Reaction mass of sodium 1-methoxy-1-oxohexadecane-2-sulphonate and sodium methyl 2-sulphooctadecanoate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was not performed according to GLP, but was well described, and basically agreed with OECD 414 (2001), although there were deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See below.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: See below

Deviations:

not specified

Principles of method if other than guideline:

It was reported that the study complied with the following guidelines: Guidelines for Toxicity Studies Required for Applications to Manufacture (Import) Drugs” Notification No. 24 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, September 11, 1989; 1990 Guidelines for Toxicity Studies of Drug Manual, First Edition (published in 1991)., Editorial Supervision by New Drugs Division Pharmaceutical Affairs Bureau, Ministry of Health and Welfare of Japan, Yakuji Nippo Ltd.
The study was performed in agreement with OECD 414 (2001), with the following deviations: the scheme for dose administration (day 7 through 17 of presumed gestation) does not agree with OECD 414 (2001), which states day 5 post mating to the day prior to scheduled caesarian section. In addition, the gravid uterine weight was not determined.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

α-sulfonated Fatty Acid Methyl Ester Sodium Salt

IUPAC Name:

α-sulfonated Fatty Acid Methyl Ester Sodium Salt

Test material form:

solid

Details on test material:

- Physical state: White solid
- Storage condition of test material: Room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: males 9 weeks, females 8 weeks
- Weight at study initiation: upon receipt (day -6) males 273–299 g, females 156–183 g
- Fasting period before study: no
- Housing: Rat cage; Quarantine period: 3–4 animals per cage; Mating period 1 male and 1 female per cage; Test period: 1 animal per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26
- Humidity (%): 30-70
- Air changes (per hr): >= 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: November 5, 1990 To: December 17, 1990

Administration / exposure

Route of administration:

dermal

Vehicle:

water

Details on exposure:

TEST SITE
- Area of exposure: 3 x 4 centimeter area
- % coverage: the entire 3 x 4 cm area was treated with 0.2 mL of test formulation
- Type of wrap if used: none
- Time intervals for shavings or clipplings: On the day before administration (Day 6 of pregnancy), the hair of animals was clipped using an electric hair clipper to create a 3 x 4 centimeter area for application. Thereafter, hair was clipped several times per week during the administration period, according to hair growth.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.2 mL
- Concentration (if solution): 1.5, 5 and 15 w/v%
- Constant volume or concentration used: yes
- For solids, paste formed: The test substance is solid at an ambient temperature. In order for it to assume the form of a uniform paste, it was melted in a water bath of approximately 70°C, then heated at approximately 40°C to prepare a study solution for administration at a concentration of 15 w/v% (calculated on the assumption of 70.3% purity), using distilled water. This preparation process was performed once a week, and the prepared solution was designated as “the preservation solution.” The preservation solution was stored at ambient temperature. Some of the solution was taken each day and diluted with distilled water to prepare 5- and 1.5-w/v% study solutions for administration. In cases in which the test substance was separated in the preservation solution at the time of preparation, the preparation was heated in warm water at approximately 40°C before it was used as the study solution.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 M with 1 F
- Length of cohabitation: overnight
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: no data.
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

from day 7 through 17 of presumed gestation.

Frequency of treatment:

Once daily

Duration of test:

21 days (Caesarian section was performed on day 20 of presumed pregnancy).

Doses / concentrations

Remarks:

Doses / Concentrations:
1.5, 5, 15
Basis:
other: % w/v active ingredient

No. of animals per sex per dose:

There were 27 or 28 rats per group that were confirmed to have mated at the time of group assignment. The number of pregnant animals was 26 in the control group and 25 in each of the treated groups.

Control animals:

yes, concurrent vehicle

Details on study design:

Three sets of doses were chosen for the study, with 15 w/v% solution being set as the maximum concentration (highest dose) based on the results of a 28-day repeated dose dermal toxicity study (study ID: 90007/90077) in which mild skin irritation was observed. The other doses were 5 w/v% and 1.5 w/v% .

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily. Observation of the topical application site was performed daily prior to application. The findings were rated in accordance with the Draize method .
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 5, 7-18 and 20 of pregnancy.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Days 0, 3, 5, 7-18 and 20 of pregnancy.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus with ovaries were excised for intrauterine examinations.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: placental weight was determined; the number of placental remnants per uterine horn was determined. Any case of remaining placenta without a fetus (i.e. a case in which placental formation was followed by resorption of fetus, leaving placenta only) was counted as a placental remnant case.

