Rhamnolipids: fermentation products of glucose with Pseudomonas putida, Pseudomonadaceae

Endpoint summary

Description of key information

Toxicokinetics, metabolism and distribution

There are no studies available in which the target substance and most of the source substances has been investigated. Therefore, in accordance with Annex VIII, Column 1, Item 8.8.1, of Regulation (EC) No 1907/2006 and with Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2016c), assessment of the toxicokinetic behavior of the substances which source and target substances is conducted to the extent that can be derived from the relevant available information. This comprises a qualitative assessment of the available substance specific data on physico-chemical and toxicological properties according to Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2016c) and considering further available information on the target and source substances.

Water-soluble substance will readily dissolve into gastrointestinal fluids.

Oral and dermal absorption of Rhamnolipids (target substance) and source substances is not anticipated because they have high molecular weights (see Read-across justification attached in Chapter 13).  Generally, the smaller the molecule the more easily it may be taken up. Molecular weights below 500 is favorable for oral absorption; molecular weights above 1000 do not favor absorption (ECHA, 2016). The molecular weight is 600 g/mol for the target substance and ranges until 1000 g/mol for the source substances. Furthermore, Lipinski rule of five for the target and source substances indicates that the substances are less to be bioavailable (OECD QSAR toolbox, 2022). In addition to molecular weight the most useful parameters providing information on the absorption potential are octanol/water partition coefficient (log P) value and the water solubility (ECHA, 2016).

With regard the source substance 4 (AM-1) a pharmacokinetic study reported that this is poorly absorbed by the oral route (Bitzer et al. 2017).

Regarding the oral toxicity data, the acute toxicity oral data for the target substance Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria, does not show any mortality and clinical signs up to LD50 of 5000 mg/kg bw.

An acute oral toxicity study was performed in female Wistar rats treated with AM-1 (purity ≥ 90% suspended in sterile water) according to OECD TG 423 (OECD, 2001) (EFSA Journal, 2021). Two groups, each of three rats, received 2000 mg/kg bw test item by gavage.

Throughout the 14-day observation period there were no deaths and no clinical signs - except for a transient piloerection at day 1 in two females. Body weight gain (13–22%) was within the normal range of variation for this strain. At necropsy, no specific findings were reported.

In an acute oral toxicity conducted in rats LD50 of Sophorolipid, hydrolysed was determined to be > 2000 kg/bw

Dermal absorption is influenced by many factors, e.g., physico-chemical properties of the substance. With regard, to the dermal absorption liquids and substances in solution are taken up more readily than dry particulates. A tiered approach for the estimation of skin absorption has proposed within a risk assessment framework (EC, 2007). Initially, basic physico-chemical information, should be considered, i.e., molecular mass and lipophilicity (log P). Following, skin absorption is generally predicted unless molecular mass is above 500 and log P is outside the range [-1, 4], in which case a value of 10% skin absorption is chosen.  Furthermore, the substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. Thus, if the water solubility is below 1 mg/L, dermal uptake is likely to be low. Between 1-100 mg/L absorption is anticipated to be low to moderate and between 100-10,000 mg/L moderate to high.  However, if the water solubility is above 10,000 mg/L and log P values below 0 the substance may be too hydrophilic to cross the lipid rich environment of the stratum corneum. Based on the above consideration, dermal uptake for these substances will be low.

Rhamnolipids are completely soluble in water and have a log P of -0.247 . Thus, the substance of interest substances does not have the potential to be absorbed via dermal route.

The acute dermal toxicity of alcohol polyglycosides (APGs) (substances source 1 and 2 are part of this group of compound) has been extensively evaluated in numerous studies with rats and rabbits.

The dermal LD50 values, tested with C8-10-alkyl glycosides and C10-16-alkyl glycosides were both above 2 g/kg.

Based on these considerations we can predict a low degree of absorption for target and source substances.

