Sophorolipids: fermentation products of glucose and fatty acids, C18 (unsaturated), glycerol esters with yeast Candida Bombicola, partially hydrolysed

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance does not meet the criteria for classification as acute toxicity or STOT SE by the oral route and no systemic effects have been observed in in vivo studies with dermal exposure (e.g. skin irritation, skin sensitisation)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The toxicological profile for systemic endpoints is expected to be similar for target substance Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed when compared to the source substance. Read-across is expected to be reliable including the data on acute oral toxicity, sensitization and gene toxicity in vitro.
For detailed information, please see the justification for read-across in chapter 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: B6 K Universal Ltd., Hull, UK
- Age at study initiation: eight to twelve weeks
- Weight at study initiation: 15 to 23 g
- Housing: suspended solid floor polypropylene cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 30-70 %
- Air changes (per hr): ca. 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethylformamide

Concentration:

undiluted, 25, 50 %

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
In a preliminary range finding test two mice were tested. The test item was applied undiluted and in a concentration of 50 % in dimethyl formamide to the dorsal surface of each ear for three consecutive days.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: The test material will be regarded as a sensitiser if at least one concentration of the test material results in a three-fold or greater increase in 3 HTdR incorporation compared to control values. Any test material failing to produce a three-fold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
- TOPICA APPLICATION
Groups of four mice were treated with the undiluted test material or the test material at concentrations of 25 or 50 % v/v in dimethyl formamide. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days. The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

- ADMINISTRATION OF 3H-METHYL THYMIDINE
Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-Methyl thymidine (3HTdR: 80 µCi/ml, specific activity 2.0 Ci/mmol, Amersham Biosciences UK Ltd.) giving a total of 20 µCi to each mouse.

- DETERMINATION OF 3HTDR
Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphixiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 ml of PBS was added to the pooled lymph nodes.
After overnight incubation at 4 °C, the precipitates were recovered by centrifugation at 2100 rpm for ten minutes, resuspended in 1 ml of TCA and transferred to ten ml scintillation fluid (Optifase "Trisafe"). 3HTdR incorporation was measured by beta-scintillation counting. The "Poly Q" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately 20 minutes. The purpose for this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes , the vials were shaken rigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system.

-FURTHER OBSERVATIONS
Clinical observations: All animals were observed twice daily on Days 1, 2 and 3 and on a dailybasis on days 4, 5 and 6. Any signs of toxicity or signs of ill health during the study were recorded.
Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

In the study with positive control substance STIMULATION INDICES of 1.52 ,2.63 and 5.07 were determined with the test item ALPHA-HEXYLCINNAMALDEHYDE at concentrations of 10 %, 25 % and 50 % (v/v) in cottonseed oil.
A test item is regarded as a sensitiser in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.
Based on these criteria, the test item ALPHA-HEXYLCINNAMALDEHYDE was found to be a non-sensitiser when tested up to 25 % (v/v) in cottonseed oil.
ALPHA-HEXYLCINNAMALDEHYDE showed an allergenic potency when tested at concentration of 50 % (v/v) cottonseed oil.

Parameter:

SI

Remarks on result:

other: see table below

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see table below

Dpm, Dpm/Node and Stimulation Index (SI)

Concentration (5 v/v) in dimethyl formamide	Dpm	Dpm/Node^a	Stimulation Index (SI)^b	Result
Vehicle	6226.30	77.29	N/A	N/A
25	5479.44	684.93	0.88	Negative
50	5445.24	680.66	0.87	Negative
100	5094.18	636.77	0.82	Negative

 

A = Dpm/node obtained by dividing the Dpm value by 8 (total number of lymph nodes)

B = Stimulation Index of 3.0 or greater indicates a positive result

N/A = Not applicable

Interpretation of results:

GHS criteria not met

Conclusions:

The substance was considered to be a non-sensitiser under the conditions of the test.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2004-11-23 to 2004-11-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Kent, UK
- Age at study initiation: eight to twelve weeks
- Weight at study initiation: 216-243 g
- Fasting period before study: overnight
- Housing: polypropylene cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 30-70 %
- Air changes (per hr): at least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continous light, twelve hours continous dark

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 6645 mg/kg bw (equvalent to 2000 mg active ingredient/kg bw)

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: Using all available information on the toxicity of the test material

Doses:

6645 mg/kg bw

No. of animals per sex per dose:

6

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: observations: 1/2, 1, 2, 4 hours after dosing and subsequently once daily for 14 days; weighing: at days 0, 7, 14
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

2 000 mg/kg bw

Based on:

act. ingr.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 6 645 mg/kg bw

Based on:

test mat.

Mortality:

no mortalty observed

Clinical signs:

other: other: no abnormal signs

Gross pathology:

No abnormalities were noted.

