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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:
25 January 2021 to September 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:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
tetrasodium 2-sulfonatododecanoate 2-sulfonatotetradecanoate
EC Number:
942-523-5
Cas Number:
2156594-77-1
Molecular formula:
Unspecified
IUPAC Name:
tetrasodium 2-sulfonatododecanoate 2-sulfonatotetradecanoate
Test material form:
solid
Specific details on test material used for the study:
Batch No. 0021238322/0021197214

Test animals

Species:
rat
Strain:
Wistar
Remarks:
The Wistar Hannover rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: females: 9 weeks old, males 11 weeks
- Weight at study initiation: females 200-225 g, males at least 336.3 g
- Fasting period before study: No, exception: As a part of the sacrificial procedure, blood samples for clinical chemistry investigations were withdrawn from the abdominal vena cava of 14 females/group, under food deprivation.
- Housing: Before and after mating, the animals were housed no more than 5 of one sex to a cage in
clear polysulfone cages measuring 59.5×38×20cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese); nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week. During the mating period, the rats were housed on the basis of 1 male to 1 female in clear polysulfone cages measuring 42.5×26.6×18.5cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese) with a stainless steel mesh lid and floor.
- Diet: ad libitum, laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei 4, 20019 Settimo Milanese (MI), Italy
- Water: ad libitum
- Acclimation period: appr. 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-2
- Humidity (%): 55+/-15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light):12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% aqueous solution of carboxymethylcellulose (CMC 0.5%)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was suspended in the vehicle. The formulation was prepared daily or up to 7 days (concentrations of 5, 20 and 80 mg/mL) according to stability data obtained from a
previous study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the formulations prepared during the study (the first and the last week of treatment) were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in ERBC SOP’s for suspension (85-115% for concentration and CV <10% for homogeneity).
Details on mating procedure:
- Impregnation procedure: cohoused
- Females were paired one to one in the home cage of the male and left overnight.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
All animals were dosed once a day from Day 6 through Day 19 post coitum.
Frequency of treatment:
daily
Duration of test:
All animals were killed on Day 20 post coitum.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
800 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 mated female rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Time of day for (rat) dam blood sampling: in the morning on day 20 post coitum

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting
from Day 0 post coitum.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 p.c.
- Organs examined: ovaries and uteri, brain, adrenals, kidney, liver, thyroid (the thyroid was also examined microsopically)

THYROID HORMONE DETERMINATION:
please refer to 'Any other information on materials and methods incl. tables'.
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
Blood sampling:
As a part of the sacrificial procedure, blood samples for clinical chemistry investigations were withdrawn from the abdominal vena cava of 14 females/group, under isofluorane anaesthesia and under condition of food deprivation. Additionally, thyroid hormone determination was performed.
- Volume collected: about 0.8 mL per animal
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Anogenital distance of all live rodent pups: yes
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by analysis of variance (one-way Anova). Inter-group differences were assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data (verified by Bartlett’s test). Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5.
Indices:
Pre-implantation loss, Post-implantation loss, Total implantation loss

