Dodecyl(2-hydroxy-3-sulphonatopropyl)dimethylammonium

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A developmental study is available which was conducted according to OECD 414. A NOAEL of 100 mg/kgbw/day was assigned for the dams and a NOAEL of 300 mg/kg bw/day was assigned for the foetuses.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

Effect on fertility: via oral route

Endpoint conclusion:

no study available

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

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 29th 2012 to October 25th 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

-Lot: 1314
-Purity: 30.8% (w/w)

Species:

rat

Strain:

other: Crl: CD(SD) SPF rat

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CHARLES RIVER LABORATORIES JAPAN, INC., Tsukuba. Females were nulliparous, nonpregnant and untreated at initiation of the study.
- Age at delivery: Males/females were 11 weeks.
- Weight at study initiation: mean weight at start of treatment was 249 (230-270) g for female.
- Housing: each animal was housed in a stainless cage (260 W x 380 D x 180 H(mm))
-Mating: Females were caged together with stock males on a one-to-one-basis up to 7 days.
- Diet: Free access to solid feed (Nosan Corporation, Japan).
- Water: Free access to Sterile water (treated by 1 μm filter and UV)
- Acclimation period: At least 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3ºC (Actual temperature: 21.6-24.2ºC)
- Humidity: 55 ± 10% (Actual humidit: 53-64%)
- Air changes: ≥ 10 per hour
- Photoperiod: 12 hours dark / 12 hours light (From 7 am to 7 pm)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- Dose volume: 10 mL/kg body weight. Actual dose volumes were calculated according to the latest
body weight.
- Preparation of test solutions: each test solution was prepared by dilution of the test substance with purified water. Each concentration was corrected by the purity of the test substance.
- Storage conditions: 2-6ºC. The amount of solution for each day was separated and stored right after the preparation.
- Storage period: 8 days because analytical results showed that the test substance was stable at least for 8 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test concentrations of two solutions, 0.05% and 10% were analyzed by HPLC on the preparation day and 8 days later. The results showed that test sollutions were stable for at least 8 days after the preparation. Analytical results are as follows; 0.053-0.055% for 0.05%, 10.8-11.0% for 10%.

Details on mating procedure:

- M/F ratio per cage: 1/1 (one female was cohabitated with one stock male)
- Age at start of mating of the females in the study: Approximately 12 weeks
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage
and/or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 postcoitum.
- After successful mating each pregnant female was caged individually.

Duration of treatment / exposure:

Females were dosed from Day 6 to Day 19 postcoitum

Frequency of treatment:

Once in a day (in the morning)

Duration of test:

Day 20 postcoitum (20 days after the pregnancy)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

-Dose-finding and composition of the study groups:
In a preliminary teratogenicity study of this test article (Study number: P12-032), the test article was administered to animals by gavage at a dose of 0 (control), 10, 30, 100 and 300 mg/kg/day from Day 6 to Day 19 of pregnancy, and the animals were subjected to Caesarean section on Day 20 of pregnancy. As a result, soft stool, diarrhea and transient salivation that occurred immediately after dosing were observed in the 300 mg/kg/day, and hypertrophy of the forestomach was observed in all of the animals. In addition, decreased food consumption and a tendency to decrease body weight gain was also observed during the early phase of pregnancy. No changes were observed in the 10, 30 and 100 mg/kg treatment groups.
From these results, 300 mg/kg/day that can cause toxic effects on dams was selected as a high dose, and 30 mg/kg/day that may not cause toxic effects was selected as a low dose. In addition, 100 mg/kg/day was also selected as a medium dose, and a total of 3 doses were established.
The study groups consisted of the vehicle control group (hereinafter referred to as the control group), 30 mg/kg/day AMPHITOL 20HD treatment group (30 mg/kg group), 100 mg/kg/day AMPHITOL 20HD treatment group (100 mg/kg/day group) and 300 mg/kg/day AMPHITOL 20HD treatment group (300 mg/kg/day group), a total of 4 groups.

Maternal examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: At least once a day after the start of treatment. Death, appearance, behavior were the ponits to be observed.

BODY WEIGHT
- Time schedule for examinations: Days 0, 3, 6, 7, 9, 12, 15, 17, 19 and 20 post-coitum. The body gain between day 0 and 6, and day 6 and 20 were calculated, respectively.

FOOD CONSUMPTION
- Days 0, 3, 6, 7, 9, 12, 15, 17, 19 and 20 post-coitum. The amount of both initial diet and the diet remained were measured.