The rate and extent of absorption of the target substance, following topical application of the test items in aqueous dilutions of five formulations to human skin, was investigated in a study conducted under GLP conditions and according to OECD TG 428: Skin Absorption: In Vitro Method (2004).

Following topical application of target substance in Test Preparation 1 to human skin in vitro, at 24 h post dose, the total absorbed dose and dermal delivery of Rha2-C10-C10through human skin in vitro were ≤0.36 μg/cm2 (≤0.53% of the applied dose) and ≤2.24 μg/cm2 (≤3.31% of the applied dose) respectively. There was no quantifiable Rha2-C10-C10 measured in receptor fluid at 24 h post dose. Following topical application of Rha2-C10-C10 in Test Preparation 2 to human skin in vitro, at 24 h post dose, the total absorbed dose and dermal delivery of Rha2-C10-C10 through human skin in vitro were ≤0.34 μg/cm² (≤0.25% of the applied dose) and ≤2.37 μg/cm² (≤1.72% of the applied dose) respectively. There was no quantifiable Rha2-C10-C10 measured in receptor fluid at 24 h post dose.

Following topical application of the source substance in Test Preparation 3 to human skin in vitro, at 24 h post dose, the total absorbed dose and dermal delivery of source substance through human skin in vitro were ≤0.36 μg/cm² (≤0.22% of the applied dose) and ≤2.48 μg/cm2(≤1.49% of the applied dose) respectively. There was no quantifiable target substance measured in receptor fluid at 24 h post dose.

Following topical application of source substance in Test Preparation 4 to human skin in vitro, at 24 h post dose, the total absorbed dose and dermal delivery of Rha2-C10-C10 through human skin in vitro were ≤0.38 μg/cm² (≤0.22% of the applied dose) and ≤2.26 μg/cm² (≤1.29% of the applied dose) respectively. There was no quantifiable source substance measured in receptor fluid at 24 h post dose.

Following topical application of source substance in Test Preparation 5 to human skin in vitro, at 24 h post dose, the total absorbed dose and dermal delivery of Rha2-C10-C10 through human skin in vitro were ≤0.36 μg/cm² (≤0.41% of the applied dose) and ≤2.24 μg/cm2 (≤2.54% of the applied dose) respectively. There was no quantifiable source substance measured in receptor fluid at 24 h post dose.

Almost all receptor values were less than the lower limit of quantification (LLOQ). The majority of stratum corneum values were <LLOQ and all skin values were <LLOQ. Where values were <LLOQ, the LLOQ value for the relevant matrix has been applied. Therefore, the total absorbed dose and dermal delivery values above are “worst-case”.

With regard to the absorption via inhalation-route the important parameters are vapor pressure, hydrolysis, and log P. Vapor pressure is a parameter for a substance to be available for inhalation as a vapor. As a general guide, highly volatile substances are those with a vapor pressure greater than 25 KPa (or a boiling point below 50°C). Substances with low volatility have a vapor pressure of less than 0.5 KPa (or a boiling point above 150°C). Both target and source substances have a low volatility. Moderate log P values (between -1 and 4) are favorable for absorption directly across the respiratory tract epithelium by passive diffusion. This is the case for the target and source substances as well.

Analytical test (Intertek confidential report, 2022) conducted on a surfactant product (cleaning product) containing RL (spray / bottle combination) showed a range of values across the devices with regards the respirable fraction of the plume. The results (see table below) generated show that %V< 10µm is less than 1%.

Results from the droplet size distribution analytical testing of surfactant product (cleaning product) containing RL (spray / bottle combination)

Bottle	Dv10 (µm)	Dv50 (µm)	Dv90 (µm)	100% Greater Than (µm)	%V< 5µm	%V< 10µm
2	38.8	74.54	147.3	0.541	0.1565	0.1565
3	39.43	74.16	139.6	10.00	0	0.002714
4	45.2	81.39	150.4	25.12	0	0

 

In addition, acute inhalation toxicity on RHAMNOLIPID BIOSURFACTANT is available. Male and female rats (5 per sex) were exposed on whole-body to a gravimetric concentration of 2.05 mg/L 9.5% rhamnolipid biosurfactant in water for four hours. Ocular and nasal discharge, irregular respiration, hunched posture, and hypoactivity were noted during exposure, and all but except ocular and nasal discharge persisted through one-hour post-exposure. All rats were active and healthy 15 hours post-exposure. The LC50 was >2.05 mg product/L (0.20 mg a.i./L) (GRAS Notice, 2016).

