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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:
key study
Study period:
12 April 2021 to 22 October 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Asphalt, sulfonated, sodium salt
EC Number:
269-212-0
EC Name:
Asphalt, sulfonated, sodium salt
Cas Number:
68201-32-1
Molecular formula:
UVCB
IUPAC Name:
Asphalt, sulfonated, sodium salt

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: Wistar (Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France.
- Age at study initiation: approximately 6-7 weeks old
- Weight at study initiation: approximately 161 to 206 g males and 109 - 152 g females
- Fasting period before study: No
- Housing: Animals were individually housed in polycarbonate cages (Makrolon type MIII, height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS-J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Cages were arranged on the racks according to a Latin-square model.
- Diet (e.g. ad libitum): Animals were fed ad libitum, except during designated procedures with SM R/M-Z (from SSNIFF® Spezialdiäten GmbH) as pellets (alternate diet may have been provided on an individual animal basis as warranted and approved by the Study Director).
- Water (e.g. ad libitum): Municipal drinking water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22ºC
- Humidity (%): 49 to 76% humidity
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

IN-LIFE DATES: From: 25th May 2021 To: 18th June 2021 (last date of necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Tap water purified by a Elix water purification system (Millipore, Bedford, MA, USA)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): n/a
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water): Water (Elix)
- Concentration in vehicle: 0, 20, 60 or 200 mg/mL
- Amount of vehicle (if gavage): 5 mL
- Lot/batch no. (if required): n/a
- Purity: n/a
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Test formulations prepared were considered homogeneous at the concentrations tested, and analysis of the accuracy revealed acceptable levels.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant arrived day 0 or 1 post-coitum
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy:
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
Day 6 to day 20 post-coitum
Frequency of treatment:
Once daily
Duration of test:
14 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 - Control (Vehicle only)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2 - SAS treatment
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3 - SAS treatment
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4 - SAS treatment
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected to be 0, 100, 300, 1000 mg/kg bw/day, based on a previously conducted Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test study (OECD 422) with oral exposure of Asphalt, Sulfonated, Sodium Salt (SAS) in Sprague-Dawley rats.
- Rationale for animal assignment (if not random): Random
- Fasting period before blood sampling for (rat) dam thyroid hormones: No fasting
- Time of day for (rat) dam blood sampling: Between 07.00 and 09.00 from the jugular vein
- Other: n/a

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes, on all animals, within their cage.
- Time schedule: Twice daily; starting on Day 6 post-coitum up to the day prior to necropsy. At 1 hour and 5 hours post dose.

DETAILED CLINICAL OBSERVATIONS: Yes, on all animals (removed from their cage)
- Time schedule: On Days 2, 6, 15 and 21 post-coitum

BODY WEIGHT: Yes, on all animals
- Time schedule for examinations: On Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, on all animals.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, over Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes, water consumption was monitored by visual inspection of the water bottles. If inter group differences were noted, consumption was assessed by weight.
- Time schedule for examinations: A regular basis throughout the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 post-coitum
- Organs examined: All animals (including animals found dead or sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. The thyroid gland was weighed at necropsy for all scheduled euthanasia animals, except for females that delivered their offspring early. Organ weights were not recorded for animals found dead, euthanized in poor condition or in extremis or that had delivered their offspring early. Paired organs were weighed together. Organ weight as a percent of body weight (using the body weight on Day 21 post-coitum) was calculated.The thyroid gland and macroscopic abnormalities were collected from all animals and preserved in 10% buffered formalin.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (not for animals found dead, sacrificed before planned necropsy or that had started to deliver)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number and distribution of live and dead fetuses: Yes
- The sex of each fetus: Based on the anogenital distance (not for animals found dead, sacrificed before planned necropsy or that had started to deliver).
Blood sampling:
- Plasma: No
- Serum: Yes, for T3, T4 and TSH analysis
- Volume collected : 1.0 mL
Fetal examinations:
- 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
- Anogenital distance of all live rodent pups: Yes, in all viable fetuses
Statistics:
- Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences will be reported as appropriate by dataset.
- Inferential statistical analysis: All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and will be reported at the 1% and 5% levels, unless otherwise noted. Parametric/Non-Parametric; Non-Parametric depending on the variables; Analysis of covariance (ANCOVA) the data corresponding to a response variable of interest and to a related covariate were submitted to ANCOVA, including only groups with at least three non-missing paired values. If found to be significant, pairwise comparisons were conducted using Dunnett’s test.
- Incidence: A Fisher’s exact test was used to conduct pairwise group comparisons of interest.
Historical control data:
Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in the Wistar Han rat from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, mean body weight gain was decreased (10.3% lower than control; not statistically significant) between Days 18-21 post-coitum, leading to a decreased trend in overall body weight between Days 6-21 post-coitum (3.3% lower than control; not statistically significant). Mean body weight adjusted for gravid uterus weight was decreased at 1000 mg/kg bw/day (9.8% lower than control; not statistically significant). Mean body weights, body weight gain and adjusted body weight gain at 300 and 100 mg/kg bw/day were considered to be in the same range as the controls over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, mean food consumption was slightly decreased at the start of treatment (Days 6-9 post-coitum; 8% lower than control, not statistically significant), and a low trend remained during the total treatment period (4.7% lower than control between Days 6-21 postcoitum; not statistically significant). Mean food consumption at 300 and 100 mg/kg bw/day was considered to be in the same range as the controls.
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
Endocrine findings:
no effects observed
Description (incidence and severity):
Mean serum levels of Total T3, Total T4 and TSH were considered to be unaffected by treatment with the test item up to 1000 mg/kg bw/day.
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):
There were no test item-related alterations in thyroid gland weights.