Ovaries and uterine content:

Animals were exsanguinated on Day 20 of pregnancy under isoflurane inhalation anesthesia and subjected to necropsy. Main organs in the thoracic and abdominal cavities were macroscopically observed. The ovary and uterus were harvested, and the number of corpus luteum in the ovary, the number of implantation in the uterus, and the weight of uterus and placenta were measured. Then, the percentage (%) of implantation [(the number of implantation/the number of corpus luteum) × 100] was calculated.

Fetal examinations:

-Number of fetuses, sex and weight of fetus
The number of live fetuses and the number of dead embryos/fetuses were counted and the embryonic/fetal mortality was calculated. Dead embryos/fetuses were classified into preimplantation deaths (implantation sites, placental remnants in a size less than a grain of rice) and postimplantation deaths (placental remnants in a size larger than a grain of rice, macerated fetuses, dead fetuses) and recorded. Live fetuses were sexed and weighed. The sex of fetuses was determined based on the distance between the anus and genital protrusion. Body weights were measured in each animal.

-External surface, internal organs and skeletal examinations
External surfaces including oral cavity were examined for any abnormalities in live fetuses. In the visceral examination, a fetus of a litter that was implanted in the left uterine horn was fixed with Buin’s solution, and the head and organs in the thoracic and abdominal cavities were macroscopically observed and observed under steroscopic microscope as necessary for any abnormalities in accordance with the Barrow & Taylor’s method1). In the skeletal examination, a fetus implanted in the right uterine horn was fixed with 95% ethanol and the skin was stripped. Then, transparent skeletal preparations were made after cleaning the skeleton with 1% potassium hydroxide by the Dawson’s method2) and staining with Alizarin Red S, and were observed under steroscopic microscope for any skeletal abnormalities, mutations and ossification. Degree of ossification (degree of advancement of the ossification process) was evaluated using the number of ossified sternabrae, caudal vertebrae, metacarpals and metatarsals as an index. A small bone fragment that was not sufficiently stained with Alizarin-red S was counted as 0.5 bones for calculation of the number of ossified bones.

Statistics:

A significant difference (a risk ratio of 5% or less) in the mean or frequency of each parameter in pregnant animals between each test article treatment group and the control group was calculated using the following method.
Body weight of dams, body weight gain, food consumption, number of corpus luteum, number of implantation, number of dead embryos/fetuses, number of live fetuses and uterine weight were analyzed using Bartlett’s variance test. As a result, when the homogeneity of variance assumption was met, then one-way ANOVA was conducted, and when there was a significant difference, Dunnett’s test was conducted to compare each treatment group to the control. When the homogeneity of variance assumption was not met, or for the data of implantation rate, embryonic/fetal mortality, sex ratio of fetuses, body weight of live fetuses, placental weight, external surface, internal organs and skeletal examination, Kruskal-Wallis rank test was conducted. Then, when there was a significant difference, Dunnett’s test was conducted to compare each treatment group to the control. Chi-square test was conducted for data from clinical observations and necropsy. For data on fetuses, the mean of a litter was determined as one sample.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 300 mg/kg group, mild salivation was transiently observed immediately after dosing in all of 25 animals from Day 7 of pregnancy (2 days after start of treatment) onwards, and soft stools were occasionally observed in 14 animals from Day 7 of pregnancy onwards. In addition to salivation and soft stools in one animal of the same group, emaciation, ptosis and soiled fur in the lower abdominal region were observed from Day 16 of pregnancy (11 days after start of treatment) onwards, piloerection, diarrhea and smudge of perioral/perinasal area were observed on Day 18 of pregnancy (13 days after start of treatment), and vaginal hemorrhage and delivery of dead fetus were observed on Day 19 of pregnancy (14 days after start of treatment). Thus, this animal was considered to have miscarriage.

No changes in clinical signs were observed in the 30 and 100 mg/kg treatment groups.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed in any treatment groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly low body weights were observed in the 300 mg/kg group from Day 9 of pregnancy (4 days after start of treatment) onwards. Significantly decreased body weight gain was observed from Days 6 to 20 of pregnancy during the treatment period.

No significant changes in body weight and body weight gain were observed at each measuring time point in the 30 and 100 mg/kg groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Significantly low food consumption was observed in the 300 mg/kg group from Days 6 to 12 of pregnancy and from Days 17 to 20 of pregnancy.

No significant changes in food consumption were observed at each measuring time point in the 30 and 100 mg/kg groups.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No significant changes in the number of corpus luteum, number of implantation, implantation rate, uterine weight and placental weight were observed in any treatment groups.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

-Observations of the inside of maternal body
Forestomach hypertrophy was observed in all of 25 animals in the 300 mg/kg group. Red spots in the stomach were observed in 2 animals including one that had miscarriage.

