Registration Dossier

Administrative data

Description of key information

Overall, although the protein hydrolysates as such are not acutely toxic, their bonding with the quaternary ammonium compound (i.e., Quab 360), which was found to cause acute toxicity and leading to ‘Acute Tox. 4’ classification, is suspected to influence the overall acute toxicity potential of the test substance, as identified in the NRU assay with the read across substance. Therefore, as a worst case the LD50 of the test substance, ‘Steardimonium hydroxypropyl hydrolysed wool’ is expected to lie in the range 300-2000 mg/kg bw (or at 541 mg/kg bw similar to Quab 360).

Key value for chemical safety assessment

Acute toxicity: via oral route

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
541 mg/kg bw
Quality of whole database:
Guideline compliant study

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The acute toxicity potential of the test substance has been assessed based on studies of read across substances as well as substances representative of the 2 main components used to manufacture the test substance, which can be categorised as hydrolysed proteins and quaternary ammonium compound (i.e., Quab 360/Quab 426). The results are presented below:

Read across studies

Study 1: An in vitro study was conducted to determine the acute toxicity potential of read across substance, 'Cocodimonium hydroxypropyl hydrolysed silk (active: 51%)', using cytotoxicity based on Neutral Red Uptake (NRU) Method, according to OECD Guideline 129, in compliance with GLP. The study was assessed in vitro using XCellR8’s internally validated Human Cell-Based Screen (Non-Regulatory Method). The test uses cultured human dermal fibroblasts in animal product free culture, NRU method and a prediction model, based on the GHS classification system for acute toxicity. After a 24 h ± 1 h exposure of 8 concentrations (i.e., 300, 200, 133.3, 88.9, 59.3, 39.5, 26.3, 17.6 µg/mL) of test substance in cell culture medium of Human Dermal Fibroblasts neonatal (HDFn), cytotoxicity was evaluated. Using a prediction model, determined previously, the IC50 value was converted to a corresponding GHS classification for oral acute toxicity. The percentage of viability for each concentration was calculated and normalised to viability results of the negative control (untreated cells) arbitrarily set to 100%. The IC50 (i.e. the concentration at which a decrease in cell viability of 50% was observed) was calculated as being 33.6 µg/mL (17.14 µg a.i./mL) in the Range Finding Experiment (RFE); 40.5 µg/mL (20.66 µg a.i./mL) in the main experiment 1 (ME1); 25.3 µg/mL (12.90 µg a.i./mL) in ME2 and 26.1 µg/mL (13.31 µg a.i./mL) in ME3. Based on the study results (IC50: 12.90 to 20.66 µg/mL), the study author concluded, the read across substance could fall in potential EU CLP category 4 (LD50: 300 to 2000 mg/kg bw) (XCellR8, 2017). However, it is known that the in vitro NRU cytotoxicity assay has a high false positive rate and, therefore, positive results cannot be readily used in a meaningful way in characterising the acutely toxic substances.

Study 2: A study was conducted to determine the acute toxicity potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed keratin' (active content not specified), according to a method described by Hagan et al 1959, in compliance with GLP. Ten albino rats (5 animals each sex), weighing 200 to 300 g, each received a single oral dose of the test substance at a dose level of 5000 mg/kg bw. Animals were observed for pharmacological activity and drug toxicity at 1, 3, 6 and 24 h after treatment and daily thereafter for a total of 14 d. Non-survivors and animals surviving the 14 d observation period were subjected to gross necropsy, with all findings noted. No significant changes in bodyweight or gross were observed in any animal. Under the study conditions, the LD50 of the read across substance was determined to be >5000 mg/kg bw (CPT 1981).

Hydrolysed proteins:

Study 1:A study was conducted to determine the acute toxicity potential of the read across substance, 'Hydrolysed keratin (active: 20%), according to a method described by Hagan et al 1959, in compliance with GLP. Ten albino rats (5 animals each sex), weighing 204 to 256 g, each received a single oral dose of the test substance at a dose level of 5000 mg/kg bw. Animals were observed for pharmacological activity and drug toxicity at 1, 3, 6 and 24 h after treatment and daily thereafter for a total of 14 d. Non-survivors and animals surviving the 14 d observation period were subjected to gross necropsy, with all findings noted. No significant changes in bodyweight were observed in any animal. Only one female animal died on the Day 9 of the observation period. No gross changes were observed in nine animals, whereas fibrous tissue encasing heart and lungs was noted in one animal. Under the study conditions, the LD50 of the read across substance was determined to be >5000 mg/kg bw ( i.e., equivalent to >1000 mg/kg bw a.i.) (CPT 1983).

Study 2: A study was conducted to determine the acute toxicity potential of the read across substance, 'Hydrolysed keratin (active content not specified)', according to OECD Guideline 401 (limit test), in compliance with GLP. Following overnight fasting, groups of five male and five female rats were administered single dose of 10 g/kg bw test substance, by oral gavage route. All animals were observed for a 14 d period for any signs of toxicity or other effects of treatment. No treatment related adverse effects or signs of toxicity and no abnormalities at necropsy were noted in all animals. No significant body weight change related to treatment was observed. Under the study conditions, the oral LD50 of the read across substance was determined to be >10 g/kg bw (TLL, 1986).

