Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

D-003 is a mixture of long-chain fatty acids isolated and purified from sugar cane wax with cholesterol-lowering properties. D003 given orally (500 and 1000 mg/kg/day) to female rats for 15 days prior to mating, through mating and gestation to day 21 of lactation and male rats for 4 weeks prior and during mating did not induce toxic effects on reproduction. There were no significant reductions in the number of animals that conceived, in the numbers of pups born to those that did conceive, in the numbers of pups that survived until weaning, and in their body weights at weaning. No treatment related effects in offspring were observed related to growth, physical and behavioral development, spontaneous activity and reproductive performance. The NOAEL is 1000 mg/kg bw.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
yes
Remarks:
only exposure of the parental (P0) generation
GLP compliance:
yes
Remarks:
conducted under the Cuban Good Laboratory Practices (GLP) guidelines
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: CENPALAB (La Habana, Cuba)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 10-12 wks; (F1) postnatal 10-12 wks
- Weight at study initiation: (P) Males: 123 g; Females: 98 g; (F1) no data
- Housing: individually
- Diet: CENPALAB-certified Rat lab chow ad libitum
- Water: tap water ad libitum
- Acclimation period: no data
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 50 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: suspension of water with 1% Acacia Gum
Remarks:
maximum dosing volume 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing suspensions were prepared fresh weekly and refrigerated after corroborating their stability in such conditions.
Details on mating procedure:
- M/F ratio per cage: P0 2 females/ 1 male; F1 1 male/afemale
- Length of cohabitation: no data
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Female rats for 15 days prior to mating, through mating and gestation to day 21 of lactation and male rats for 4 weeks prior and during mating
Frequency of treatment:
daily
Details on study schedule:
- F1 parental animals not mated until 10-12 weeks after selected from the F1 litters.
- Selection of parents from F1 generation at weaning, when pups were 21 days of age.
- Age at mating of the mated animals in the study:10-12 weeks
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
30 females and 15 males
Control animals:
yes, concurrent vehicle
Details on study design:
Only exposure of the P0 animals
Dose selection rationale: 1000 mg/kg bw is the acceptable upper limit dose by ICH/OECD guidelines.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes daily

DETAILED CLINICAL OBSERVATIONS: no data

BODY WEIGHT: Yes regulary at least on day 0, 6, 13 and 20 of gestation and at day 1, 4, 7, 14 and 21 of lactation

FOOD CONSUMPTION: Yes, regulary

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
not investigated
Sperm parameters (parental animals):
not investigated
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4/sex/litter excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring: post natal day 1 number, weight, external examination and sex
F1 offspring: survival, weight and locomotor activity on day 4, 7, 14 and 21 (for F1 animals selected for mating: body weight weekly)
F1 offspring: day 4 unfolding of the pinnae and righting in air on day 4; day 10 hair growth; day 12 eruption of the incisors; day 13 opening of the ear; day 14 visual placing and auditory startle; day 15 opening of the eyes ; day 17 pupilar, corneal and parpebral reflexes; day 21 activity measurements
such as squares entered, urination/defecation and standing on hindlegs; day 25 testicular descent; day 37 vaginal canalization

GROSS EXAMINATION OF DEAD PUPS: no data

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not investigated

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not investigated
Postmortem examinations (parental animals):
not specified
Postmortem examinations (offspring):
not specified
Statistics:
Maternal initial body weight, maternal weight gain, fetal and pup body weights were analyzed using a parametric analysis of variance followed by Tukey's multiple comparison test. Data on dead and alive fetuses, sex ratio, litter size, survival through the weaning period and percentage of pups showing physical and reflex development were analyzed by the Kruskal–Wallis (nonparametric) test followed by Mann–Whitney U-test to determine which treatment groups differed from the control.
Trend analysis via ANOVA, Jonckheere's test and the Cochran–Armitage test.
Reproductive indices:
not specified
Offspring viability indices:
not specified
Clinical signs:
no effects observed
Mortality:
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see attached table
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
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):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
see attached table
Pregnancy rate: 30/30. 28/30 and 29/30 at 0, 500 and 1000 mg/kg bw
see table
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of effects
Critical effects observed:
no
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see figure
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
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):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not specified
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no details except for body weight
Critical effects observed:
no
Clinical signs:
not specified
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
see table (live pups and dead pups)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see table
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
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
Sexual maturation:
no effects observed
Description (incidence and severity):
see table (no effects on testes and vaginal opening)
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
no effects on behavior and development (see table)
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
see table
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no effects observed
Critical effects observed:
no
Mortality / viability:
not specified
Description (incidence and severity):
no treatment related effects
Body weight and weight changes:
no effects observed
Description (incidence and severity):
body weight on day 1 did not show any treatment related effects
Gross pathological findings:
no effects observed
Description (incidence and severity):
no treatment related effects
There were no treatment-related effects on mortality, weight, or external morphology of the F2 pups
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: absence of effects on mortality, body weight and external examinations
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
No effects on reproductive performance in the exposed parental generation were observed at 1000 mg/kg bw.
No effects on development and reproductive performance of the F1 generation were observed at 1000 mg/kg bw.
Executive summary:

D-003 is a mixture of long-chain fatty acids isolated and purified from sugar cane wax with cholesterol-lowering properties. D003 given orally (500 and 1000 mg/kg/day) to female rats for 15 days prior to mating, through mating and gestation to day 21 of lactation and male rats for 4 weeks prior and during mating did not induce toxic effects on reproduction. There were no significant reductions in the number of animals that conceived, in the numbers of pups born to those that did conceive, in the numbers of pups that survived until weaning, and in their body weights at weaning. No treatment related effects in offspring were observed related to growth, physical and behavioral development, spontaneous activity and reproductive performance.

The NOAEL is 1000 mg/kg bw.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
the study is on long-chain fatty acids that are expected to be analogue to the long-chain fatty acids formed after hydrolysis of the substance
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

D003 was shown not to influence reproduction and development at concentrations of 1000 mg/kg bw. The substance was tested in a 2-generation study in rats and a developmental toxicity study in rabbits. In both studies no adverse effects were observed in parental animals or offspring. No effects on fertility were found in the rat study. More in general saturated fatty acids are not expected to exhibit any effects on reproduction and development.

In a dermal developmental toxicity study with ethylene glycol in mice, no toxicity was noted in the maternal animals and no effects on development were reported up to a dose of ethylene glycol of ca 3400 mg/kg bw (Tyl 1995).

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:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Remarks:
conducted under the Cuban Good Laboratory Practices (GLP) guidelines
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: CENPALAB (La Habana, Cuba)
- Age at study initiation: no data
- Weight at study initiation: 2.5-3.5 kg
- Housing: individually
- Diet: CENPALAB-certified Rabbit Lab chow ad libitum
- Water: tap water ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 50%
- Air changes (per hr): not indicate
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: suspension of water with 1% Acacia Gum
Remarks:
maximum dosing volume 5 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing suspensions were prepared fresh weekly and refrigerated after corroborating their stability in such
conditions.
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: no data
- Proof of mating: indicated as copulation observed
Duration of treatment / exposure:
days 6 through 18 of gestation
Frequency of treatment:
daily
Duration of test:
until day 29 of gestation
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
27 mated females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 1000 mg/kg bw is considered an acceptable upper limit dose by ICH/OECD guidelines.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes daily

BODY WEIGHT: Yes on regular basis and at least on day 0, 6, 18 and 29

FOOD CONSUMPTION Yes, on regular basis

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: not specified
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
All fetuses: body weight, sex

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter(examined by freehand coronal sectioning)
Statistics:
Maternal initial body weight, maternal weight gain, fetal body weights were analyzed using a parametric analysis of variance followed by Tukey's multiple comparison test. Data on numbers of corpora lutea, implantations, resorptions, dead and alive fetuses, sex ratio and litter size were analyzed by the Kruskal–Wallis (nonparametric) test followed by Mann–Whitney U-test to determine which treatment groups differed from the control. The incidence of fetuses and litters with malformations was compared to that of the control group by using Chi square and Fischer's exact probability test, respectively.
Trend analysis via ANOVA, Jonckheere's test and the Cochran–Armitage test.
Indices:
no data
Historical control data:
NA
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
1 female at 500 mg/kg bw and 1 female at 1000 mg/kg bw
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see table
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
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
Description (incidence and severity):
statement in the publication
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
in both treament groups and in controls 2 abortions/group were reported (see table)
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Preimplantation loss ( [(Corpora lutea minus implant sites)/corpora lutea] x100): 24, 13 and 11 at 0, 500 and 1000 mg/kg bw
Postimplantation loss ([(Implant sites minus viable fetuses)/implant sites] x 100): 14, 23 and 22 at 0, 500 and 1000 mg/kg bw
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
total resorptions : 2, 1 and 1 at 0, 500 and 1000 mg/kg bw
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
Early 0.3 ± 0.6, 0.8 ± 1.5 and 0.5 ± 0.5 at 0, 500 and 1000 mg/kg bw
Late 0.05 ± 0.2, 0.05 ± 0.2 and 0.2 ± 0.5 at 0, 500 and 1000 mg/kg bw
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Dead fetuses 0.2 ± 1.1, 0 and 0.2 ± 0.7 at 0, 500 and 1000 mg/kg bw
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
pregnancy rate: 23/27, 21/27 and 21/27 at 0, 500 and 1000 mg/kg bw
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: absence of effects
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
fetal body weight (mean/litter) 35.0 ± 5.5, 36.8 ± 5.9 and 33.8 ± 8.7 g at 0, 500 and 1000 mg/kg bw
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Live fetuses 4.3 ± 1.8, 4.2 ± 1.5 and 4.8 ± 2.2 at 0, 500 and 1000 mg/kg bw
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio (M/F) 44/46, 44/35 and 43/48 at 0, 500 and 1000 mg/kg bw
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
see table
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
delayed ossification in controls and in both treatment groups (see table)
Visceral malformations:
no effects observed
Description (incidence and severity):
see table
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
The substance does not induce any developmental or teratogenic effects in rabbits.The NOAEL is 1000 mg/kg bw
Executive summary:

Pregnant New Zealand rabbits were given D-003 as oral doses of 500 and 1000 mg/kg/day on days 6 through 18 of gestation without any evidence of embryotoxicity or teratogenicity. The no-observed-effect dose in these two experimental studies was 1000 mg/kg/day. After assessment of the potential of high doses of D-003 to act on developing embryo, no evidence supports the conclusion that D-003 is a developmental toxicant/teratogen.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
purity 100 w/w%
Species:
mouse
Strain:
CD-1
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Kingston, NY)
- Age at study initiation: (Males 59 days) females 53 days
- Average weight at study initiation: females 24-25 g
- Fasting period before study: no (fasted during exposure)
- Housing: individually (in restrainer during exposure)
- Diet: ad libitum (except during exposure)
- Water: ad libitum (except during exposure)
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS: no details
- Temperature (°C): room temeprature
- Humidity (%): monitored
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
dermal
Vehicle:
water
Remarks:
for controls, low and mid dose
Details on exposure:
TEST SITE
- Area of exposure: 3x3 cm (dorsum clipped and shaved)
- Type of wrap if used: occlusion (sterile gauze and masking tape)
- Time intervals for shavings or clipplings: when necessary during the study

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes with water and sterile gauze
- Time after start of exposure: after each 6 hour exposure period

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 uL
- Concentration (if solution): 0, 12.5, 50 and 100%
- Constant volume used: yes

VEHICLE: deionized water

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (see picture in attached document)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC
Linear regression curve used for calculation of doses (no details provided)

The dosing solutions were stable for at least 19 days, so they were formulated once and used throughout the study. Cutaneous dosing formulations (low and mid) were 99.2-101.2% of target concentrations; the gavage solution was 106.1% of the target. No ethylene glycol was detected in the vehicle control solution with a minimum detection limit of 0.01 mg/ml.
The neat EG solution was not analyzed for ethylene glycol concentration.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: no details
- Proof of pregnancy: vaginal plug referred to as day 0 of gestation
Duration of treatment / exposure:
day 6-15 of gestation
Frequency of treatment:
daily during 6 hours in restrainer
Duration of test:
18 days
Dose / conc.:
404 mg/kg bw/day (actual dose received)
Remarks:
12.5%
Dose / conc.:
1 677 mg/kg bw/day (actual dose received)
Remarks:
50%
Dose / conc.:
3 549 mg/kg bw/day (actual dose received)
Remarks:
100%
No. of animals per sex per dose:
30 females/group with proof of successful mating
Control animals:
yes, concurrent vehicle
other: positive controls
Details on study design:
- Dose selection rationale: the route was selected to investigate route specific effects
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes (no details)

BODY WEIGHT: Yes (no details)

FOOD CONSUMPTION: No

WATER CONSUMPTION Yes (no details)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day18
- Organs examined: liver, paired kidneys, and gravid uterus weight; histopathological examination of kidneys of control, 100% and gavage treated mice; non-gravid uteri examined with Salewski staining

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:
Live and dead fetuses; fetal weight, sex
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
The unit of comparison was the pregnant female or the litter (Weil, 1970). Results of the quantitative continuous variables (e.g., maternal body weights, organ weights, fetal weights, etc.) were compared for the three ethylene glycol cutaneous exposure groups or the positive control group versus the vehicle control group by use of Levene's test for equal variances (Levene, 1960), analysis of variance (ANOVA), and / tests with Bonferroni probabilities.
The t tests were used when the F value from the ANOVA was significant When Levene's test indicated homogeneous variances, and the ANOVA was
significant, the pooled (test was used for pairwise comparisons. When Levene's test indicated heterogeneous variances, all groups were compared by an
ANOVA for unequal variances (Brown and Forsythe, 1974) followed, when necessary, by the separate variance t test.
Nonparametric data obtained following laparotomy were statistically treated using the Kruskal-Wallis test (Sokal and Rohlf, 1969) followed by the Mann-Whitney U test (Sokal and Rohlf, 1969) when appropriate.
Incidence data were compared using Fisher's exact test (Sokal and Rohlf, 1969). Statistical evaluation of the incidence of fetal malformations and variations was performed with the unit of comparison of the number of litters with one or more affected fetuses. For all statistical tests, the significance level was p « 0.05 (two-tailed).
Indices:
not reported
Historical control data:
no data, comparison with earlier studies on ethylene glycol via oral and inhalation exposure
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Maternal clinical signs included those limited to the restrained groups and associated with the restraint conditions, e.g., facial swelling and ocular discharge (from animals struggling during the period of restraint). In the positive control gavage group, clinical signs with increased incidences were death, hypoactivity, cold extremities, hunched posture, urogenital discharge, and tissue on paperboard (from abortions).
Dermal irritation (if dermal study):
no effects observed
Description (incidence and severity):
No dams which were cutaneously dosed exhibited any erythema or edema at the dosing site at any time during or after the dosing period.
Mortality:
no mortality observed
Description (incidence):
8 females died in the gavage treated positive control group
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Maternal body weights and weight gain were equivalent across all groups at all time points and intervals measured (see attached tables)
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption, expressed as grams/animal/day (see attached tables), exhibited a significant treatment-related increase only in the positive control gavage group and only for GD 6-9. Water consumption also appeared increased for GD 9-12, 12-15 and 6-15 (treatment period) in the positive control gavage group but the values were not statistically significantly different from those in the vehicle control group.
Ophthalmological findings:
not examined
Haematological findings:
not examined
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):
liver and kidney weights (absolute or relative to body weight), and gravid uterine weights were not affected by treatment (see attached tables)
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
In the positive control gavage group, there was a statistically significant incidence of lesions characterized as tubular nephrosis and tubular cell degeneration involving mainly the proximal tubules of the outer to middle cortex. Changes included necrosis and sloughing into the lumina of epithelial lining cells, dilated tubules lined by thinned, flattened, sparsely populated epithelium, and dilated tubules. lined by hyperplastic regenerating epithelium. Tubular cell degeneration was characterized by individual cell necrosis. No oxalate crystals were observed in normal or affected tubules.

The minimal-grade tubular lesions in 3 mice (of 30) in the high-cutaneous-dose group, similar to those in the positive control gavage group, may represent treatment-related effects or incidental findings
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
1, 0, 2 and 0 at control, 12.5, 50 and 100% (positive controls 0) (see attached tables)
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
see attached tables
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
see attached tables
Early or late resorptions:
no effects observed
Description (incidence and severity):
see attached tables
Dead fetuses:
no effects observed
Description (incidence and severity):
0.4, 0.1, 0.3 and 0.3 per litter at control, 12.5, 50 and 100% (positive controls 0.9/litter) (see attached tables)
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
25, 26, 23 and 22 females were pregnant at control, 12.5, 50 and 100% (positive controls 17) (see attached tables)
Other effects:
not specified
Details on maternal toxic effects:
Dosing by gavage at 3000 mg/kg/day (positive control group) produced maternal toxicity (deaths, increased water consumption, clinical signs of toxicity, and renal tubular lesions) . These effects are absent when ethylene glycol is applied dermally.
Dose descriptor:
NOAEL
Effect level:
3 549 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: absence of effects
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Average fetal body weights: 1.241, 1.255, 1.238 and 1.243 g at control 12.5, 50 and 100% (positive controls 0.99 g (sign decreased)) (see attached tables)
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
average percentage live fetuses/litter: 80.8, 71.8, 63.7 and 70.9 at control 12.5, 50 and 100% (positive controls 71.3%) (see attached tables)
Changes in sex ratio:
no effects observed
Description (incidence and severity):
percentage males: 47.1, 48.9, 47.3 and 50 at control 12.5, 50 and 100% (positive controls 47.7%) (see attached tables)
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
incidentally: protruding tongue (see attached tables)
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations include increased incidences of fusion of thoracic, lumbar, and sacral arches and/or centra, malaligned thoracic centra, missing thoracic arches, short rib (other than the 13th, which is a common variation), and fusion of 2—12 ribs (sse attached tables).