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

-Effect on pregnant dams
As the result of mating, there were one each of unmated animals in the 30 and 100 mg/kg groups. At the time of Caesarean section, one non-pregnant animal was observed in the 100 mg/kg group. There were 25 pregnant animals in the control group, 24 in the 30 mg/kg group, 23 in the 100 mg/kg group and 25 in the 300 mg/kg group. The effect of AMPHITOL 20HD on these animals are shown as follows.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly reduced weight changes of male and female fetuses was only observed in the 300 mg/kg group.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly decreased number of live female fetuses was only observed in the 30 mg/kg group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No significant changes were observed in any treatment groups.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The external surface including oral cavity was inspected in 348 fetuses of the control group, 315 of the 30 mg/kg group, 324 of the 100 mg/kg group and 326 of the 300 mg/kg group.
One dwarf fetus each (an incidence of 0.3%) was observed in the control and the 30 mg/kg groups, and 2 acaudal fetuses (0.6%) were observed in the control group, but there was no significant change.
No fetuses with external abnormalities were observed in the 100 and 300 mg/kg treatment groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal examination was conducted in 181 fetuses of the control group, 161 of the 30 mg/kg group, 183 of the 100 mg/kg group and 164 of the 300 mg/kg group.

As skeletal abnormalities, sacral deficit was observed in one of 2 acaudal fetuses in the control group. Thus, this fetus was considered to have sacrococcygeal deficit (an incidence of 1.0%) and another fetus was considered to have coccygeal deficit (0.6%).
As skeletal mutations, dumbbell-shaped thoracic vertebrae (an incidence of 3.8 to 13.4%), separation of the thoracic vertebral body (2.3 to 3.1%) and lumbar rib (3.7 to 12.1%) were observed in each treatment group including the control group. Separated sternabrae (0.5%), shortened 13th rib (0.4%) and the 14th rib (0.4%) in the control group, dumbbell-shaped lumbar vertebrae (0.7%) in the 30 mg/kg group, cervical rib (0.6%) in the 100 mg/kg group, and cervical rib (0.4%) and dumbbell-shaped lumbar vertebrae (0.5%) in the 300 mg/kg group were observed. The number of fetuses with any of these skeletal mutations is shown as follows: 18 fetuses (11.0%) in the control group, 44 (27.1%) in the 30 mg/kg group, 24 (14.5%) in the 100 mg/kg group and 25 (13.4%) in the 300 mg/kg group. There was no significant difference in the incidence between each treatment group and the control group, and a dose-dependent increasing trend for the incidence was not also observed. As for an incidence of skeletal mutations by type, there was no significant difference between the control and test article treatment groups, and a dose-dependent increasing trend for the incidence was not also observed.

As for ossification, significantly increased number of ossified sternabrae and ossified metacarpals were observed in the 300 mg/kg group. No significant changes in the number of ossified coccygeal vertebrae and ossified metatarsals were observed in any treatment groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral examination was conducted in 167 fetuses of the control group, 154 of the 30 mg/kg group, 141 of the 100 mg/kg group and 162 of the 300 mg/kg group.

No fetuses with visceral abnormalities were observed in the control group and the treatment groups.

Twisted ureter (an incidence of 1.2 to 2.1%) were observed in each treatment group including the control group as visceral mutation. In addition, residual thymus in the cervical region (0.6 to 1.4%) was observed in each treatment group except the 100 mg/kg group. Left umbilical artery remnant (2.5% and 0.5%) was observed in the control and 100 mg/kg groups. Pyelectasia (0.8% and 1.1%) was observed in the 30 and 300 mg/kg groups. The number (incidence) of fetuses with any visceral mutations is shown as follows: 8 fetuses (3.9%) in the control group, 4 (2.4%) in the 30 mg/kg group, 3 (1.8%) in the 100 mg/kg group and 5 (3.2%) in the 300 mg/kg group. There was no significant difference in the incidence between each treatment group and the control group, and a dose-dependent increasing trend for the incidence was not also observed. As an incidence of visceral mutations by type, left umbilical artery remnant was observed in 2 fetuses of the control group but was not observed in the 30 and 300 mg/kg groups. Thus, there was a statistically significant difference between these groups. However, there was no significant difference in other visceral mutations between the control and test article treatment groups, and a dose-dependent increasing trend for the incidence was not also observed.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Highest concentration dosed.