Mean gravid uterus weight at 1000 mg/kg bw/day was slightly lower (5.9% lower than control; not statistically significant). This was mostly caused by Female Nos. 71 and 79 with 3 and 5 fetuses, respectively. As all individual weights were within the range of biological variation, this was considered not test-item related. Mean gravid uterus weight at 300 and 100 mg/kg bw/day was considered unaffected by treatment with the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations in the thyroid glands. The recorded macroscopic finding of the thyroid gland in a single animal (bilateral enlargement at 1000 mg/kg bw/day) had no microscopic correlate and was within the range of background gross observations encountered in rats of this age and strain. The only remaining finding was scabbing of the skin in a single female in the control group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
All pregnant females had live fetuses. Therefore, the number of females with viable litters for evaluation was 21, 21, 21 and 19 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The mean percentage pre-implantation loss in the control and test groups were considered in the range of normal biological variation.
The mean percentage of post-implantation loss at 300 mg/kg bw/day was slightly higher compared to control (6.05% vs. 3.09%; not statistically significant) but remained within the range of the available Historical Control Data. This was mostly due to Female Nos. 47 and 58 with a relative high number of early resorptions compared to total number of implantations (n=3/9 and 4/9, respectively). In the absence of a dose-related effect, this was considered not test item-related.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
The mean number of early or late resorptions in the control and test groups were considered in the range of normal biological variation.
Dead fetuses:
no effects observed
Description (incidence and severity):
The mean number of dead fetuses in the control and test groups were considered in the range of normal biological variation.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The mean number of corpora lutea at 1000 mg/kg bw/day was slightly lower (10% lower than control), although not statistically significant. This was mostly due to Female Nos. 71 and 79 with a relatively low number of corpora lutea (n=3 and n=5, respectively) compared to available Historical Control Data. Accordingly, mean numbers of implants and live fetuses at 1000 mg/kg bw/day were slightly lower (8% and 7% below control, respectively; not statistically significant). However, since treatment with the test item did not start before implantation of the conceptus was completed, these lower numbers in corpora lutea and consequently lower litter sizes were considered to reflect normal biological variation.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean fetal body weights (male, female and combined) of all treated groups were considered unaffected by treatment with the test item.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was considered unaffected by treatment with the test item up to 1000 mg/kg bw/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter size was considered unaffected by treatment with the test item.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
Mean fetal anogenital distance (both sexes) of all treated groups was considered unaffected by treatment with the test item.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
External malformations were observed in one fetus of each of the low- and high-dose groups. The low-dose fetus (No. 42-R5) had no lower jaw, whereas the lower jaw of the high dose fetus (70-L5) was small. Due to single occurrence and the resemblance, these malformations were considered chance findings. There were no external variations observed in any groups.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Both fetuses of which the lower jaw was externally affected (Nos. 42-R5; low dose and 70-L5; high dose), had matching (small mandibles) and relating skull malformations. Additionally, skeletal malformations occurred in two other fetuses of the high-dose group. Fetus No. 85-R5 was also noted with lower jaw malformations (fused mandibles with a single incisor socket) despite a normal jaw externally. The malformations affecting the jaw were considered not test item-related due to the low incidence and absence of a clear dose-response. The other high-dose fetus (No. 75-L5) had lumbar vertebral malformations that were considered chance findings due to their single occurrence.

All skeletal variations occurred in the absence of a dose-related incidence trend, infrequently, and/or in control fetuses only. Therefore, they were considered not test item-related.
Visceral malformations:
no effects observed
Description (incidence and severity):
Visceral malformations were not observed in this study, and the visceral variations that were noted affected the liver (supernumerary lobes) and ureters (convoluted). The low incidences and group distribution of these findings did not indicate any test item-relationship.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test item-related changes were noted in any of the developmental parameters investigated (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations and developmental variations).

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results of this prenatal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, in time-mated female Wistar Han rats, the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Asphalt, Sulfonated, Sodium Salt (SAS) were established to be greater than or equal to 1000 mg/kg bw/day.