Alcohols, C11-15-secondary, ethoxylated

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 September 2015 to 22 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Conducted to GLP standards (ENV/MC/CHEM (98) 17)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

January 22, 2001

Deviations:

not specified

GLP compliance:

yes

Remarks:

Certificate not included

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, secondary C11-15, ethoxylated

EC Number:

614-295-4

Cas Number:

68131-40-8

Molecular formula:

C(11-15) H(23-31) O (C2H4O)xH where n= approximately 3

IUPAC Name:

Alcohols, secondary C11-15, ethoxylated

Details on test material:

- Name of test material (as cited in study report): Softanol 30
- Physical state: Slightly yellow clear liquid
- Analytical purity: 100%
- Lot/batch No.: 9G28W1
- Expiration date of the lot/batch: end of July 2010
- Storage condition of test material: Room temperature: +4°C in the dark

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor, Batch 15E25
- Expiration date of the lot/batch: Not reported
- Purity test date: <99%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Room temperature (21.6-25.0), protected form light and in airtight
container
- Stability under test conditions: in refrigerator for 7 days + 24h at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: soluble in corn oil
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): NOne
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid:N/A

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan Inc.
- Age at study initiation: 12 weeks for females; 23-26 weeks for males
- Weight at study initiation: 201-205g on receipt
- Fasting period before study: none
- Housing: single housing
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 15 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 41-53%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 30 September 2015 To: 02 January 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): poo solubility in water
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bodyweight
- Lot/batch no. (if required): V4T4854
- Purity: not reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of dosing solutions was by LC/MS/MS method using a 1290 Infinity LC system (Aglient Technol
ogies) with detection by API 4000, AB SCIEX MS/MS system. the gtest material was used as the refere
nce standard and corn oil as the control vehicle.
Dosing solutions of all concentrations prepared at the first and final preparation were analyzed for the
concentration of the test article. In the 20, 60, and 200 mg/mL dosing solutions, the contents of the test
article were 101.0%, 93.5%, and 90.5%, respectively, at the first preparation, and 106.0%, 107.5%, and
109.0%, respectively, at the final preparation; the relative standard deviations were 2.9%, 3.3%, and
1.1%, respectively, at the first preparation, and 2.8%, 0.5%, and 0.6%, respectively, at the final preparati
on. All these values were within the acceptance criteria of concentration (100% ± 15.0%) and standard
deviation (not more than 15.0%).
Stability of the 5 and 200 mg/mL preparations after 7-day storage (the day of preparation was defined as
day 0) in a refrigerator followed by 24-hrs storage at room temperature was confirmed in a related study
(SR15065)

Details on mating procedure:

Timing for mating: After completion of quarantine, mating started on quarantine and acclimatization day
15.
Mating period: Mating was performed for 9 days, until the required number (22 females/group) of suc
cessfully mated females were obtained. Clinical condition of females was observed once daily until
copulation was observed. Males for mating were observed for clinical condition once daily during the
mating period, and excluded from the study on the final day of mating period, and handled according to
the Standard Operating Procedures.
Mating procedures: Vaginal smear was observed by the vaginal smear method, and the females showing
proestrous vaginal smears were transferred to males’ cages in the early evening and paired overnight w
ith males on a 1:1 basis. On the following morning, successful mating was confirmed by the
presence of sperm in vaginal smears, and the day of confirmation of mating was defined as gestation day
0.
Group assignment: On the day of confirmation of mating, females were weighed and assigned to test
groups so that group mean body weight and standard deviations were as comparable as possible using
the computer system (MiTOX, Mitsui Zosen Systems Research Inc.).

Duration of treatment / exposure:

Treatment was from gestation days 6 to 19

Frequency of treatment:

Once daily from gestaton days 6 to 19

Duration of test:

21 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Remarks:

Individual dose volume was calculated based on the body
weight on gestation day 6

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Remarks:

Individual dose volume was calculated based on the body
weight on gestation day 6

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Individual dose volume was calculated based on the body
weight on gestation day 6

No. of animals per sex per dose:

22 females/dose group

Details on study design:

Dose selection rationale: In the preliminary teratogenicity study of Alcohols, C11-15-secondary, eth
oxylated in rats (dose levels: 100, 300 and 1000 mg/kg/day)1), soft feces, a decrease or suppression of
body weight gain, and a decrease in food consumption were noted in the 1000 mg/kg group. In fetuses,
no changes attributable to the test article administration were noted. Based on these results, 1000 mg/
kg that causes toxic changes in dams was selected as the high dose level, and 300 and 100 mg/kg were
selected by dividing the high dose level by a common ratio of approximately 3.
- Rationale for animal assignment (if not random): On the day of confirmation of mating, females were we
ighed and assigned to test groups so that group mean body weight and standard deviations were as co
mparable as possible using the computer system (MiTOX, Mitsui Zosen Systems Research Inc.).

