Registration Dossier

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-referenceopen allclose all
Reason / purpose:
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
Please see the attached document “Sophorolipid_Justification for read-across_Tox_2015-04-158828144136649115863”.
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances, both Sophorolipids, have similar toxicological properties, based on underlying similar generic structure and similar metabolism pathways. There are only differences in the ratio of the different structures (differentiation between acid and lactone form).
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
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/Nodea

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.
Reason / purpose:
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:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals 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:
no abnormal signs
Body weight:
All animals showed expected gains in body weight over the study period
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).

Reason / purpose:
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:
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 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:
reduced locomotion, sedation, hyperventilation, weakness of the extremities, loose stool, diarrhoea, piloerection
Body weight:
not reported
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.

 

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion