Reaction mass of 2,4-bis(xylylazo)resorcinol and 2,4-bis[(4-dodecylphenyl)azo]resorcinol and 2-[(4-dodecylphenyl)azo]-4-(2,4-xylylazo)resorcinol

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics, other

Type of information:

other: Written assessment based on the available information

Adequacy of study:

key study

Study period:

December 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: non-GLP assessment

Materials and methods

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2,4-bis(xylylazo)resorcinol and 2,4-bis[(4-dodecylphenyl)azo]resorcinol and 2-[(4-dodecylphenyl)azo]-4-(2,4-xylylazo)resorcinol

EC Number:

915-586-1

Molecular formula:

Variable; substance is a UVCB.

IUPAC Name:

Reaction mass of 2,4-bis(xylylazo)resorcinol and 2,4-bis[(4-dodecylphenyl)azo]resorcinol and 2-[(4-dodecylphenyl)azo]-4-(2,4-xylylazo)resorcinol

Results and discussion

Any other information on results incl. tables

Summary and discussion of toxicokinetics

Introduction

This assessment of the toxicokinetic properties of Solvent Yellow 175 is based on the results obtained for the toxicological end‑points listed below with simultaneous reference to relevant physico-chemical data:

-         Acute oral toxicity

-         Skin irritation

-         Eye irritation

-         Skin sensitisation

-         Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test

-         Bacterial reverse mutation test

-         In vitro chromosome aberration test

Toxicological Profile

Solvent Yellow 175 is a UVCB substance primarily containing three main constituents (origin: organic). The substance has a molecular weight range of 374 - 696, a water solubility of <1.0 x 10 -5g/L at 20 °C, a partition coefficient (n-octanol/water) (log Kow) of > 6.5 and a vapour pressure of 3.4 x 10-4Pa at 25 °C.

Solvent Yellow 175 was administered to fasted male/female Sprague-Dawley rats via oral gavage with mineral oil at a single dose of 5 g/kg bw. Treated animals were then observed for a period of 14 days for signs of toxicity and mortality, gross necropsy was performed following the end of the observation period. Under the conditions of the study, no mortality occurred and all treated animals were in good health with normal weight gain. Clinic signs comprised of hunched posture and loose stool which occurred on days 1 and 2 only. Gross necropsy did not reveal any treatment related effects. Based on the results of the study, the acute oral LD50 was determined to be > 5 g/kg bw.

Skin irritation for Solvent Yellow 175 was assessed by applying 0.5 g of the substance to an unabraded site of 6 male albino rabbits for a period of 4 hours and then observed for 72 hours. The were no effects relating to skin irritation noted throughout the study and had a calculated Primary Irritation Index of 0 / 8.

Evaluation of the eye irritation potential for Solvent Yellow 175 was done with the use of six New Zealand Albino rabbits at a dose of 0.1g. The substance was instilled into the conjunctival sac of each animal and observed for 72 hours following instillation. Conjunctival irritation (erythema) was noted in 4/6 treated eyes with discharge being observed in 1/6 eyes treated, effects were seen to be fully reversible within 24 and 48 respectively. The mean group score was calculated to be 2.0/110.0.

Sensitisation potential of Solvent Yellow 175 was assessed with the use of Buehler test. The substance was administered undiluted to a shaved area on the left trunk of 10 albino guinea pigs with the use of a Hilltop Chamber. Following the initial dose period of 24 hours, nine succeeding induction doses were unwrapped after a 6 hour exposure period. This procedure was repeated three times weekly (with at least one day intervening between treatments) for a total of ten applications. Two weeks after the final application the animals received a topical challenge dose (24 hour contact) at a naive site located on the right trunk. Animals were scored for irritation 24 and 48 hours after each application. Using the same regimen, six guinea pigs received 0.5 ml of a 0.1 % solution of 1-chloro 2,4 dinitrobenzene in (80% aqueous ethanol) to serve as a positive control. Four guinea pigs were observed as a negative control. Two negative control animals were also challenged using the test material. The initial dose of Solvent Yellow 175 or 1-chloro 2,4 dinitrobenzene produced no positive irritation scores in any of the guinea pigs. The ten dose average irritation score was 0 for Solvent Yellow 175. The ten dose average irritation score for 1-chloro 2,4 dinitrobenzene was 0.39. Average scores for the challenge dose were 0 for Solvent Yellow 175 and 0 for the challenged negative controls. The average score for the challenge dose of 1-chloro 2,4 dinitrobenzene was 1.08. The challenge dose of Solvent Yellow 175 and two negative control animals challenged with Solvent Yellow 175, did not elicit a positive response. The challenge dose of 1-chloro 2,4 dinitrobenzene resulted in a positive response in 6/6 guinea pigs, characterized by a grade 1 for erythema and/or edema. Based on comparisons of the initial test dose response to the challenge test dose and to the reactions elicited by the positive and negative controls, Solvent Yellow 175 not be considered a dermal sensitizing agent.

 

Solvent Yellow 175 was administered orally (by gavage) to male and female Wistar rats once a day for 14 consecutive days at 100, 500 and 1000 mg/kg/day body weight in a dose range toxicity study. There were no mortalities or test-item-related clinical signs throughout the study (other than colored feces/urine in cage, related with the test item color, or salivation related with the test item palatability). Food consumption and body weight were considered unaffected by treatment. No relevant test-item-related effects were seen in the water consumption. No macroscopic lesions were recorded at the end of the study. Staining in adipose tissue and stomach were observed, related with the test item color. There were no effects in organ weights, although males showed a slight increase in mean liver and kidney weight when increasing the dose. Under the conditions of this study, the daily oral gavage administration of Solvent Yellow 175 to Wistar rats daily for 14 consecutive days at dose levels up to 1000 mg/kg/day caused no signs of evident toxicity. No target organ has been identified within the confines of this study.

 

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Study in the Wistar Rat by Oral Gavage Administration In this study, oral administration (by gavage) of Solvent Yellow 175 was undertaken at the doses of 100, 300 and 1000 mg/kg/day for two weeks prior to mating and up to the day before sacrifice inclusive (males) or up to days 13-15 of lactation (females) was well tolerated.

 

Mortality was observed only in two females at 300 mg/kg/day and 1000 mg/kg/day, who were sacrified for welfare reasons between gestation days 22 and 23, showing signs of difficulty during parturition – it is thought that a direct toxic effect of the test item was observed in the stomach and occasionally in the duodenum and cecum and was considered to have been the main factor contributing to the unscheduled deaths. In addition, one female at 100 mg/kg/day, three females at 300 mg/kg/day and six females at 1000 mg/kg/day were sacrificed as they lost their corresponding litters during late gestation and early lactation phases.

 

Behavioural parameters (sensory reactivity, grip strength and motor activity) were unaffected in surviving animals and there were no overt signs of toxicity from the clinical signs. Dark feces and yellow urine were considered related to the test item color. Salivation recorded during the administration period was related to taste aversion (from gavage dosing) (Hood, R.D. 2006).

 

No treatment related, statistically significant effects were observed in body weight, food consumption, blood chemistry.

 

Regarding T4 concentrations in adult males, statistically significant lower mean values were observed in the test-item-administered groups when compared to Control. Although these values were within the historical control data, minimal follicular cell hypertrophy was observed in all test-item-treated male groups, in correlation with the increased thyroid and parathyroid weights in males compared to Controls, with a higher incidence at 1000 mg/kg.

 

Given the concomitant increase in liver weights, follicular cell hypertrophy was most probably compensatory, secondary to a test-item-related non-adverse hepatic enzyme induction and clearance of thyroid hormones (Chandra et al., 2013). The fact that no centrilobular hypertrophy suggestive of enzyme induction was seen in the histological examination does not rule out its occurrence.

 

The minor increase in hyaline droplets in the kidneys of males given 1000 mg/kg/day is consistent with treatment-related accumulation of α-2u-globulin, a common finding in untreated male rats (Khan et al., 2002). Hyaline droplet accumulation is specific to the male rat and is not generally considered to be significant in man. In this study, this was not accompanied by any other histologic features of pathology, and was therefore considered to be non-adverse.

 

The kidneys of one female treated at 1000 mg/kg/day showed histological changes which correlated with gross pallor. The single case of marked tubular necrosis in a high dose female (1000 mg/kg/day) is of unclear pathogenesis and its relation with the treatment cannot be completely ruled out.

 

There were minimal to severe treatment-related changes in the thymus of both sexes at 300 or 1000 mg/kg/day, with a higher incidence and severity in females. They correlated with gross findings at necropsy and/or lower organ weights recorded in males, and were interpreted as stress-related (Everds et al., 2013). In this study, other findings indicative of stress were occasionally found in decedent females treated at 300 or 1000 mg/kg/day (spleen, lymph nodes and pancreas). A minimal degree of decreased lymphocyte cellularity in the thymus can be normally seen among pregnant/lactating females. Thus, the minimal changes seen in two females given 100 mg/kg, in the absence of concurrent changes in the stomach or cecum, were considered to be a result of normal individual variation. A single female given 100 mg/kg had a small thymus. However, no thymic tissue was found at histological examination.

 

The other organ weight changes reported at the end of treatment (female brain and oviducts/uterus/cervix) did not have a correlate at histopathology. The increased weight in the reproductive organs of females given 300 mg/kg was explained by the incidental increase in cervical mucification of a single female. The increase in epididymides (mainly at 1000 mg/kg/day) was considered not relevant, taking into consideration that there were no histopathology findings corroborating it and as mean values are within the historical control data.

 

In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted in the examined males treated at 1000 mg/kg/day.

 

Estrous cycles (during treatment and at termination) and reproductive parameters of pre-coital interval, mating performance, conception or fertility were unaffected by treatment. The duration of the gestation length was increased in the animals administered the test item as well as the gestation index was reduced in a dose relation, when compared with the Control group (mainly at 300 and 1000 mg/kg/day).

 

The number of live-born fetuses was reduced in a dose-related manner, as was the gestation index, in which the mean values of 300 and 1000 mg/kg/day were statistically significant. There was no effect in offspring mean body weights. Regarding survival indices, there were lower mean values in post-implantation survival, live birth and viability indices when compared to Control, showing statistically significant differences at 300 and 1000 mg/kg/day. In addition, at 1000 mg/kg/day, lactation index on day 13 was reduced with respect toControl values. Consequently, litter size in the test-item-administered groups was lower than that recorded in the Control group (mainly at 300 and 1000 mg/kg/day in which statistically significant differences were recorded). There were no offspring clinical or necropsy signs  indicative of a reaction to Solvent Yellow 175, and there was no relevant effect on sex ratio or nipple areolae. Females at 300 and 1000 mg/kg/day showed significantly higher ano-genital distance mean values with respect to Control which were considered not relevant taking into account the Historical Control Data. No Observed Adverse Effect Level (NOAEL) could be established at 100 mg/kg/day.

 

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations. The vehicle (acetone) control plates gave counts of revertant colonies within the historical control range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A yellow colour (becoming orange/red with increasing test material concentration) was noted from 50 μg/plate. A red test material precipitate was also observed at and above 150 μg/plate. Neither of these observations prevented the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

Solvent Yellow 175 was evaluated in the Prival modification of the Ames/Salmonella Liquid pre-incubation Assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella typhimuriurn in the presence and absence of an exogenous metabolic activation system (S9). Toxicity of Solvent Yellow 175 was first evaluated in a prescreen by treating duplicate cultures of strains TA1538 and TA100 with five doses of Solvent Yellow 175 in the absence of S9. Results of the prescreen indicated Solvent Yellow 175 was not toxic to either strain at a dose of 50.0 ug/plate. Inhibited growth (characterized by a reduced background lawn and/or the presence of pindot colonies) was observed in both strains at a dose of 167 ug/plate and in strain TA100 at a dose of 500 ug/plate. Complete toxicity was observed in strain TA1538 at a dose of 500 ug/plate and in both strains at doses of 1670 and 5000 ug/plate. In addition, the test article precipitated from solution/formed oily droplets at all doses. Based upon these findings, Solvent Yellow 175 was evaluated in triplicate cultures in strains TA1535, TA1537, TA1538, TA98 and TA100 in the presence and absence of S9 at doses of 1.67, 5.00, 16.7, 50.0, 167 and 500 ug/plate. Six dose levels of Solvent Yellow 175 were evaluated with and without S9 in the event of unacceptably high toxicity at the highest dose levels. The S9 mixture included 30% (v/v) un-induced male Syrian Golden hamster liver homogenate with the appropriate buffer and cofactors. The test article was found to be incompletely soluble at doses of greater than or equal to 50.0 ug/plate. Inhibited growth was observed in all tester strains at doses of 167 and 500 ug/plate without S9. Revertant frequencies for all doses of Solvent Yellow 175 in all strains without S9, and in strains TA1535, TA1538, TA98 and TA100 with S9, approximated or were less than those observed in the concurrent solvent control cultures. In contrast, statistically significant and apparently dose-dependent increases in revertant frequencies, to approximately 1.8-fold control values, were observed in strain TA1537 with S9. However, the increases observed were within historical control values, and dose-dependent decreases in revertant frequencies were observed over the dose range of 1.67-500 ug/plate. Therefore, Solvent Yellow 175 was re-evaluated in strain TA1537 at doses of 0.0500, 0.167, 0.500, 1.67, 5.00, 16.7, 50.0, 167 and 500 ug/plate with S9. Revertant frequencies for all doses of Solvent Yellow 175 in strain TA1537 in the re-test approximated those observed in the concurrent solvent control values. Thus, the slight increases observed in strain TA1537 in the original assay are considered to be statistical abberations due to random fluctuation of the spontaneous revertant frequency. All positive and negative control values in both assays were within acceptable limits. Therefore, the results for Solvent Yellow 175 were negative in the Prival modification of the Ames/Salmonella Liquid Pre-incubation Assay under the conditions, and according to the criteria, of the test protocol.

