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

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: see below

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

yes

Remarks:

No analytical verification was done on the doses.

Principles of method if other than guideline:

Partial Revisions of Methods of Testing New Chemical Substances. December 5, 1986. Notices issued by the Directors of the Planning and Coordination Bureau, Environment Agency; the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare.

No analytical verification was done on the doses.

GLP compliance:

yes

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 6 weeks
- Weight at study initiation: Males: 172–185 g; females: 136–147 g
- Fasting period before study: no
- Housing: Rat cage; Quarantine period: 2–3 animals 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: July 4, 1990 To: August 1, 1990

Administration / exposure

Type of coverage:

open

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: Several hours prior to application, the hair of animals was clipped using an electric hair clipper. Thereafter, hair was clipped several times per week during the application period. The first hair clipping occurred at one day before application

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 room temperature. In order for it to become 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 application at a concentration of 15 w/v% (calculated based on the purity of the test substance was 70.3%), using distilled water. This test solution (15% α-SFMe solution) was prepared once a week as a mother solution, and the solution was stored at room temperature. Some of the mother solution was taken each day and diluted with distilled water to prepare 5- and 1.5 w/v% study solutions for application on a daily basis. In the case where the test article was separated out in the mother solution at the time of preparation, the preparation was heated in warm water at approximately 40°C before it was used as the test solution.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Treatment was carried out for a period of 28 days in males and 29 days in females.

Frequency of treatment:

Once daily.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1.5 other: w/v% active ingredient

Doses / concentrations 2

Dose / conc.:

5 other: w/v% active ingredient

Doses / concentrations 3

Dose / conc.:

15 other: w/v% active ingredient

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In the dose finding study (Study ID: 90007/90076: at concentrations of 10%, 20%, and 30% α-SFMe for 5 days), moderate skin irritation was observed in the 20% and 30% concentrations. With the 10% concentration, very mild irritation was found. From these results, 15% concentration thought to cause mild skin irritation in a 28-day repeated-dose investigation was set as the highest dose in this study, and the two other doses (1.5% and 2%) were calculated using a common ratio of 1/3.

Positive control:

None.

Examinations

Observations and examinations performed and frequency:

- Symptoms and application sites were observed and evaluated for skin reactions every day before application and rated in accordance to Draize.
- The body weight of each animal was determined before application, once weekly during the application period, and just before autopsy.
- The amount of feed consumed was measured five times altogether, once a week for each cage. The amount of daily feed consumption per animal was determined in terms of the difference in the feed given and the amount of feed remaining in the feed box.
- Urinalysis: Fresh urine was collected on Day 20 (male) and on Day 22 (female) and tested for urine volume, urine color, specific gravity, pH, occult blood, protein, glucose, ketone body, urobilinogen, bilirubin and urine sediment (visual observation).
- Blood was sampled from all animals during autopsy for the blood tests after overnight fasting and under anesthetic using pentobarbital sodium. Hematological tests and Biochemical examination included: RBC, WBC, PLT, HLT, CHr, WBC, RET%, GOT, GPT, gamma-GPT, alkaline phosphatase, blood glucose, urea nitrogen, total cholesterol, total protein, albumin, A/G ratio, creatinine, Triglyceride, Ca, P, Na, K and Cl
- Histopathological tests: The weight of the following organs was measured in each animal: liver, kidneys, adrenal glands, testes, ovaries, brain
The following organs and tissues obtained from the animals were fixed in 10% formalin and stored: skin (area applied with sample and untreated area), mammary gland, lymph node (cervical spine and mesenterium), submandibular gland, femur, thymic gland, trachea, lungs/bronchial tube, heart, thyroid gland/parathyroid, tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, spleen, kidney, adrenal gland, bladder, seminal vesicle, prostrate, testis, ovary, womb, vagina, brain, pituitary gland, spinal cord, eyeball/harderian gland, and other organs showing macroscopic changes at autopsy.
The eyes were separately fixed in formalin-glutal aldehyde mixture, and stored in 10% formalin solution.
Of the above-listed organs and tissues that were removed, paraffin blocks were prepared for the skin (area applied with sample and untreated areas), heart, liver, spleen, kidneys, and adrenal glands. Paraffin blocks were also prepared for organs and tissues showing change in macroscopic findings, organ weight, etc.
For animals treated at the highest dose and the control group, slide specimens were prepared from the paraffin block, hematoxylin and eosin (H.E.) staining was performed according to the normal procedure, and histopathological tests were conducted.
Histopathological tests were performed on all males and females for the area to which the sample had been applied and untreated skin areas. In Group 4 (highest dose) and Group 1 (control), histopathological tests were carried out on the heart, liver, spleen, kidneys, and adrenal glands of all males and females. In these two groups, organs showing gross lesions in autopsy were also examined histopathologically.
As significant increase in organ weight was observed in the kidneys and adrenal glands, histopathological tests were conducted on both left and right organs of all males and females.
There were changes in macroscopic findings in the lungs in groups 4 and 1. The histopathological results revealed a high tendency for bleeding, infiltration of macrophages and mononuclear cells, and sedimentation of hemosiderin in Group 4 compared with group 1. Histopathological tests were hence performed on the males and females of all groups.