Fetal examinations:

- External examinations: Yes: [all per litter]; fetuses were also weighed and sex was determined
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes, external examination

Statistics:

Animals that showed no sign of pregnancy during the test period and were confirmed not to be pregnant on Day 20 or those that gave birth before the scheduled cesarean operation on Day 20 were excluded from the analysis. Measurements obtained in this study were first checked by Bartlett test for homoscedasticity at a 1% level of significance. If they were found to be homoscedastic, multiple comparisons were performed using Dunnett's test between the control group and each study substance group; if there was no homoscedasticity, attribution to an outlier was checked. If an outlier was excluded from statistical analysis, results were dismissed and the homoscedasticity check was performed again, followed by multiple comparisons between the control group and each of the study substance groups using Dunnett's test. If an outlier could not be excluded from the statistical analysis, Dunnett's test was still performed for multiple comparisons between the control group and each of the study substance groups.
For rates of occurrence of anomalies and alternations in fetuses, rates of implantations, and embryonic/fetal mortality rates, multiple comparisons were performed between the control group and each of study substance groups using Steel's test.
To examine the sex ratio, a chi-square test was performed.
Measurements regarding fetuses were based on a litter as a sample unit.

Indices:

The following rates (indices) were calculated:
Implantation rate = (No. of implantations / corpora lutea counts) x 100
Embryonic/fetal mortality rate = (No. of embryonic/fetal deaths / No. of implantations) x 100
Rate of implantation sites = (No. of implantation sites / No. of implantations) x 100
Rate of placental remnants = (No. of placental remnants / No. of implantations) x 100
Rate for early (late) fetus resorption = (No. of fetuses resorbed / No. of implantations) x 100
Pre-implantation mortality = [(corpora lutea counts - No. of implantations) / corpora lutea counts] x 100
Post-implantation mortality = (No. of embryonic/fetal deaths / No. of implantations) x 100

Historical control data:

No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects. Remark: No systemic maternal toxicity, but mild skin reactions were observed at high dose (see below).

Details on maternal toxic effects:
At 5% w/v, very mild skin reactions (max Draize score 0.12) were observed in dams from day 10-12 of pregnancy. At 15% w/v, mild skin reactions (max Draize score 1.20) were observed in dams from day 8-14 of pregnancy.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEC

Effect level:

>= 15 other: % w/v

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 101.3 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

3

Dose descriptor:

NOAEC

Effect level:

>= 15 other: % w/v

Based on:

act. ingr.

Basis for effect level:

other: developmental toxicity

4

Key result

Dose descriptor:

NOAEL

Effect level:

>= 101.3 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: developmental toxicity

5

Dose descriptor:

NOAEC

Effect level:

5 other: % w/v

Based on:

act. ingr.

Basis for effect level:

other: see 'Remarks'

6

Key result

Dose descriptor:

NOAEL

Effect level:

33.2 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: see 'Remarks'

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

>= 101.3 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Basis for effect level:

other: no embryotoxic/teratogenic effects observed.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Maternal toxicity.

No deaths were observed in any of the study groups. No changes were observed in general toxicity symptoms during pregnancy. Skin reactions caused by primary irritation of the study substance were observed at the administration site of animals in the medium (5 w/v%) and high dose (15 w/v%) groups. At 5% w/v, very mild skin reactions (max Draize score 0.12) were observed in dams from day 10-12 of pregnancy. As these observations were very mild and very occasionally, they were not seen as significant. At 15% w/v, mild skin reactions (max Draize score 1.20) were observed in dams from day 8-14 of pregnancy. Body weight, body weight gain and feed consumption of female animals during pregnancy were comparable across all study groups. Autopsy of female animals on day 20 of pregnancy showed no influence of α-SFMe.

Intra-uterine data.

There were no statistically significant differences observed between the control and α-SFMe groups for the following: number of corpora lutea; number of implantations (implantation rate); number of live fetuses; number of embryonic/fetal deaths (embryonic/fetal mortality rate) and their classification [number of implantation sites (rate of implantation sites); number of placental remnants (rate of placental remnants); number of resorbed fetuses (rates for early and late fetus resorption)]; and pre- and post-implantation mortalities.