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Hairloss was occasionally recorded in all groups including controls and was considered incidental.
Salivation was occasionally observed during the last days of gestation in 5 out of 25 females of the high dose group and, althought the short appearance, this sign was considered treatmet related.
Other findings recorded in one mid-dose animal (cyphosis, emaciated and teeth missing) and one high dose animal (piloerection, rales, semiclosed eyes) were considered as incidental or related to in-life procedures and were, thus, deemed as minor clinical signs, not related to treatment with the test item.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A trend of decrease in body weight starting from Days 12 post coitum, and body weight gain starting from Days 9 post coitum was detected in the high dose group when compared to controls and was considered to be related to treatment.
No treatment-related differences in body weight or body weight gain were noted in the low and mid-dose treated groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Slight statistically significant reduction in food consumption was noted in the high-dose females from Day 9 to Day 20 post coitum. This reduction was considered related to the exposure of the test item, indicating a slight maternal toxicity.
No significant changes were noted in the low and mid-dose groups, when compared to controls.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
High dose animals showed an increase of alanine aminotransferase (38%) and a decrease of cholesterol (30%) and triglycerides (65%). The changes of alanine aminotransferase and cholesterol were within the range of expected biological variation and therefore considered to be incidental. The decrease of triglycerides was mostly due to the high triglycerides values recorded in control animals and in those receiving 50 (low dose) and 200 mg/kg/day (mid-dose). This is due to the gestational hypertriglyceridemia, therefore the findings observed were considered to be unrelated to treatment.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
No differences between control and treated animals were recorded. Triiodothyronine (T3) was statistically significantly lower than controls in high dose females (29%). Most of the individual values were within the range of the control group data, were judged to be due to individual changes or due to biological variation and no other related changes were recorded (thyroxine and/or thyroid stimulating hormone changes, histopathological findings), therefore the T3 decrease was considered to be incidental.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No differences were detected in the gravid uterus weight between control and treated groups.
A statistically significant decrease in terminal body weight, corrected maternal body weight (terminal body weight at necropsy minus gravid uterine weight) and corrected body weight gain (terminal body weight at necropsy minus gravid uterine weight, minus body weight at Day 6 post coitum) was noted in high females. These decreases were considered related to the reduced increment in body weight noted in this group during the whole treatment period, indicating a slight maternal toxicity.
No treatment-related differences were observed in the remaining groups compared to controls. There was no effect of on the absolute and relative (to brain) organ weights in treated females when compared to controls.
Any organ weight variations were within the range of expected variations in Wistar rats of the same age and considered incidental and unrelated to treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At post mortem examination, no treatment-related macroscopic observations were noted in treated females when compared to controls.
Any macroscopic observations (alopecia) were within the range of expected spontaneous findings in Wistar rats of the same age considered incidental and unrelated to the test item.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Minimal follicular distension was found in the thyroid of one control animal. No treatment-related changes were noted in thyroid gland of all treated females when compared to controls. The microscopic observations corresponding to the macroscopic observations in control and treated females (alopecia) were consistent with those known to occur spontaneously in untreated Wistar rats of the same age and were considered incidental and unrelated to treatment.
Histopathological findings: neoplastic:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The percentage of post-implantation losses was significantly increased in the mid dose group. Total implanation losses were significantly increased in all groups except for the control. These statistically significant variations in total implantation loss of treated groups were considered incidental since they were not dose related or depended on the pre-implantation loss that occur prior to treatment.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The statistically increase in uterine death noted in the mid-dose group was considered not treatment related as mostly due to the loss of a single animal that showed signs of discomfort during the in vivo phase.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 7 females were found not pregnant at necropsy: 1 each in the control and low
dose groups, 2 in the mid-dose group and 3 in the high dose group. This finding can not be related to the treatment as dosing started on day 6 p.c..

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 6 foetuses were small (bodyweight<2.7g): 2 in the control group, 1 in the mid-dose group and 3 in the high dose group. The mean foetal weights per dose group were not significantly different from the control.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related differences were noted in the mean values of the anogenital distance of foetuses of both sexes maternally exposed at all dose levels compared to the control group. The statistically significant increase (approximately 13%) noted in female foetuses of low dose group compared to the control was considered incidental since the difference was slight and without any dose-relationship.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Hindlimb with abnormal shape as bent were noted in one foetus of the control group and 2 foetuses of the same litter in the high dose group. These findings were not confirmed as skeletal alteration at the skeletal examination of foetuses.
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In this Developmental Toxicity Study exposure to the test substance resulted in signs of systemic toxicity at a concentration of 800 mg/kg bw/d in the pregnant rats, such as reduced food consumption and impaired body weight development. Thus, the no observed adverse effect level (NOAEL) for maternal toxicity was 200 mg/kg bw/d. The no observed adverse effect level (NOAEL) for developmental toxicity was 800 mg/kg bw/d.
Executive summary:

The effects of the test substance were investigated, after oral administration in female Wistar Hannover rats during pregnancy and embryo-foetal development, from gestation Day 6 through Day 19. Females were mated with sexually mature males of the same strain and then assigned to 4 groups of 25 females each. The test item was applied at the following concentrations (CMC 0.5 % served as vehicle): test group 1 (control): 0 mg/kg bw/d, test group 2 (low dose): 50 mg/kg bw/d, test group 3 (mid dose): 200 mg/kg bw/d, test group 4 (high dose): 800 mg/kg bw/d.

Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All surviving females were caesarean-sectioned onDay 20 post coitum and subjected to post mortem examination. Blood collection for clinical chemistry and hormone determination was performed on Day 20 post coitum. Organ weigtht (brain, adrenals, kidney, liver and thyroid) was performed in all females. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, liver and uterusweights, foetalweight and sexwere recorded. The anogenital distance (AGD) in all live foetuses was recorded. All foetuses were examined for external abnormalities. Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

No animal died during the study. Clinical signs (salivation) noted in few high dose females were considered to be treatment related. A trend of decrease in body weight and body weight gain was detected in the high dose females, together with a slight statistically significant reduction in food consumption when compared to controls, indicating a slight maternal toxicity. No treatment-related effects concerning clinical biochemistry investigations or thyroid hormone determination were noted. There was no effect of on the absolute and relative (to brain) organ weights in treated females when compared to controls.

No treatment related effects were seen in litter data and sex ratios. No treatment related differences were noted in the anogenital distance in both sexes. The incidences of foetuses or litters with external findings did not indicate test item-related effect. No skeletal malformations were noted in foetuses.