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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:
From March 07, 2011 to June 30, 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
OECD 414 guideline study without any deviation. However the Certificate of Analysis is not available and the analytical verification of doses is not specified. Alkyl Ether Carboxylate is considered adequate for read-across purpose (see §"Toxicokinetics").

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: transparent liquid
- Storage condition of test material: Stored in the dark at room temperature, at the study laboratory.
- Stability: stable, under storage conditions

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl: CD (SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Tsukuba Breeding Center, Charles River Laboratories Japan, Inc. (955, Kamibayashi, Ishioka-shi, Ibaraki Prefecture)
- Age at study initiation: no data
- Weight at study initiation: 239 to 285 g on Day 0 of gestation
- Housing: upon arrival and until paring, individually housed in in a stainless metal cage [260W × 380D × 180H (mm)]. For mating, each pair of male and female was housed together in a cage overnight for the maximum of 7 days.
- Diet (e.g. ad libitum): ad libitum, feedstuff (solid feed Labo MR Stock, lot No. 20110161, Nosan Corporation)
- Water (e.g. ad libitum): ad libitum, sterilized water ( prepared by filtration of tap water through a cartridge filter of 1-μm pore size, followed by ultraviolet light irradiation)
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): >= 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light.

IN-LIFE DATES: From: March 10, 2011 To: April 12, 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Purified water of Japanese Pharmacopoeia grade.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily using the test item as supplied by the Sponsor. The test substance, in an amount calculated for each dose, was weighed into a volumetric flask, and made up to volume by adding the vehicle while dissolving the test substance and swirling the solution. The dose formulations were prepared after correcting for the purity of the test substance (21.9%). That the dosing solutions were prepared at the prescribed concentrations was confirmed by the measurement of the solid content once during the study period.

VEHICLE
- Justification for use and choice of vehicle : had been prooved as appriopriate in previous studies
- Amount of vehicle: dose volume = 10 mL/kg body weight
- Lot/batch no.: lot No. 760513
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for the maximum of 7 days.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From GD6 through GD19
Frequency of treatment:
Once daily
Duration of test:
14 days
Doses / concentrationsopen allclose all
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: provided by the sponsor

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Visually inspected daily for morbidity, mortality and evidence of reaction to treatment or ill-health


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily from day 0 through 20 of gestation.

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days 0 and 3-20 (daily)
Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 0-6, 6-19 and 19-20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Individual food consumption was recorded on gestation day 0 and 3-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weigh change intervals .

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No, not a drinking water study

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gross observations of organs, maternal tissues were preserved


