Reaction mass of 2-O-α-L-Rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid and α-L-Rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-14 to 2018-01-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit (05.06.2015)

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-O-α-L-Rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid and α-L-Rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid

EC Number:

604-610-3

Cas Number:

147858-26-2

IUPAC Name:

Reaction mass of 2-O-α-L-Rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid and α-L-Rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoic acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Storage condition of test material: 2-8 °C; in closed packaging
- Final preparation of a solid: In order to get the test item in a solution or suspension, which is applicable to the animals, aqua ad injectionem was evaluated as vehicle and was considered to be adequate. The dose formulations were made shortly before each dosing occasion. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before each dose administration.

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Step 1, animal no. 1 - 3: 8 - 9 weeks; Step 2, animal no. 4 - 6: 9 - 10 weeks
- Weight at study initiation: Step 1, animal no. 1 - 3: 153 - 169 g; Step 2, animal no. 4 - 6: 161 - 172 g
- Fasting period before study: Prior to the administration food was withheld from the test animals for 18 to 19 hours (access to water was permitted).
- Housing: IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: Adequate acclimatisation period (at least five days) under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 0.2 g/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Justification for choice of vehicle: The vehicle aqua ad injectionem was chosen due to its non-toxic characteristics.
- Lot/batch no. (if required): Aqua ad injectionem (DELTAMEDICA, lot no. 705820, expiry date: 30/04/2020).
- Purity: not specified

MAXIMUM DOSE VOLUME APPLIED: a dose of 2000 mg/kg body weight at a dose volume of 10 mL/kg body weight

Doses:

2000 mg/kg body weight

No. of animals per sex per dose:

6 female rats

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: A careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes and with special attention given during the first 4 hours post-dose). As soon as symptoms were noticed they were recorded. Thereafter, the animals were observed for clinical signs once daily until the end of the observation period. All abnormalities were recorded. The animals were weighed on day 1 (prior to the administration) and on days 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: Cageside observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Particular attention was directed to observations of tremor, convulsions, salivation, diarrhoea, lethargy, sleep and coma. All animals were subjected to gross necropsy and examined macroscopically for gross pathological changes. In the absence of gross pathological changes no tissues were preserved for a possible histopathological evaluation.

Statistics:

no data

Results and discussion

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50

Remarks:

cut-off

Effect level:

2 500 mg/kg bw

Based on:

test mat.

Mortality:

The test item showed mortality in 1 of 6 animals and acute oral toxicity characteristics in all animals after a single dose administration.

Clinical signs:

other: Detailed clinical findings of the study are presented in Table 2 (below).

Gross pathology:

No specific gross pathological changes were recorded for any animal.

Any other information on results incl. tables

Table 2. Clinical signs - individual data.

Step	Animal No. / Sex	Starting Dose (mg/kg bw)	Timepoint	Observations
1	1 / Female	2000	0 - 60 min	nsf
			60 - 120 min	Moderately reduced spontaneous activity, slight piloerection, hunched posture
			120 - 180 min	Moderately reduced spontaneous activity, hunched posture, moderate piloerection, slight ataxia, slow movements
			180 - 240 min	Slightly reduced spontaneous activity, hunched posture, moderate piloerection, slight ataxia
			240 min - d 2	Slightly reduced spontaneous activity, hunched posture, slight piloerection
			d 2 - 15	nsf
	2 / Female	2000	0 - 60 min	nsf
			60 - 120 min	Moderately reduced spontaneous activity, slight ataxia, slight piloerection, hunched posture
			120 - 180 min	Moderately reduced spontaneous activity, hunched posture, moderate piloerection, half eyelid-closure, slight ataxia, slow movements
			180 - 240 min	Slightly reduced spontaneous activity, hunched posture, moderate piloerection, slight ataxia
			240 min - d 2	Slightly reduced spontaneous activity, hunched posture, slight piloerection
			d 2 - 15	nsf
	3 / Female	2000	0 - 60 min	nsf
			60 - 120 min	Moderately reduced spontaneous activity, slight ataxia, slight piloerection, hunched posture
			120 - 180 min	Moderately reduced spontaneous activity, hunched posture, moderate piloerection, slight ataxia, slow movements
			180 - 240 min	Slightly reduced spontaneous activity, hunched posture, moderate piloerection, slight ataxia
			240 min - d 2	Slightly reduced spontaneous activity, hunched posture, slight piloerection
			d 2 - 15	nsf
2	4 / Female	2000	0 - 30 min	nsf
			30 - 60 min	Slightly reduced spontaneous activity, slight piloerection
			60 - 120 min	Slightly reduced spontaneous activity, hunched posture, slight piloerection
			120 – 180 min	Moderately reduced spontaneous activity, slight ataxia, moderate piloerection, half eyelid-closure, severe diarrhoea, hunched posture, sunken flunks
			180 min - d 2	Moderately reduced spontaneous activity, hunched posture, moderate piloerection, half eyelid-closure, moderate diarrhoea, slight ataxia, sunken flunks
			d 2	Found dead
	5 / Female	2000	0 - 30 min	nsf
			30 - 60 min	Slightly reduced spontaneous activity, slight piloerection
			60 - 120 min	Slightly reduced spontaneous activity, hunched posture, slight piloerection
			120 - 180 min	Slightly reduced spontaneous activity, slight piloerection, slight diarrhoea, hunched posture
			180 - 240 min	Slightly reduced spontaneous activity, hunched posture, slight piloerection, half eyelid-closure, slight diarrhoea
			240 min - d 2	Slightly reduced spontaneous activity, hunched posture, slight piloerection, slight diarrhoea
			d 2 -3	Slightly reduced spontaneous activity, moderate piloerection
			d 3 - 4	Slight piloerection
			d 4 - 15	nsf
	6 / Female	2000	0 - 30 min	nsf
			30 - 60 min	Slightly reduced spontaneous activity, slight piloerection
			60 - 120 min	Slightly reduced spontaneous activity, hunched posture, slight piloerection
			120 - 180 min	Slightly reduced spontaneous activity, slight piloerection, slight diarrhoea, hunched posture
			180 - 240 min	Slightly reduced spontaneous activity, hunched posture, slight piloerection, half eyelid-closure, slight diarrhoea
			240 min - d 2	Slightly reduced spontaneous activity, hunched posture, slight piloerection, slight diarrhoea
			d 2 -3	Slightly reduced spontaneous activity, moderate piloerection
			d 3 - 4	Slight piloerection
			d 4 - 15	nsf