Assessment of the potential mutagenicity of Solvent Yellow 175 on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line was conducted. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline. One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (acetone), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows: 0.31, 0.63, 4-hour with S9 (2%) 1.25, 2.5, 5, 10 μg/mL. The maximum dose levels in the Mutagenicity Test were limited by the presence of test item precipitate in all three of the exposure groups, as recommended by the OECD 490 guideline. The vehicle control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups. The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

The peripheral erythrocyte micronucleus (MN) assay was used to evaluate the clastogenic potential of Solvent Yellow 175. Doses of 0, 1200, 2500, and 5000 mg/kg BW of Solvent Yellow 175 in males produced PCE/RBC values of 1.68, 1.80, 1.58, and 1.62%, respectively. In females, the same doses produced PCE/RBC values of 1.58, 1.50, 1.37, and 1.54%, respectively. In males, doses of 1200, 2500, and 5000 mg/kg BW yielded 0.06, 0.22, and 0.16% PCE with MN, respectively, compared with a vehicle control value of 0.20%. In females, the same doses yielded 0.22, 0.20, and 0.24% PCE with MN, respectively, compared with a vehicle control value of 0.26%. In contrast, benzene yielded 3.90% PCE with MN in male mice. None of the doses of Solvent Yellow 175 induced a statistically significant increase in the micronucleus frequency. On the basis of the results, it is concluded that Solvent Yellow 175 does not induce MN in polychromatic erythrocytes from swiss-Webster mice under the conditions of this assay.

Toxicokinetic parameters

Absorption

Oral

All male and female rats given 100, 300 or 1000 mg/kg/day had a yellow discoloration of various tissues throughout the body attributed to the presence of the test material. The yellow discoloration was most prominent in adipose tissue. Histopathology revealed changes that were considered related to treatment with the test item in male thyroids and kidneys, female stomach and cecum, and thymus of both males and females. Changes observed in males, i.e. decrease in thymus weight and increase in thyroids and parathyroids, correlate with these findings. No treatment-related effects were detected in the reproductive organs or mammary glands. Regarding offspringsurvival indices, post implantation and live birth indexes decreased in a dose-related manner, and lactation index at 1000 mg/kg/day was lower with respect to Control. This implies that the substance and/or its metabolites are absorbed following oral ingestion, but the incidence of oral availability is unknown given the toxicity observed. The test substance has water solubility of <1.0 x 10-5g/L at 20 °C and a log Kow of > 6.5. The absorption of highly lipophilic substances (log Kow ≥ 4) may be limited by the inability of such substances to dissolve in gastrointestinal fluids and therefore make contact with the mucosal surface. However, the absorption of such substances will be increased if they undergo micellular solubilisation by bile salts. As a worst case, for risk assessment purposesthe oral absorption of the test substance is set at 100%. This is due to the fact that the absorption is unknown, hence it is appropriate to consider worst case for the purposes of derivation of DNEL.

 

Dermal

The results obtained from the skin irritation and skin sensitisation studies do not provide any evidence to imply that there will be significant absorption via the dermal route. Minimalerythema and/or edema was observed in the challenge in a Buehler test, a Primary Irritation Index of 0/8 was calculated in the skin irritation study.

 

The n-octanol/water partition coefficient of the test substance is > 6.5 and the molecular weight is moderate (374 – 696 (<100 being optimal for dermal update > being too large) therefore the dermal absorption of the substance is expected to be limited based on the high log Kow value and molecular weight. At log Kow values above 4, the rate of penetration may be limited by the rate of transfer between the stratum corneum and the epidermis, but uptake into the stratum corneum is likely to be high. Maximum dermal absorption is often associated with values of log Kow between +1 and +2 (ECETOC (European Centre for Ecotoxicology and Toxicology of Chemicals). Monograph No, 20; Percutaneous absorption. August 1993). In addition, the substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. The test substance has a water solubility of <1.0 x 10-5g/L at 20 °C therefore dermal uptake is likely to be low. In conclusion, dermal absorption of the substance is expected to be low. In the absence of significant absorption, due consideration of the absorption needs to be carefully considered. 