Variations:
The incidence of 56 skeletal variations (out of 170 different findings observed) exhibited statistically significantly different values in one or more treatment groups relative to the vehicle control group. Fifty-five of these were different for the positive control gavage group only. Fifty-one involved increased incidences of poorly ossified and unossified cervical thoracic, lumbar, and sacral centra, poorly ossified anterior arch of the atlas, extra thoracic centrum and arch, extra ribs (thoracic and lumbar), poorly ossified and unossified bones of the forelimbs and hind limbs in the paws (metatarsals and proximal phalanges of the hindlimbs), and of fused sternebrae, enlarged sagittal suture (one finding), and skewed thoracic centra. In addition, four findings exhibited a significantly decreased incidence in the positive control gavage group, including poorly ossified intermediate phalanges of the forelimb and hind limb, including some (1-4) or majority (5-7). These decreased findings were most probably because the incidence of all intermediate poorly ossified/unossified phalanges was increased in this group, but the incidence was not statistically significant.

The group cutaneously exposed to 100% ethylene glycol exhibited a statistically significantly increased incidence of two skeletal variations: poorly ossified parietal skull bone (also significantly increased in the positive control gavage group) and majority (6-9) of the intermediate phalanges of the hindlimb unossified. The latter increased incidence was observed only in this group. The incidence of pooled skeletal variations by fetus and by litter was 100% in all groups (i.e., all fetuses in all litters exhibited at least one skeletal finding) and the incidence of all variations was therefore also 100%
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Dilation of the lateral ventricles of the brain with tissue compression was observed incidentally (significantly increased in 9/17 litters of the positive controls) (see attached tables)
No treatmentrelated variations were observed
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
In the positive control (treated with ethylene glycol via gavage) significantly increased incidence of the visceral and skeletal malformations mentioned above were observed (see attached tables)
Dose descriptor:
NOAEL
Effect level:
3 549 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no
Executive summary:

Ethylene glycol (EG; CAS No. 107-21-1) is teratogenic to mice by whole-body exposure to an aerosol at high concentrations, but results were confounded by possible exposure from ingestion after grooming and/or from percutaneous absorption. Therefore, CD-I mice were exposed to EG on Gestational Days (GD) 6 through 15, 6 hr/day by occluded cutaneous application at 0, 12.5, 50, or 100% (undiluted) EG (0.1 ml/animal, equivalent to ~0, 404,1677,or 3549 mg/kg/day, respectively) or by gavage on GD 6 through 15 at 3000 mg/kg/day [10 ml/kg, positive control gavage group (PCGG)], 30 females/group. Dams were weighed and evaluated daily (including application site) for clinical signs and water consumption throughout gestation.

On GD 18, maternal uterus, liver, and paired kidneys were weighed; kidneys of 0 and 100% and the PCGG were examined microscopically. Corpora lutea and implantation sites were recorded. Live fetuses were weighed, sexed, and examined for structural alterations. For cutaneously exposed dams, there was no treatment-related maternal toxicity, no differences in pre- or postimplantation loss or in fetal body weights/ litter, and no increased incidences of any fetal malformations. Two skeletal variations, increased at 100%, may represent effects of restraint stress and/or findings due to chance. In the PCGG, 8 females (26.7%) died, water consumption was increased, fetal body weights/litter were reduced, and fetal malformations and variations were increased. PCGG kidneys exhibited tubular nephrosis and tubular cell degeneration, with no oxalate crystals, documenting renal toxicity at this oral dose in mice. Minimal-grade renal tubular lesions observed in 3 mice (of 30) at 100% EG may represent treatment-related or incidental findings. Therefore, exposure of pregnant CD-1 mice to 0, 12.5, 50, or 100% EG during organo-genesis by occluded cutaneous application resulted in minimal or no observable maternal or developmental toxicity at 100% ( ca. 3549 mg/kg/day), the NOAEL.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
mouse
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The hydrolysis product's analogue long-chain fatty acids (montanic acid, C28) was shown not to influence reproduction and development at concentrations of 1000 mg/kg bw. In general saturated fatty acids are not expected to exhibit any effects on reproduction and development.

The other hydrolysis product ethylene glycol when applied dermally to mice during day 6-15 of gestation, gave no effects on development at maternally non-toxic doses (NOAEL 3460 mg/kg bw).

Based on the results of the studies by Neeper Bradley (1999) it can be concluded that ethylene glycol could exhibit developmental toxicity. For the target substance the contribution of ethylene glycol is expected to be low, as exposure to the target substance is via the dermal route.

Justification for classification or non-classification

Based on the information available no classification according to Regulation EC 1272/2008 (CLP) is considered necessary.

Additional information