Key result

Developmental effects observed:

no

Discussion

As the effect of AMPHITOL 20HD on pregnant dams, transient salivation that occurred immediately after dosing and hypertrophy of the forestomach were observed in the 300 mg/kg group. These changes were also observed in the preliminary teratogenicity study in pregnant rats by oral gavage. It has been confirmed that AMPHITOL 20HD had an irritating effect on the eye mucous membrane of rabbits (Product safety data sheet by Kao Corporation). Thus, salivation and hypertrophy of the forestomach observed in this study might be changes attributable to the local irritating effect of the test article solution. In relation to these findings, soft stools, decreased food consumption and decreased body weight gain during the treatment period (pregnancy period) might also be observed in the same group. Similar changes were also observed in the previous preliminary teratogenicity study. No changes attributable to such a local irritating effect of this test article was observed in the 13 -week dietary repeated-dose study in which rats were fed diet containing this test article at the highest concentration of 0.5%. Miscarriage was also observed in one animal of the 300 mg/kg treatment groups.

As the effect on fetuses, significantly decreased number of live female fetuses (6.1) was observed in the 30 mg/kg treatment group. However this change was considered to be an incidental change without any relation to treatment with the test article since the number of live fetuses per litter was within the reference value [number of live female fetuses: 3.17 to 11.89 (mean, 7.53)] in our laboratory’s background data and there was no dose-dependency. Significantly increased mean body weight of male and female fetuses (male: 4.03 g, female: 3.82 g) observed in the 300 mg/kg group was not considered to be indicative of the toxicological effect but was considered to be an incidental change, since the mean body weight of male and female fetuses per litter was within the reference values [mean male fetal body weight: 3.13 to 4.43 g (mean 3.78 g), mean female fetal body weight: 2.96 to 4.22 g (mean 3.59 g)] in the background data except one each of male and female fetuses of which the mean body weight exceeded the reference value.

No test article-related changes in the number of dead embryos/fetuses, embryonic/fetal mortality, sex ratio and body weight of live fetuses were observed.

In the fetal examination, external abnormality that was considered to occur spontaneously was only observed in one fetus of the 30 mg/kg group, and no visceral or skeletal abnormalities were observed in any test article treatment groups. Visceral and skeletal mutations occasionally observed in each group were spontaneous changes, and no test article-related changes were observed.

As for ossification, increased number of ossified sternabrae (5.5) and metacarpals (3.3) was observed in the 300 mg/kg group. However, it was considered to be an incidental change related to slightly higher mean fetal body weights (both males and females in this group) since the value per litter was within the reference values [the number of ossified sternabrae: 4.38 to 6.15 (mean, 5.27), and the number of ossified metacarpals: 2.93 to 3.82 (mean, 3.37)] in our laboratory’s background data and there was also no dose-dependent increasing trend.

From these results, it was considered that the no-observed adverse effect level (NOAEL) of AMPHITOL 20HD was 100 mg/kg/day for dams and 300 mg/kg/day for fetuses under the conditions of this experiment. No changes indicative of teratogenicity was observed in fetuses even when the test article was administered to dams at a dose up to 300 mg/kg/day.

 

Conclusions:

Based on experiemntal results, the no observed adverse effect level (NOAEL) of Amphitol 20HD was determined to be 100 mg/kg/day for dams and 300 mg/kg/day for fetuses in the present teratogenicity study in rats. No change suggesting teratogenicity in fetuses was observed when up to 300 mg/kg/day of the test article was administered to dams.

Executive summary:

To investigate the teratogenicity of Amphitol 20HD, the test article was orally administered at 0 (control; purified water), 30, 100, or 300 mg/kg bw/d using a gastric tube to SD (Crl: CD [SD]) rats on Days 6 to 19 of pregnancy. The animals were subjected to caesarean section on Day 20 of pregnancy. Twenty-five female animals per dose group were mated, resulting in pregnancy in 25, 24, 23, and 25 animals in the control, 30, 100, and 300 mg/kg bw/d groups, respectively.  For the effects on pregnant dams, transient salivation and forestomach thickening were observed immediately after administration in the 300 mg/kg group, which were associated with soft faeces, reduced food consumption, and suppressed body weight gain during the administration (pregnancy) period. One animal in the 300 mg/kg bw/d group had an abortion. No toxic effect on dams was observed in the 30 or 100 mg/kg bw/d groups.  For the effects on foetuses, no toxic effect was observed in any test article dose group for the number of live foetuses, number/rate of embryonic/foetal death, sex ratio, body weight of live foetuses; or external, visceral or skeletal examination findings.  Based on these results, the no observed adverse effect level (NOAEL) of Amphitol 20HD was determined to be 100 mg/kg bw/d for dams and 300 mg/kg bw/d for foetuses in the present teratogenicity study in rats. No change suggesting teratogenicity in foetuses was observed when up to 300 mg/kg bw/d of the test article was administered to dams.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available