress conditions. Therefore, it can be stated that no teratogenic effects were observed in this study and the maternal and developmental NOAEC is considered to be 600 mg/m³.
Executive summary:

In a developmental toxicity study (OECD 414), 2-ethylhexyl lactate (98.2% purity) was administered to 12 female Wistar rats per dose level in clean air (nose-only exposure for 6 hours/day) at concentration levels of 0, 200 and 600 mg/m³ from day 6 through day 15 of gestation. On day 21 of gestation the animals were sacrificed. There were no treatment-related effects on mortality, clinical signs, body weight or Casarean parameters. Food consumption of the groups was statistically significantly decreased in comparison to the control group animals. As no differences were noticed in body weight change between control and treated animals this effect was classified as not biologically adverse. Based on the results, the maternal NOAEC is considered to be 600 mg/m³. Moreover, no treatment related effects were noted in developmental parameters, except slightly retarded ossification. This is considered to be a minor developmental effect, most attributable to the stress conditions. Thus, the developmental NOAEC is 600 mg/m³.

This developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in rat.