Sacrifice and pathology:

See above.

Statistics:

Homoscedasity was tested by comparing weight, feed consumption, urinalysis (quantitative parameters), blood tests, and absolute and relative organ weights among groups, according to the Bartlett method. If distribution was found to be consistent among groups, they were subsequently subject to one-way analysis of variance (ANOVA). If ANOVA results indicated significant difference between the groups (P < 0.05), the parameters were further analyzed by Dunnet’s multiple comparison between the control and three test groups. The parameters showing non-homoscedasity and results of urinalysis (qualitative examination) were analyzed by Kruskal-Wallis test. If significant difference was observed between the groups (P < 0.05), the parameters were compared between the control and three test groups using the Dunnet method.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

The results are summarised in the Table below. The changes are represented either as percentage change relative to the control (e..g -35 = 35% reduction, +10 = 10% increase) or as incidence (e.g. 3/10 represents 3 out of 10 rats). Where a dose relationship was observed, this is indicated in the last column. Under the table with findings, a discussion of the study results is provided and the NOAEL is derived.

The following findings are considered to be unrelated to the treatment and/or of no toxicological relevance:
·        Increased feed consumption during week 3 in females of all treated groups (incidental finding in one sex only without a dose relationship).
·        Reduced gamma-GPT (females, mid and high dose) and reduced glucose (females, mid dose): no dose-relationship, not confirmed by histopathological findings in the liver).

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

5 other: % w/v

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Local effects: based on primary skin reaction with histopathological changes.

2

Dose descriptor:

NOAEL

Effect level:

37 mg/kg bw/day

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: Local effects: based on thickened epidermis and dose response

3

Dose descriptor:

NOAEL

Effect level:

55 mg/kg bw/day

Based on:

act. ingr.

Sex:

female

Basis for effect level:

other: Local effects: based on parakeratosis with dose response

4

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

15 other: % w/v

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Absence of relevant systemic effects

5

Key result

Dose descriptor:

NOAEL

Effect level:

111 mg/kg bw/day

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: Absence of relevant systemic effects

6

Key result

Dose descriptor:

NOAEL

Effect level:

161 mg/kg bw/day

Based on:

act. ingr.