As a worst case, for risk assessment purposes thedermal absorption of the test substance is set at 100%. 

 

Inhalation

The substance has a predicted low vapour pressure (3.4 x 10-4Pa at 25 °C) therefore a significant inhalation exposure to vapours is not expected. The substance is not marketed in a physical form that allows exposures to dusts, so is not potentially respirable. Moderate log Kow values (between -1 and 4) are favourable for absorption directly across the respiratory tract epithelium by passive diffusion. The test substance has a high log Kow value (> 6.5) therefore it may be taken up by micellular solubilisation particularly as the substance is poorly soluble in water (<1.0 x 10-5g/L).The aromatic component amines of azo dyes may be absorbed intothe body through the lungs, the gastrointestinal tract or the skin (ECDIN, 1993).

As a worst case, for risk assessment purposes theinhalation absorption of the test substance is set at 100%.

Distribution

The combined repeated dose/reproductive toxicity oral study using Solvent Yellow 175, revealed effects in the rat. The majority of effects noted were considered to be adaptive, but were inclusive of vital organs such as the thyroids, kidneys, thymus, stomach, cecum. Moreover organs were shown to be stained with the colour of the test item, preferencing the adipose tissues.The test substance is lipophilic therefore it is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration particularly in fatty tissues. Metabolites released following ingestion of the test substance in the gastrointestinal tract are likely to be widely distributed in the body. Substances with high log Kow values tend to have longer half-lives unless their large volume of distribution is counterbalanced by a high clearance. Once exposure stops, the concentration within the body will decline at a rate determined by the half-life of the substance. However, as detailed below, the test substance is likely to undergo metabolism following absorption so potentially only transient exposure to the parent compound is expected. 

 

Metabolism and excretion

The azo linkage is the most labile portion of an azo dye molecule and may easily undergo enzymatic breakdown in mammalian organisms, including man. The azo linkage may be reduced and cleaved, resulting in the splitting of the molecule in two parts (Brown & DeVito, 1993). The anaerobic environment of the lower gastrointestinal tract of mammals is well suited for azo-reduction. Several anaerobic intestinal bacteria are capable of reducing the azo linkage. The majority of these bacteria belong to the genera Clostridium and Eubacterium. They contain an enzyme associated with the cytochrome P 450, also termed azo-reductase. It is a non-specific enzyme, found in various micro-organisms and in all tested mammals (NIOSH, 1980). In mammalian organisms azo-reductases are, with different activities, present in various organs like liver, kidney, lung, heart, brain, spleen and muscle tissues. The azo-reductase of the liver, followed by the azo-reductase of the kidneys possess the greatest enzymatic activity.

Although reduction and cleavage of the azo-linkage is the major metabolic pathway of azo dyes in mammals, other metabolic pathways may take place. Major routes of detoxifying metabolism of azo dyes and aromatic amines are ring hydroxylation and glucuronide conjugation. After cleavage of the azo-linkage, the component aromatic amines are absorbed in the intestine and excreted in the urine (Brown & DeVito, 1993). However, the polarity of azo dyes influences the metabolism and consequently the excretion.

The majority of azo dyes requires metabolic activation, namely reduction and cleavage of the azo linkage to the component aromatic amines to show mutagenicityin vitrotest systems. Therefore the majority of azo dyes, if highly purified, will, at least without metabolic activation, be negative in such tests (Arcos & Argus, 1994).

 

After cleavage of the azo-linkage, the component aromatic amines are absorbed in the intestine and excreted in the urine (Brown & DeVito, 1993)

 

Conclusion

In conclusion, there is evidence that the substance is absorbed on ingestion and distributed widely throughout the body, the substance is then metabolised via understood paths for azo dyes such as azo reduction. Such metabolites are subsequently eliminated within urine. Consequently, the substance is considered to have low bioaccumulation potential.

Applicant's summary and conclusion

Conclusions:

In conclusion, there is evidence that the substance is absorbed on ingestion and distributed widely throughout the body, the substance is then metabolised via understood paths for azo dyes such as azo reduction. Such metabolites are subsequently eliminated within urine. Consequently, the substance is considered to have low bioaccumulation potential.