d = day (day 1 = day of administration); min = minute(s); nsf = no specific findings

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, a single oral application of the test item 'Decanoic acid, 3-[[6-deoxy-2-O-(6-deoxy-a-L-mannopyranosyl)-a-L-mannopyranosil-1-(carboxymethyl)octyl ester, mixture with 1-(carboxymethyl)octyl 3-[(6-deoxy-a-L-mannopyranosyl)oxy]decanoate' to rats at a dose of 2000 mg/kg body weight was associated with signs of toxicity in all animals and mortality in 1 of 6 animals.
The median lethal dose of 'Decanoic acid, 3-[[6-deoxy-2-O-(6-deoxy-a-L-mannopyranosyl)-a-Lmannopyranosil-1-(carboxymethyl)octyl ester, mixture with 1-(carboxymethyl)octyl 3-[(6-deoxy-a-Lmannopyranosyl)oxy]decanoate' after a single oral administration to female rats, observed over a period of 14 days is:
LD50 cut-off (rat): 2500 mg/ kg bw

Executive summary:

The acute oral toxicity of the test item 'Decanoic acid, 3-[[6-deoxy-2-O-(6-deoxy-a-L-mannopyranosyl)-a-L-mannopyranosil-1-(carboxymethyl)octyl ester, mixture with 1-(carboxymethyl)octyl 3-[(6-deoxy-a-L-mannopyranosyl)oxy]decanoate' in rats after oral administration via gavage was studied according to OECD TG 423.

Two groups, each of three female WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg body weight. The test item was suspended with the vehicle aqua ad injectionem (sterile water) at a concentration of 0.2 g/mL and administered at a dose volume of 10 mL/kg. All animals used in the study after their entrance at BSL were allowed to acclimatise to the laboratory conditions for at least 5 days. The animals were observed on delivery, on inclusion in the study and before administration for mortality/morbidity and other clinical signs. All animals were examined for clinical signs several times on the day of dosing and once daily until the end of the observation period. Their body weights were recorded on day 1 (prior to the administration) and on days 8 and 15. All animals were necropsied and examined macroscopically.

One animal of the second group treated with the test item at a dose of 2000 mg/kg died spontaneously the day after treatment. All remaining animals survived until the end of the study while temporarily showing signs of toxicity. The most relevant clinical findings in the animals treated with the test item at a dose of 2000 mg/kg bw were reduced spontaneous activity, piloerection, half eyelid-closure, ataxia, slow movements, sunken flunks, hunched posture and diarrhoea. All symptoms recovered within up to 4 days postdose. Throughout the 14-day observation period, the weight gain of the surviving animals was within the normal range of variation for this strain. At necropsy, no macroscopic findings were observed in any animal of any step.

Therefore, a LD50 cut-off value of 2500 mg/kg bw can be derived from this study.