Sex:

female

Basis for effect level:

other: Absence of relevant systemic effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. Results from 28-day dermal toxicity study withα-sulfonated fatty acid methyl ester sodium salt in rats

applied concentration (% w/v)	0	0	1.5	1.5	5	5	15	15	DR
Sex	m	f	m	f	m	f	m	f
Mortality	none
Clinical signs	no treatment related findings
Skin reactions at appln site							+	+
Body weight	no treatment related findings
Feed consumption	no treatment related findings
Urinalysis	no treatment related findings
Haematology	no treatment related findings
Clinical chemistry	no treatment related findings
Organ weight	See remarks
kidney (right)							+14(a) +15(r)	+24(a)** +15(r)**
kidney (left)							+9(a) +10(r)	+19(a)** +10(r)
adrenal (right)				+20(a)* +19(r)		+18(a) +15(r)		+26(a)** +18(r)
adrenal (left)				+16(a) +15(r)		+13(a) +9(r)		+22(a) +14(r)
Pathology
Macroscopy	no treatment related findings
Microscopy^(A)
skin (treated site)
- mild parakeratosis			1/5			1/5	1/5	4/5	X
- thickened epidermis					1/5		3/5	5/5	X

DR          dose related

*              Statistically significant difference from water control at 5% level

**            Statistically significant difference from water control at 1% level

(a)/(r)     absolute/relative organ weight

+             Skin reactions were observed; mild skin reactions (max mean score 1.4 for both sexes) in males from day 2-7 and in females from day 3-7. Mean score is erythema + edema/no. of animals

(A)          Empty cells: finding not observed. Findings other than those in the table were observed but not included in the above table, since they also occurred in controls at comparable frequency and or a dose relationship was absent.

Evaluation 

The following changes are considered to be treatment related.

·        Primary skin reactions: Mild irritation at the application site, accompanied by histopathogical changes (mild parakeratosis and thickened epidermis in high dose males and females). Thickened epidermis was also observed in one mid-dose male and mild keratosis in one low-dose male and one mid-dose female.

·        Systemic toxicity consisted of increased absolute and relative kidney weights of high-dose males and females. The increases of absolute and relative kidney weight in males were not statistically significant, but due to their magnitude (mean increase for combined left and right kidney >10%) a relationship to treatment cannot be excluded, considering also the more pronounced increases in the kidney weight of high dose female rats. There were no treatment related histological findings in the kidneys of females and only mild to very mild histological findings were recorded in the kidneys of 1-2 high dose males (basophilic degeneration of tubular epithelium, dilation and hyaline cast of renal tubule and haemostasis). As these histological changes are not deemed to be relevant to kidney weight increases, the NOAEL was set to the highest dose.

Remarks:

 Absolute and relative adrenal weights were increased in females of all treatment groups, with statistical significance for the increase of absolute right adrenal weights at the low and high dose. It should be taken into consideration that there was no dose relationship, that the weight of this organ is very small, which can easily lead to errors in the determination of the weight, and that there were no histopathological findings in the adrenals. Moreover, in a 28-day repeated dose oral study (see study 1 of this section), the NOEL was 2000 ppm nominal equivalent to 214.8 mg/kg bw/day for females, with no effects on adrenal weight at the highest dose of 8000 ppm nominal (hence equivalent to approximately 850 mg/kg bw/day for females). For these reasons, the increased adrenals weights recorded in the present dermal toxicity study at dose levels which are much lower (for females 17, 55 and 161 mg/kg bw/day, with probably even far lower systemic exposure levels) are considered to be an incidental finding and unrelated to the treatment.

 

The NOAEL for local effects is 5% (equivalent to 37 mg/kg bw/day for males and 55 mg/kg bw/day for females) based on thickened epidermis in both sexes at 15% and the dose response.

Based on the absence of relevant effects, the NOAEL for systemic effects was set to 15%, equivalent to 111 mg/kg bw/day for males and 160 mg/kg bw/day for females.

 

Applicant's summary and conclusion

Conclusions:

The NOAEL for local effects is 5% (equivalent to 37 mg/kg bw/day for males and 55 mg/kg bw/day for females) based on thickened epidermis in both sexes at 15% and a dose response . As the effects seen at the high dose were not considered as relevant, the NOAEL for systemic effects is 15%, equivalent to 111 mg/kg bw/day for males and 161 mg/kg bw/day for females.