Tripotassium hydrogen ethylenediaminetetraacetate

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

1

Reference substance name:

trans-cyclohexane-1,2-dinitrilotetraacetic acid

EC Number:

236-308-9

EC Name:

trans-cyclohexane-1,2-dinitrilotetraacetic acid

Cas Number:

13291-61-7

Molecular formula:

C14H22N2O8

IUPAC Name:

2,2',2'',2'''-(cyclohexane-1,2-diyldinitrilo)tetraacetic acid

Test material form:

solid: particulate/powder

Details on test material:

Purity: > 99 %

Specific details on test material used for the study:

The test item provided by the sponsor was in the powdered form. Hence, the test item was moistened with distilled water and a paste was made and applied on the clipped area of the rat skin.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Total 50 Males + 50 Females (15 male + 15 females for DRF study), (20 males + 20 females for Main groups, 10 males + 10 females for recovery group and 5 males + 5 females for randomization purpose) were received.

Sex:

male/female

Administration / exposure

Type of coverage:

occlusive

Vehicle:

water

Details on exposure:

Required quantity of the test item was calculated based on individual animal’s recent body weight and then test item was applied on the clipped area (approximately 10% of the body surface area). After application of the test item that area of skin was covered with cotton gauze and held in place with non-irritating adhesive occlusive (crepe bandage). For control group only distilled water was applied and same procedure was carried out. The test item was held in contact with the skin for at least 6 hour and the patch was removed after completion of exposure period. At the end of the contact period, residual test item was washed using soaked absorbent cotton in distilled water and dried using absorbent cotton.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

The animals were treated with the test item for at least 6 hours daily for a minimum period of 14 days for DRF study and 28 days for main and recovery group animals. After Day 28, the test application for recovery group animals was stopped and these animals were observed for additional 14 days in order to detect delayed occurrence, if any, or recovery from toxic effects.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

400 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

800 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males, 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

For the DRF study the following doses were applied: 0, 250, 500, 750, 1000 mg/kg bw.
The dose groups for the main study were choosen due to effects in the highest dose group of the DRF study.

Examinations

Sacrifice and pathology:

Animals sacrificed by using over dose of CO2 and examined externally.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment related clinical signs were observed in either sex upto 1000 mg/kg body weight in Dose Range Finding (DRF) study and upto 800 mg/kg body weight in Main study.

Dermal irritation:

no effects observed

Description (incidence and severity):

Skin scoring did not reveal erythyma or oedema in any of the groups throughout the study period for both DRF as well as main study.

Mortality:

no mortality observed

Description (incidence):

No treatment related mortality/morbidity were noted in either sex upto 1000 mg/kg body weight in Dose Range Finding (DRF) study and upto 800 mg/kg body weight in Main study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights for the DRF study showed a statistically significant decrease in the G4 and G5 male. Even in the % body weight change, a reduction was observed in the G5 males. Due to this result obtained, for the main study 1000 mg/kg body weight dose was not selected. Even though this reduction was obtained it can not be certainly said that it is test item related as the number of animals in these groups were 3 and even if one animal shows a reduction (which could be incidental), will give statistical significant result.
Mean body weights remained comparable among the main groups and also between the recovery groups for every time point recorded in both the sexes during and after the exposure period.
Nonetheless, a slight statistically significant increase was observed in % body weight change of G3 female at day 8 when compared to the control group animals (G1). But this difference was not seen till the end of the study period and hence the result can be regarded as inconsequential. Decrease in % body weight of G4R male was also observed at day 8 and day 28 however the same was not observed during the additional 14 days recovery period of these animals and hence this result does not affect the inference of the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The feed consumed per animal per day remained statistically comparable among the experimental groups within each sex at every point of observation for DRF and the main study animals.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Eye examinations through ophthalmoscope did not reveal any abnormalities in the control or high-dose groups in either sex during the last week of treatment for DRF and Main group and recovery group animals.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The hematology parameters revealed a statistically significant increase in mean corpuscle haemoglobin concentration (MCHC) of G3 males when compared to the control animals. Platelet count was also found to be increased in G3 and G4 males in comparison to the control group however the values obtained were still within the biological range. In female only the Mean Corpuscular Hemoglobin (MCH) parameter decreased significantly for the G4 group animals when compared to the control animals. In view of the results available from the present study, the increased haemoglobin content in females does not seem to be a biologically significant outcome of test-item application. Female recovery group depicted a statistically significant increase in the platelet count, prothrombin time and activated prothrombin time.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry parameters revealed statiscally significant increase in the creatinine kinase enzyme level in the G2 male when compared to the G1 control goup animals, however in G2 group only one animal showed that increase and should be considered as incidental. In main group of females none of the parameters showed any change that was significant. However, G4 high dose recovery female group (G4R) showed an increase in the AST levels when compared to the control recovery group (G1R). Though a siginificant decrease has been observed in AST levels, the values obtained for recovery group are well within the biological range and in accordance with the liver histology thus has no impact on the results of the study. The high dose male recovery group (G4R) depicted a statistically significant decrease in the total protein as well as in the globulin level when compared to the male control recovery group (G1R), because of which the A/G ratio increased significantly. Even for both these parameters, the level of total protein as well as globulin falls under the biological range and the decrease has no biological significance to the outcome of test-item application.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant change was observed in the absolute organ weight of all animals of either sex. In the organ weight relative to body weight parameter, G3 (mid-dose) male group showed statistically significant increase in the adrenal when compared to the control group (G1). This observation was inconsistent with the dose levels and therefore not of great informative value, in the context of the current study. An increase in testes was also noted in the G4 recovery male (G4R) group in comparison with the control male recovery group (G1R). A statistically significant increase was also observed in the lungs of female (high dose) recovery group (G4R) when compared to the control recovery group (G1R). The differences in mean weight of lungs is not a cause for concern as far as test-item effects from this study are concerned.