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily during administration period and once daily during rest of study
- Cage side observations for mortality, external alteration, behavior and other changes.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: on gestatoin days 0, 3, 6, 9, 12, 15, 18 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: ovaries and uterus

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: No data

Fetal examinations:

- External examinations: Yes: all live fetuses per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

Statistical analyses were performed using the computer system (MiTOX). The data in the test article
groups and those in the control group were compared using the procedures shown below. Fetal data we
re processed using the litter as the unit of analysis. As animal Nos. 50164 and 50260 were non-pregnant,
all data of these animals were excluded from calculation of all examination parameters.
Group means and standard deviations were calculated for the maternal body weight, adjusted body
weight, body weight gain, food consumption, gravid uterine weight, numbers of corpora lutea, impl
antations, live fetuses, total postimplantation loss, and early and late deaths, body weight and placental
weight of live fetuses, and degree of ossification, which were analyzed for homogeneity of variances by
the Bartlett test. When the variances were homogeneous (p ≥ 0.05), the One-way analysis of variance
(ANOVA) was used, and when the variances were heterogeneous (p < 0.05), the Kruskal-Wallis test was
used to detect significant differences among groups. When the One-way ANOVA indicated a significant
difference (p < 0.10), Dunnett’s test was used for comparison with the control group. When the Kruska
l-Wallis test indicated a significant difference (p < 0.10), Steel’s test was used for comparison with the c
ontrol group.
The preimplantation loss (%), implantation index, post-implantation loss (%), early and late deaths (%
), incidences of fetal external anomalies or variations, and incidence of anomalous placentas were
analyzed by the Steel’s test to detect significant differences between the test article and control groups,
using the litter as the unit of analysis.
The sex ratio, and the incidences of dams with fetal external anomalies and variations and dams with
anomalous placentas were analyzed by Fisher’s exact probability test.
For comparative analysis with the control group, a p value of less than 5% was considered statistically
significant.

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, treatment-related

Description (incidence and severity):

n the 1000 mg/kg group, body weight gain was significantly low from gestation day 9, and body weight was significantly low on gestation days 9, 18, and 20. At study termination, no significant differences were noted in gravid uterine
weight compared to the control group; however, adjusted body weight was significantly lower than that in the control group.
In the 300 mg/kg group, body weight gain was significantly lower than that in the control group from gestation day 15; however, no significant differences were noted in the body weight. At study termination, in the 300 mg/kg group, gravid uterine weight was low in 4 dams, which was due to the small number of live fetuses (less than 10) of these animals.
No significant differences were noted in body weight or body weight gain in the 100 mg/kg group compared to the control group.No significant differences were noted in gravid uterine weight or adjusted body weight in the 100 mg/kg group compared to the control group at study termination.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

n the 1000 mg/kg group, food consumption was significantly lower than that in the control group from gestation days 9 to 18.
No significant differences were noted in food consumption in the 100 or 300 mg/kg group compared to the control group.

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:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

not examined

Other effects:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

<= 300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Fetal body weight by sex and their total in the 1000 mg/kg group were significantly lower than those in the control group.
No significant differences were noted in live fetal body weight by sex in the 100 or 300 mg/kg group compared to the control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One fetus in the 100 mg/kg group showed omphalocele and one fetus in the 1000 mg/kg group was conjoined twins.
There were no significant differences in the incidence of fetal external anomalies or dams with fetal external anomalies in the 100, 300, or 1000 mg/kg group compared to the control group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No significant differences were noted in the incidence of fetal skeletal variations in the 100, 300 or 1000 mg/kg group, or dams with fetal skeletal variations in the 100 or 1000 mg/kg group compared to the control group.
In the 300 mg/kg group, incidence of dams with fetal skeletal variation was significantly lower than that in the control group; this event was unmeaning. Skeletal variations noted in the test article groups were bipartite ossification, unilateral ossification, dumbbell ossification, and asymmetric ossification of sternebra, cervical supernumerary rib, short thoracolumbar supernumerary rib, unossified, bipartite ossification, and dumbbell ossification of thoracic centrum, and lumbarization of sacral vertebra. Incidences of these findings were not significantly different from those in the control group. In the control group, short 13th rib, full thoracolumbar supernumerary rib, and unilateral ossification of thoracic centrum were noted. Skeletal anomalies were not noted in any test article or control group.
In the degree of ossification, no significant differences were noted in the sacrocaudal centrum, sternebra, metacarpus, or metatarsus in any test article group compared to the control group.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No significant differences were noted in the incidence of fetal visceral anomalies or dams with fetal visceral anomalies in the 100, 300, or 1000 mg/kg group compared to the control group. Other visceral findings were thymic remnant in the neck in all groups including the control group, dilated cerebral ventricle and bilateral or left umbilical artery in few fetuses in the test article groups, and ventricular septum defect and dilated renal pelvis in the control group.

Other effects:

not examined

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

<= 300 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

The result tables for this study are provided below in the document attached as background material.

Applicant's summary and conclusion

Conclusions:

The No-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg/day in both dams and
fetuses. It was also concluded that the test article induces no teratogenicity up to the dose level of 1000 mg/kg/day.

Executive summary:

Alcohols, C11-15-secondary, ethoxylated was administered to 21 ~ 22 pregnant Crl:CD(SD) rats per group by oral gavage at doses of 100, 300, and 1000 mg/kg once daily from gestation days 6 to 19, and its potential effects on dams and embryos and fetuses were investigated.

In dams, body weight, body weight gain, and food consumption were low throughout the administration period, and adjusted body weight was also low in the 1000 mg/kg group. No effects of the test article administration were noted in clinical condition or necropsy. Live fetal body weight of both sexes was low in the 1000 mg/kg group. No effects of the test article administration were noted in the number of corpora lutea or implantations, implantation index, preimplantation loss (%), number of embryofetal deaths, postimplantation loss (%), number, sex ratio, or weight or state of the placenta of live fetuses up to the dose of 1000 mg/kg. No morphologic anomalies attributable to the test article administration were noted in the external, visceral, or skeletal examination of live fetuses up to the dose of 1000 mg/kg, and no changes caused by the test article administration were noted in the degree of ossification.

As described above, body weight and food consumption were low in dams at 1000 mg/kg/day, and body weight of fetuses was also low at the same dose level; therefore, the No-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg/day in both dams and fetuses. It was also concluded that the test article induces no teratogenicity up to the dose level of 1000 mg/kg/day.