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Classification & Labelling & PBT assessment

PBT assessment

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PBT assessment: overall result

PBT status:
the substance is not PBT / vPvB
Justification:

Persistence (P) assessment

One study, according to the OECD Guideline 301C (modified MITI Test (I)) performed under GLP, is available on the biodegradability of AMBER CORE by microorganisms. The concentration of test substance was 100 mg/L, the concentration of activated sludge was 30 mg/L and the cultivating duration was 28 days. The measurement of biochemical oxygen demand (BOD) was performed and the results show that the percentage biodegradation of the test substance was 3% on the average. AMBER CORE was not biodegraded under these test conditions. Furthermore, hydrolysis studies were performed according to the Directive 84/449/EEC Part C Method C.07, under GLP. In the definitive study, the hydrolysis reaction of AMBER CORE at 25°C and pH 4 occurred with a half-life time of 9 days. At pH 7 and 25°C, the hydrolysis reaction of AMBER CORE occurred with an estimated half-life of 27 days and at pH 9 and 25°C, an estimated half-life of 25 days. These half-lives obtained are compared to persistence criteria of Annex XIII (i.e. a substance fulfils the P(vP) criterion if T1/2> 40(60) days).

In conclusion AMBER CORE is not readily biodegradable and in the hydrolysis studies, the half-lives are lower than criteria for P. This substance is not considered Persistent (P) or very Persistent (vP).

Bioaccumulation (B) assessment

For the B and vB assessment a screening criterion has been established, which is a BCF > 2000 L/Kg (B criterion) or > 5000 L/Kg (vB criterion) or log Kow greater than 4.5.

 

One study is available to assess the bioaccumulation of AMBER CORE on Carp (Cyprinus carpio). This study was performed in a flow through system under GLP and according to the "Method for Testing the Degree of Accumulation of Chemical Substances in Fish Body" stipulated in the "Testing Methods for New Chemical Substances" (July, 1974) and is based on OECD Guideline 305 (May, 1981). An acute toxicity test was performed before the bioaccumulation test. This preliminary test result for 48 -hour LC50 was used to decide on the test concentrations of the test substance in the bioaccumulation study. Two test medium concentration levels were used: 0.050 mg/L and 0.0050 mg/L. The temperature of test water was maintained at 25 +/- 2°C with concentrations of dissolved oxygen in test tank comprised between 6.4 and 7.8 mg/L (corresponding to > 60% saturation). BCFs of the test substance were estimated to have reached equilibrium after 8 weeks of exposure. The bioaccumulation potential of test substance in carp was an approximately similar value at both levels. However, to consider the worst case, the value used for chemical safety assessment was the highest BCF obtained: 173.

 

Furthermore, the partition coefficient of AMBER CORE was estimated using QSAR calculations. Three models (KowWin, SPARC and VCClab), using different methodologies, were run, one was considered as valid (KowWin), the two others not assignable. A total of 10 consistent results were obtained, with a log Kow within the range 2.73-4.05. The arithmetical mean value of these 10 results is 3.70 +/-0.38. However, if we discard the extreme lower value (2.73), the standard deviation lowers to +/-0.18, so the corresponding mean of 3.81 will be retained as key value.

Additionally, to provide supplementary evidence, the bioconcentration factor was also estimated using QSAR calculations. With EPI Suite – BCFBAF v3.01, the log BCF from the regression-based method was 2.340, corresponding to a BCF of 218.8 L/Kg and the log BCF from Arnot-Gobas method (upper trophic) was 2.483, corresponding to a BCF of 304.1 L/Kg. With the CAESAR model, the log BCF was estimated to be 1.43, corresponding to a BCF of 27 L/Kg.

According to all results obtained, the BCF is below than B criterion and the log Kow value is below 4.5. It is assumed that the affinity for the lipids of an organism is insufficient to exceed the B criterion. In conclusion, AMBER CORE is not considered potentially Bioaccumulable (B) or very Bioaccumulable (vB).