The available inhalation toxicity data of a structural similar substance RHAMNOLIPID BIOSURFACTANT did not show any mortality and transient clinical signs.

Hydrolysis data are not available for target and source substances.

Based on the physicochemical properties of target and source substances and data on acute inhalation toxicity of the target and source substances, the absorption via the lung is not expected.

Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2016c) states that if the substance is a skin irritant or corrosive, damage to the skin surface may enhance penetration. Target and source resulted not to be corrosive to skin. Therefore, enhanced penetration of the substance due to local skin damage can be excluded.

Taken all together, based on the above consideration we can predict the target and source substances are unlike to be absorbed via dermal route.

Overall, it can be concluded that, target and source substances are not expected to be absorbed in the respiratory and gastro-intestinal tracts.

Distribution within the body through the circulatory system depends on the molecular weight, the log P and water solubility. In general, the smaller the molecule, the wider is the distribution. If the molecule has a log P> 0 (lipophilic) it is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration particularly in fatty tissues.

As explained in Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2016c) lipophilic substances have the potential to accumulate within the body depending on the conditions of the exposure. If the dosing interval is shorter than 4 times the whole half-life of the substance, then there is the potential for the substance to accumulate. It is generally the case that substances with high log P values have long biological half-lives. Based on these considerations, daily exposure to a substance with a log P value of around 4 or higher may results in a build-up of that substance within the body. Substances with a log P values of 3 or less would be unlikely to accumulate with the repeated intermittent exposure patters normally encountered in the workplace but may accumulate if exposures are continuous. Once exposure to the substance stops, the substance will be gradually eliminated at a rate dependent on the half-life of the substance.

Highly lipophilic substances (log P between 4 and 6) that come into contact with the skin can readily penetrate the lipid rich stratum corneum but are not well absorbed systemcally. Although they may persist in the stratum corneum, they will eventually be cleared as the stratum corneum is sloughed off. Thus, based on the fact that the target substance has a log Pow higher than 4 then a high bioaccumulation potential is expected for the target substance.

Based on the close structural similarities, the same metabolic pathways are expected to be relevant for the target and source substances, resulting in similar metabolites.

Alkyl polyglycosides have a common metabolic fate that involves hydrolysis of the α- and β-glycosidic bond to the fatty alcohol and glucose. Glucose and glucose oligomers enter the carbohydrate metabolic pathway and are catabolised into pyruvate and subsequently to the major extent into acetyl-CoA, which is introduced into the citric acid cycle with the aim to generate reduction equivalents for energy generation in the oxidative phosphorylation. Fatty alcohols, representing the main difference in the structure of different alkyl polyglycosides, are oxidized to the corresponding fatty acid and fed into the physiological pathway of β-oxidation, where they are also oxidised to acetyl-CoA. In addition to its function in the generation of energy by catabolic processes acetyl-CoA can also be used in anabolic processes like lipid synthesis, which is important for the storage of energy in form of large high-energy macromolecules.

For target and the source substance Sophorolipids, the main route of excretion is expected to be by expired air as CO2 after metabolic degradation. The second route of excretion is expected to be by biliary excretion with the faeces. For the cleavage products, the main routes are renal excretion via the urine due to the low molecular weight and the high-water solubility and exhalation as CO2.

With regard the source substance AM-1 a pharmacokinetic study revealed that this is primarily eliminated via faeces without absorption.

Based on the available data no additional conclusions can be drawn on the distribution, metabolism and excretion of target and source substances.