Study 3: A study was conducted to determine the acute toxicity potential of the read across substance, 'Hydrolysed keratin from wool (active: 25%)', according to OECD Guideline 401 (limit test), in compliance with GLP. Following overnight fasting, groups of five male and five female rats were administered the test substance at 2000 mg/kg bw, by oral gavage route. All animals were observed for a 14 d period for any signs of toxicity or other effects of treatment. One female animal did exhibit piloerection immediately after dosing. No other signs of toxicity were observed and minor abnormalities were noted in two animals at necropsy. Under the study conditions, the oral LD50 of the read across substance was determined to be >2000 mg/kg bw (i.e., equivalent to >500 mg/kg bw a.i.) (TLL,1988).

Further, the hydrolysed proteins differences across the read across substances is not expected to have an impact, as the proteins in general are not toxic and form and play an important part in the living organisms (such as enzymes, antibodies, hormones, transport or structural proteins, essential elements of the motile and contractile systems) (Lehninger, 1983). The proteins that are found in food and eaten by human beings and mammals are normally degraded metabolically by means of enzymatic processes to give rise to more simple metabolites (peptides and amino acids) that are used by the live cells for the biosynthesis of new specific proteins. The hydrolysed proteins coming from the enzymatic hydrolysis of the animal tissues, therefore, do not cause any danger to human beings and mammals in general. Proteins appear in all biochemical processes that take place in every live cell being, this way they are considered as essential compounds for human life. Moreover, the hydrolysed proteins are authorized by the EU in order to be used as attractant in the elaboration of baits in combination with appropriate insecticides of the Organic Farming (Regulation EC 1488/97 annex 2, part B). This shows the innocuousness of these compounds, since the practice of this kind of agriculture is very demanding with the use of products that can be harmful to human beings. Also in Regulation (EC) No 1774/2002 of the European Parliament and the Council of 3 October 2002 laying down health rules concerning animal by-products not intended for human consumption, hydrolysed proteins (molecular weight <10,000 Dalton) have been allowed in feed animal products, ensuring the safety and the toxicology harmlessness of them.

Quaternary ammonium compounds (QAC)

A study was conducted to determine the acute toxicity potential of the read across substance, Quab 360 ( (3-chloro-2-hydroxypropyl)-cocoalkyl dimethylammonium chloride; active: 35%) when administered by oral gavage to Wistar rats (Bor: WISW) according to OECD Guideline 401. The test was performed with undiluted substance. Four groups of 5 male rats were dosed with 693, 838, 1015, 1230 mg a.i./kg bw and three groups of 5 females were dosed with 389, 573, 838 mg/kg bw of the read across substance; doses were corrected for the 35% active content of the read across substance. Following signs of toxicity were noted: piloerection, restrained gait, stilted gait, diarrhea, sunken sides, body weight decrease, individual chromodacryorrhoea, and tremor, premortal general loss of reflexes. First signs of intoxication symptoms were seen 2 to 5 h post administration and in the low dose 1 to 2 d post administration. Mortality was observed between Day 1 and 4. Gross pathological findings showed red-coloured mucous membrane of the stomach and duodenum. Stomach tympanic and stomach and abdomen filled with liquid. Under the study conditions, the oral LD50 of the read across substance was determined at 541 mg/kg bw (95% confidence intervals: 354 -827) for female rats and at 912 mg/kg bw (95% confidence intervals: 742 -1121) for male rats (Degussa, 2007). Further, a single acute oral toxicity study available on the test substance, Quab 426 (or (3-chloro-2-hydroxypropyl)dimethyloctadecylammonium chloride; active not specified) revealed an oral LD50 value of 2590 mg/kg bw (95% confidence interval: 1400-47900) (Carpenter et al., 1978). However, in absence of active ingredient specification in the study with Quab 426, the lower oral LD50 value from study with Quab 360, has been considered further for hazard assessment.

Overall, although the protein hydrolysates as such are not acutely toxic, their bonding with the quaternary ammonium compound, which was found to cause acute toxicity and leading to ‘Acute Tox. 4’ classification, is suspected to influence the overall acute toxicity potential of the test substance, as identified in the NRU assay with the read across substance. Therefore, as a worst case the LD50 of the test substance, ‘Steardimonium hydroxypropyl hydrolysed wool’ is expected to lie in the range 300-2000 mg/kg bw.

References:

1) Principles of biochemistry by Albert L Lehninger. pp 1011. Worth Publishers, New York, January, 1983.

2) Carpenter CP, Weil CS and Smyth HF 1974. Range-finding toxicity data: List VIII. Toxicology and Applied Pharmacology, 28: 313-319.

Justification for classification or non-classification

Based on the weight of evidence information, the test substance, 'Steardimonium hydroxypropyl hydrolysed wool' is concluded to warrant 'Acute Tox.4, H302: Harmful if swallowed' classification according to EU CLP criteria (Regulation EC 1272/2008).