 

Toxicity (T) assessment

 

Environmental toxicity

 

Acute and chronic data, based on international guidelines, are available for three trophic levels: Algae, Invertebrates and Fish. Among all the species tested, a toxic effect was found at the three trophic levels, and the most sensitive species was fish with a measured 33 d NOEC of 0.22 mg/L (Pimephales promelas).

In conclusion, AMBER CORE does not meet the criterion for toxicity (T) stipulated in REACH Annex XIII because the lowest NOEC was greater than 0.01 mg/L.

Human Health Hazards Assessment (T)

 

Carcinogenesis:

 

No information was available

 

Mutagenicity:

 

Ø Genetic toxicity in vitro:

In a GLP study performed according to OECD guideline 471, AMBER CORE was tested for mutagenicity usingSalmonella typhimuriumTA 1535, TA 1537, TA 98, TA 100 andEscherichia coliWP2 uvrA-with the preincubation method (20 min) in the presence and absence of metabolic activation system (S-9 mix). A preliminary test was performed using concentrations between 9.8 and 5000 µg/plate with and without S-9. Due to cytotoxicity, the following concentration ranges were tested in the 2 main tests, using the preincubation method (20 min at 37°C) with and without S-9. The positive controls induced the appropriate responses in the corresponding strains. AMBER CORE showed no substantial increases in revertant colony numbers over control count obtained with any of the tested strains at any concentrations in either presence or absence of S-9. Under the test conditions, AMBER CORE is not considered as mutagenic in this bacterial system according to the criteria of the Annex VI of the Regulation (EC) No 1272/2008 (CLP) and the Annex VI of the Directive 67/548/EEC. In a chromosomal aberration assay in mammalian cells, performed according to the OECD No.473, and in compliance with the GLP, AMBER CORE diluted in DMSO was tested in female Chinese Hamster lung (CHL) cells in the presence and the absence of mammalian metabolic activation (S9) at concentrations of varying from 22.5 to 700 µg/mL. AMBER CORE was incubated with the cells for 6, 24 or 48 hours and the cells were analysed for the presence of chromosomal aberrations 18 hours (in the case of the 6 hrs exposure period) or immediately after the end of the exposure period (in the case of the 24 or 48 hrs exposure period). Mitomycin C and Benzo(a)pyrene were used as positive controls and induced appropriate responses. Cytotoxicity was observed at 700 µg/mL with metabolic activation in the first assay. For the second test, the only maximum dose level (100 µg/mL direct assay, 700 µg/mL for the metabolic activation with S9) of the first test was repeated to test because the negative results were obtained in the first test. No increase in the occurence of chromatid or chromosome aberrations was observed with and without metabolic activation at any tested concentration and for all exposure period tested. Under the test conditions, AMBER CORE did not show any cytogenic activity in the chromosomal aberrations test using CHL cells according to the criteria of the Annex VI of the Regulation (EC) No 1272/2008 (CLP) and the Annex VI of the Directive 67/548/EC. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 473.

 

Ø Genetic toxicity in vivo:

In anin vivomicronucleus assay performed according to the OECD guideline No. 474, and in compliance with the GLP, AMBER CORE was administrated via intraperitoneal route to male albino Hsd:ICR (CD-1) mice (7 animals/dose level). In a preliminary test, two mice (one female, one male) were dosed once only at the appropriate dose level by gavage using a metal cannula or with a hypodermic needle attached to a graduated syringe. In the preliminary test, the test material showed no marked difference in its toxicity to male or female mice; it was therefore considered to be acceptable to use males only for the main test. No evidence of toxicity was observed in animals dosed with test material via the oral route and, therefore systemic absorption could not be confirmed using this dose route. Adequate evidence of test material toxicity was demonstrated via the intraperitoneal route of administration which was therefore selected in the main test. The maximum recommended dose of the test material, 2000 mg/kg bw, was selected for use in the main test, with 1000 and 500 mg/kg bw as the lower dose levels. In the main, test, animals were therefore treated with AMBER CORE via one intraperitoneal injection. 24 or 48 Hours after the treatment, animals were killed and bone marrow was collected for further analysis. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated in order to determine the toxicity of the test material. A modest decrease (but not statistically significant) was observed in the PCE/NCE ratio in both the 24 and 48h test material dose groups when compared to their concurrent control groups. This, together with the observation of clinical signs (Hunched posture, ataxia, lethargy, ptosis and splayed gait) was considered to indicate that systemic absorption occurred and exposure to the target tissue was achieved. Furthermore, there was no statistically significant increase in the incidence of micronucleated PCE.