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:
- External examinations: Yes: all per litter
- Visceral examination: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
- Soft tissue examinations (head): Yes: half per litter
Statistics:
The significant difference (significance level ≤5%) of the mean and frequency of each index parameter obtained from pregnant animals between each test substance group and the control group was calculated according to the following methods:
- Parametric data (body weight of mother animals, amount of body weight increase, food consumption, number of corpora lutea, number of implantations, number of dead embryos/fetuses, number of live fetuses, uterine weight) were subjected to Bartlett’s variance test. If the variance was uniform, data were subjected to one-way analysis of variance and, if significant difference was observed, subjected to comparison of each group to the control group by Dunnett’s test.
- Data with non-uniform variance and nonparametric data (implantation rate, embryonic/fetal mortality, sex ratio of fetuses, fetal body weight, placental weight, and data of external, visceral, and skeletal examination) were subjected to Kruskal-Wallis rank test. If a significant difference was observed, data were subjected to comparison of each group to the control group by Dunnett type test.
- Categorical data (observation for clinical signs, necropsy) were subjected to Fisher’s exact test. For the data of fetuses, the mean value obtained from litter mates was handled as that of 1 sample.
Indices:
- Postimplantation loss/litter= (No. dead fetuses, resorptions (Early/Late)/Group) / No. Gravid Females/Group.
- Summation per group (%) = Sum of postimplantation loss/litter (%) / No. litters/group.
- Postimplantation loss/litter (%) = (No. dead fetuses, resorptions (Early/Late)/litter) / No. implantation sites/litter * 100
- Viable fetuses affected/litter (%) = (No. viable fetused affected/litter) / No. viable fetuses/litter * 100
Historical control data:
No data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- At 1000 mg/kg bw/day: mild decrease in locomotor activity in 12 of 25 animals, loose stool in 24, and soiled lower abdomen due to urine in 23 animals, on and after gestation day 7 (2 days after the start of administration); and transient mild salivation immediately after administration, on and after gestation day 6 (1 day after the start of administration). The decreased locomotor activity resolved around 4 hours after administration.
- At 60 and 250 mg/kg bw/day: no changes in clinical signs were observed (See Attached Table 7.8.2.2).
Mortality:
no mortality observed
Description (incidence):
No death was observed in any of the groups (See Attached Table 7.8.2.2).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 1000 mg/kg bw/day: decreased weight gain was observed during the gestation period. A significant decrease was observed in body weight from gestation day 9 through 20 and in body weight gain during the administration period.
- At 60 and 250 mg/kg bw/day: no significant changes were observed.
- No test substance-related effects on mean body weights, body weight gains, net body weight gains or gravid uterine weights were observed (See Attached Table 7.8.2.3).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- At 1000 mg/kg bw/day: a significant decrease in food consumption was observed from gestation day 6 through 20.
- Animals in 60 and 250 mg/kg bw/day groups did not show any significant change (See Attached Table 7.8.2.4).
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:
effects observed, treatment-related
Description (incidence and severity):
- Animals in 1000 mg/kg bw/day group showed a significant decrease in placental weight but did not show any significant change in other test parameters.
- Animals in 60 and 250 mg/kg bw/day groups did not show any significant change in uterine weight, or placental weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Necropsy revealed mild thickening of forestomach in 23 of 25 animals in 1000 mg/kg bw/day group. Mother animals in the control group, 60 mg/kg bw/day group, and 250 mg/kg bw/day group did not show gross abnormalities in the visceral organs, showing no abnormalities in the uterus, such as amniotic fluid, either (See Attached Table 7.8.2.5).
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):
See Attached Table 7.8.2.6.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
See Attached Table 7.8.2.6.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See Attached Table 7.8.2.6.
Early or late resorptions:
no effects observed
Description (incidence and severity):
See Attached Table 7.8.2.6.
Dead fetuses:
no effects observed
Description (incidence and severity):
See Attached Table 7.8.2.6.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
See Attached Table 7.8.2.6.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
The number of pregnant animals was 23 in the control group, 25 in 60 mg/kg bw/day group, 24 in 250 mg/kg bw/day group, and 25 in 1000 mg/kg bw/day group (See Attached Table 7.8.2.6).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Remarks on result:
other: Clinical signs, decreased body weight gain and food consumption observed at 1000 mg/kg bw/day