Individual body weights and weekly body weight changes

Dose level (mg/kg bw)	Animal number and sex	Body weight (g) at day			Body weight gain during week
		0	7	14	1	2
6645*	2-0 Female	241	276	290	35	14
	2-1 Female	243	265	288	22	23
	2-2 Female	216	235	257	19	16
	3-0 Female	240	270	288	30	18
	3-1 Female	237	264	274	27	10
	3-2 Female	244	270	275	26	5

*: = equivalent to 2000 mg active ingredient/kg bw

Interpretation of results:

GHS criteria not met

Conclusions:

No mortality, signs of systemic toxicity or abnormalities at necropsy were observed. All animals showed expected gains in body weight over the study period.

Executive summary:

The acute oral toxicity of the substance to rats was investigated in a study according to OECD Guideline 423 (Acute oral toxicity - acute toxic class method; adopted 17 December 2001). A group of three fasted females was treated with the test material at a dose level of 6645 mg/kg bw (eqivalent to 2000 mg active ingredient/kg bw). This was followed b a further group of threee fasted females at the same dose level.

The test material was administered undiluted. Clinical signs and body weight development were monitored during the study. All animals were subjected to necropsy. No mortality, signs of systemic toxicity or abnormalities at necropsy were observed. All animals showed expected gains in body weight over the study period.

It can be concluded that the acute oral median lethal dose (LD50) of the test material in the female Sprague-Dawley CD strain was estimated as being greater than 6645 mg/kg bw (equivalent to 2000 mg active ingredient/kg bw).

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

yes

Remarks:

7 days observation

GLP compliance:

no

Test type:

standard acute method

Species:

mouse

Strain:

ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shizuoka Prefecture Agricultural Cooperative Association for Laboratory Animals
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 25-29 g
- Fasting period before study: yes, 16-18 hours before the administration
- Housing: polycarbonate cages with wood chip floor covering and 5 animals per cage
- Diet: solid feed (CE-2, CLEA Japan, Inc.) ad libitum
- Water: tap water ad libitum
- Acclimation period: not reported
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2 °C
- Humidity (%): 55 ± 10 %,
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): not reported

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 37.5 w/v suspension
- Amount of vehicle (if gavage): not specified

Doses:

7230, 8680, 10420, 12500 and 15000 mg/kg bw

No. of animals per sex per dose:

10 males per dose level

Control animals:

no

Details on study design:

- Duration of observation period following administration: 7 days
- Necropsy of survivors performed: yes

Statistics:

LD50 value was calculated with the Litchfield and Wilcoxon method using the mortality rate.

Preliminary study:

not applicable

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

ca. 12 500 mg/kg bw

Based on:

test mat.

95% CL:

>= 11 060 - <= 14 130

Mortality:

At the highest dose level of 15000 mg/kg bw, all 10 animals died in between 1 day after administration.
At the dose level of 12500 mg/kg bw, 5 animals died in between 1 day after administration.
At the dose level of 10420 mg/kg bw, 1 animal died in between 1 day after administration.
No other deaths were reported in the 7-day observation period.

Clinical signs:

other: other: reduced locomotion, sedation, hyperventilation, weakness of the extremities, loose stool, diarrhoea, piloerection

Gross pathology:

Individuals which had died showed hyperemia or mild bleeding from the pyloric region of the stomach to the small intestine. No specific abnormalities could be found in the surviving individuals.

Table 1: Deaths of mice and LD₅₀ after oral installation of the test item.

	Number of dead animals during observation period (day)
Dose level [mg/kg bw]	0-1	2-4	5-7	Lethality [%]	LD50 and 95 % CL [mg/kg bw]
7230	0	0	0	0	12500 (11060 – 14130)
8680	0	0	0	0
10420	1	0	0	10
12500	5	0	0	50
15000	10	0	0	100

 

Interpretation of results:

GHS criteria not met

Conclusions:

The oral LD50 of the test item in male mice was determined to be 12500 mg/kg bw.

Executive summary:

The publication by Ikeda et al. (1986) reports the determination of the LD₅₀ value of Sophorolipid in its lactone form. The study has not been conducted according to an OECD guideline, but generally meets the principle for determination of acute oral toxicity and has been described in sufficient detail. The study is therefore considered as acceptable for classification purposes.

In this study, 10 fasted male mice per dose group were administered the test item p.o. via gavage. The dose groups were 7230, 8680, 10420, 12500 and 15000 mg/kg bw. The animals were observed for 7 days. Necropsy was conducted after death or in all surviving animals after the observation period. Clinical signs were reduced locomotion, sedation, hyperventilation, weakness of the extremities, loose stool, diarrhoea and piloerection.

The LD₅₀ of Sophorolipid, lactone form, was calculated to be 12500 mg/kg based on test material. However, the percentage of active ingredient in the test item is not reported. The test item is described as a semi-solid colloid which may indicate rather high content of active ingredient. Taking into account that the Sophorolipid raw material after filtration and decanting is a solution consisting of approximately 50-60 % Sophorolipids mainly in the lactone form, the LD₅₀ would here still be in a range that does not need classification.