Fetal examinations

Fetal weights were slightly increased compared to the control at mid and high dose (by 6% and 9%, respectively, for males and by 6% and 8%, respectively, for females), but the differences were not statistically significant and the change is not an adverse effect. No statistically significant differences were observed in the sex ratio of living fetuses between the control and treated rats. No statistically significant differences were observed in weight of placenta of living fetuses between the control and treated rats.

Fetal external examination

Fetal external examination was performed on 25-26 litters and 370-385 fetuses per group. Fetal external anomalies were observed in 2, 1, 3 and 4 cases of the control and low, mid and high dose group, respectively. All of them had low incidence (≤0.8%), and the report stated that the external anomalies concerned may be considered to be naturally occurring (historical control data were however not reported). There was no statistically significant difference between the control and treated rats.

Fetal visceral examination

Fetal visceral examination was performed on 25-26 litters and 169-178 fetuses per group. Fetal external anomalies were observed in 1, 1, 1 and 3 cases of the control and low, mid and high dose group, respectively. All of them had low incidence (≤1.18%), and the report stated that the visceral anomalies concerned may be considered to be naturally occurring (historical control data were however not reported). There was no statistically significant difference between the control and treated rats.

Fetal skeletal examination

Fetal skeletal examination was performed on 25-26 litters and 195-207 fetuses per group. Fetal skeletal anomalies were observed in 0, 0, 2 (1.0%) and 1 (0.5%) cases of the control and low, mid and high dose group, respectively. There was no statistically significant difference observed between the control and treated rats. Fetal skeletal alterations were observed in 41, 35, 37 and 19 cases of the control and low, mid and high dose group, respectively. Skeletal alternations were found across study groups, including isolation, division, or shrinkage of cervical, thoracic, and lumbar vertebral bodies, shrinkage of the 13th rib, cervical ribs, and lumbar ribs. There was no statistically significant difference between the control and treated rats, and there were no remarkable trends for a possible treatment related non-statistically significant effect.

Degrees of ossification examined based on the ossification of bone throughout the bodies of fetuses were lower in the control group than the low, mid and high dose groups for the trunk, vertebral column, 4^thcaudal vertebral body (ossification 53%, 73%, 75% and 81% respectively), 5^thcaudal vertebral body (ossification 14%, 14%, 22% and 33% respectively), sternum, 5^thand 6^thsterna ossification centre (ossification 33-46%, 61-62%, 63-66% and 64-74% respectively), for the right forelimb, 5^thmetacarpal bone (ossification 36%, 57%, 69% and 77% respectively), digit coffin bones (ossification 60-64%, 86-87%, 88-91% and 91-97% respectively), and for the right forelimb, coffin bones of the toe (ossification 45-46%, 66%, 71-72% and 83-86% respectively). The author of the report stated the following: “Rats, however, are born with incomplete ossification that is completed after birth; thus, the regions mentioned above are all prone to varying degrees of ossification in fetuses at the end of pregnancy. The time differences across autopsies on the day of cesarean operations may also result in observation of varying degrees of ossification. It is also possible that litters with more fetuses can yield lower body weights and delayed development for fetuses, resulting in partially delayed ossification. In the present study, fetuses in the control group tended to have slightly lower (although not statistically significant) body weights compared with those in α-SFMe groups, which indicates that there were varying degrees of ossification. It should be noted that the degrees of ossification for fetuses in the α-SFMe groups were within the normal range, based on our background data.” This is an acceptable explanation. Moreover, increased degree of ossification is not an adverse effect.

The maternal NOAEL for local effects is 5% (equivalent to 33.2 mg/kg bw/day) based on mild irritation at the application site. The maternal NOAEL for systemic effects is ≥15%, equivalent to ≥101.3 mg/kg bw/day in the absence of maternally toxic effects at the highest test dose.The developmental NOAEL is also ≥15 % w/v equivalent to ≥101.3 mg/kg bw/day, in the absence of embryofetal toxic effects at the highest test dose.

 

 

Applicant's summary and conclusion

Conclusions:

Developmental toxicity, dermal treatment (treatment site not covered) from day 7-17 of gestation: The maternal NOAEL for local effects is 5% (equivalent to 33.2 mg/kg bw/day) based on mild irritation at the application site. The maternal NOAEL for systemic effects is ≥15%, equivalent to ≥101.3 mg/kg bw/day in the absence of maternally toxic effects at the highest test dose. The developmental NOAEL is also ≥15 % w/v equivalent to ≥101.3 mg/kg bw/day, in the absence of embryofetal toxic effects at the highest test dose.