Maternal abnormalities

Key result
Abnormalities:
not specified

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No significant changes were observed in body weight of live fetuses in any of the test substance groups (See Attached Table 7.8.2.6).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No significant changes were observed in the number of live fetuses in any of the test substance groups (See Attached Table 7.8.2.6).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No significant changes were observed in the sex ratio in any of the test substance groups (See Attached Table 7.8.2.6).
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No significant changes were observed in any of the test substance groups (See Attached Table 7.8.2.6).
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- External examination including oral examination was conducted in 324 animals in the control group, 375 in 60 mg/kg bw/day group, 359 in 250 mg/kg bw/day group, and 360 in 1000 mg/kg bw/day group. Dwarf fetuses were observed as follows: 3 (incidence 0.9%) in the control group, 2 (0.7 %) in 60 mg/kg group, 3 (0.8 %) in 250 mg/kg bw/day group, and 1 (0.3 %) in 1000 mg/kg bw/day group. One (0.3%) each of cleft-lip fetus and tail-less fetus was observed in 1000 mg/kg bw/day group.
- There was no significant difference between the control group and each test substance group in the incidence of fetuses with external abnormality or in the incidence of each type of external abnormality (See Attached Table 7.8.2.7).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was no significant difference between the control group and each test substance group in the incidence of skeletal variations (See Attached Table 7.8.2.8) .
Skeletal examination was conducted in 160 fetuses in the control group, 186 in 60 mg/kg bw/day group, 172 in 250 mg/kg bw/day group, and 193 in 1000 mg/kg bw/day group.
- Skeletal anomalies observed were fused cervical vertebral arch and fused thoracic vertebral arch in 1 fetus each, 2 in total (incidence 1.0%) in the control group. In the test substance groups, the only skeletal anomaly observed was partially missing sacral vertebral bones in 1 fetus (0.5%) in 1000 mg/kg bw/day group.
- The following skeletal variations were observed in each group: Cervical rib (incidence 0-3.4%), asymmetric sternebrae (0-0.5%), dumbbell-shaped thoracic vertebral body (1.7-5.2%), split of the thoracic vertebral body (0.3-2.3%), short thoracic vertebral arch (0-0.5%), small thoracic vertebral body (0-1.0%), short 13th rib (0-1.6%), 14th rib (0-0.6%), lumbar rib (4.5-9.8%), dumbbell-shaped lumbar vertebral body (0-0.5%), sacralization of lumbar vertebral arch (0-0.5%), variation of the number of lumbar spinal bones (0-0.5%), split of the lumbar vertebral body (0-0.5%), small lumbar vertebral body (0-0.5%), and lumbar vertebral deformity (0-0.5%).
- The number of fetuses with any of these skeletal variations (incidence) was 21 (13.8 %) in the control group, 27 (13.7 %) in 60 mg/kg bw/day group, 16 (8.9 %) in 250 mg/kg bw/day group, and 31 (17.5 %) in 1000 mg/kg bw/day group, with the incidence showing no significant difference between the control group and each test substance group or no tendency of dose-dependent increase. The incidence of each type of skeletal variation did not show any significant difference between the control group and each test substance group, nor did it show any tendency of dose-dependent increase.
- As for ossification, all parameters including the numbers of ossified sternebral bones, caudal vertebral bones, metacarpal bones, and metatarsal bones were comparable between the control group and each test substance group, showing no significant changes. In 250 mg/kg bw/day group, one fetus (incidence 0.5%) showed delayed ossification of cervical vertebral arch and 3 fetuses (1.4%) showed delayed ossification of ischial bones, but none of them were significant.
Visceral malformations:
no effects observed
Description (incidence and severity):
Visceral examination was conducted on 164 fetuses in the control group, 189 in 60 mg/kg bw/day group, 187 in 250 mg/kg bw/day group, and 167 in 1000 mg/kg bw/day group (See Table 7.8.2.9)
- No fetuses with visceral anomalies were observed either in the control group or in the test substance groups.
- Visceral variations observed were thymic cervical residue (incidence 0.5%-1.3%) in all treatment groups and persistent left umbilical artery (1.7%) in 1000 mg/kg bw/day group.
- The number of fetuses with either of these visceral variations (incidence) was 2 (1.1%) in the control group, 2 (1.2%) in 60 mg/kg bw/day group, 3 (1.3%) in 250 mg/kg bw/day group, and 3 (2.1%) in 1000 mg/kg bw/day group, with no significant difference in the incidence between the control group and each test substance group. Also, no significant difference was observed in the incidence of each type of visceral variation between the control group and each test substance group.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were observed.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the NOAEL for maternal systemic toxicity was 1000 mg /kg bw/day based on clinical signs, decreased body weight gain and food consumption observed at 1000 mg/kg bw/day that are due to the irritant potential of the test substance. The NOAEL for developmental toxicity was 1000 mg/kg bw/day as no effects were observed at all tested doses.
Executive summary:

In a developmental toxicity study conducted according to the OECD guideline No. 414 and in compliance with GLP, the test substance diluted in deionized water was administered to 25 females SD [Crl: CD (SD)] rats/dose by gavage at dose levels of 0, 60, 250 or 1000 mg/kg bw/day from days 6 through 19 of gestation (dose volume = 10 mL/kg bw).

 

All animals were observed twice daily for appearance and behavior. Clinical observations, bodyweight and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female. The uteri and ovaries were examined, and the number of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighted, sexed and examined for external, visceral and skeletal malformations and developmental variation.

 

All animals survived to the scheduled necropsy on GD20. Animals at 1000 mg/kg bw/day showed changes in clinical signs including decreased locomotor activity, loose stool, soiled lower abdomen due to urine, and transient salivation immediately after administration, as well as decreased weight gain, decreased food consumption, thickening of forestomach, and decreased placental weight. No effects were observed in 60 and 250 mg/kg bw/day groups.

There were no test article-related internal findings at the scheduled necropsy. For the effect on fetuses, no significant changes were observed in the number of live fetuses, embryo/fetal mortality, sex ratio, body weight of live fetuses, or in the external, visceral, or skeletal examination of fetuses, at all tested animals. No test article-related fetal malformations or developmental variations were noted at any dose level in this study. No other signs of developmental toxicity were noted.

Based on the results of this study, the dose level of 1000 mg/kg bw/day was considered to be the NOAEL(systemic effects) for maternal toxicity and the NOAEL for developmental toxicity.

Under the test conditions, the test substance is not classified according to Regulation (EC) No.1272/2008 (CLP) criteria.

This study is acceptable and satisfies the requirement for developmental toxicity endpoint.

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