In conclusion, under the test conditions, AMBER CORE is not considered as genotoxic in thisin vivomicronucleus test. Therefore, AMBER CORE is not classified as genotoxic according to the criteria of the Annex VI of the Regulation (EC) No 1272/2008 (CLP) and the Annex VI of the Directive 67/548/EC. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 474.

 

Reproductive toxicity:

The effects of Amber core (P#620) on reproductive function and pre-natal and post­natal developments were assessed according to OECD guideline 415. The test material was administered by gavage to three groups of 24 male and 24 female Wistar rats each, at dose levels of 20, 100 and 500 mg/kg bw/day. A control group of 24 males and 24 females was dosed with vehicle alone (0.5% sodium carboxymethyl cellulose/0.5% Tween 80).

Clinical signs, bodyweight, dietary intake and water consumption were monitored. After ten weeks of treatment for males and two weeks of treatment for females, pairing of animals within each dose group was undertaken on a one male: one female basis, to produce litters. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size. Litter weights and individual offspring weights were also recorded on specific days post partum. All surviving females and offspring were terminated on Day 21 post partum with the males being terminated after the majority of the females and litters had been killed. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

There was no unscheduled death, no relevant clinical sign, no effect on bodyweight and food consumption. Increased water intake was observed for both sexes at 500 mg/kg bw/day and males at 100 mg/kg bw/day (likely to reflect unpalatable, or slightly irritant, test material formulation). Mating performance, fertility and gestation length were considered to have been unaffected by treatment. No treatment-related macroscopic abnormalities were detected for adults or offspring.

Histopathological examinations of adult tissues revealed centrilobular hepatocyte enlargement for both sexes at 500 mg/kg bw/day and a greater incidence and severity of globular accumulations of eosinophilic material in the tubular epithelium of male rats at 500 mg/kg bw/day, with an associated greater incidence and severity of groups of basophilic tubules also being present. Additionally tubular necrosis and affected isolated tubules in the outer medulla immediately adjacent to the renal cortex was observed for 10 males at this dosage level.

It is assumed that the centrilobular hepatocyte enlargement observed, occurs as an induction of the microsomal drug metabolizing enzyme systems caused by the treatment of several compounds and is considered to be cellular adaptation phenomena. In the case of Amber core, the adaptative response is important as it was already demonstrated in the 28 -day repeated oral dose toxicity study described in this dossier (see § 7.5.1).

Moreover, the changes of both the renal tubular epithelium and basophilic tubules are lesions known to be spontaneous in male rats only and are not observed in other species. Therefore, this effect is specific to male rat, and is not relevant for the risk assessment in human.

There was no obvious adverse effect of treatment of the corpora lutea and implantation counts, litter size at birth and subsequent survival and bodyweight of the offspring to weaning or on sex ratio.

Therefore, it can be assumed that no test substance related effect was observed in the highest dose group of adult animals considering that the changes observed in the liver and in the kidney resulted from cellular adaptation phenomena and species specificity, respectively.

Hence, histopathological examinations of liver and kidneys at 20 or 100 mg/kg bw/day were not performed even if increased absolute and relative liver weights in males were recorded assuming that in the absence of any other effect, the liver weight changes was likely related to cellular adaptation phenomena. Therefore, it is assumed that the highest dose (500 mg/kg bw/d) is a NOAEL for systemic effect in the adult animals.

Moreover, there were no effects observed for reproductive parameters and a clear NOEL for reproductive toxicity including the survival, growth and development of the offspring was established at 500 mg/kg bw/day.