Gross pathological findings:

no effects observed

Description (incidence and severity):

External examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance for both DRF as well as for main study. Internal examination of the rats of control and other treated groups did not reveal any abnormality of pathological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination of control group and treatment group at 800mg/kg revealed varying degree of pathological changes in different organs. This includes. Liver: focal mild hepatocellular neutrophilic infiltration (female:G4:1/5), Lungs: multifocal minimal to mild, lymphocytic infiltration (female: G1: 1/5; G4: 2/5). Thyroid: focal minimal ultimobranchial cyst (male: 1/5).
The observed lesions in lungs, liver and thyroid in treated rats are of low occurrence rate and compared well with the rats of control groups in either sex. Such lesions usually develop in the different laboratory animals to certain extent, hence are considered to be spontaneous / incidental in nature. These lesions are also reported by previous workers during toxicological studies (Boorman et al., 1990; Greaves, 2007).

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No neoplastic histopathological findings were observed.

Other effects:

no effects observed

Details on results:

Based on the above-stated results and under the experimental conditions used in this study, it is hereby concluded that the No Observed Adverse Effect Level (NOAEL) of the test item, CDTA acid (Trade name: Goldmann CDTA HHQ) (CAS No.: 13291-61-7) is found to be 800 mg/kg body weight, when administred for 28 days to Wistar Rats with a 14 days recovery period.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

dermal irritation

food consumption and compound intake

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

Remarks on result:

other: no effects were observed that can be assigned to the toxicity of the test substance.

Target system / organ toxicity

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

800 mg/kg bw/day (nominal)

Applicant's summary and conclusion

Conclusions:

Based on the above-stated results and under the experimental conditions used in this study, it is hereby concluded that the No Observed Adverse Effect Level (NOAEL) of the test item, CDTA acid (Trade name: Goldmann CDTA HHQ) (CAS No.: 13291-61-7) is found to be 800 mg/kg body weight, when administred for 28 days to Wistar Rats with a 14 days recovery period.

Executive summary:

A total of 60 Wistar rats (30 males and 30 females) were randomly allocated to six different dose groups for Main study. Each group consisted of 5 animals/sex for main group and 5 animals/sex for recovery group. Main Study doses were selected based on the results of DRF study. The animals allocated to Group G1/G1R, G2, G3, and G4/G4R received 0, 200, 400 and 800 mg/kg body weight of CDTA acid (Trade name: Goldmann CDTA HHQ) for 28 consecutive days. The animals of G1 group were applied distilled water for 28 consecutive days.

Observations on the animals encompassed mortality/morbidity, onset of any clinical signs/symptoms, skin scoring for erythma and oedema, body weight, body weight changes, feed consumption, ophthalmological examination, hematology, clinical biochemistry, gross pathology and histopathology.

The results of this study reveal that CDTA acid does not cause mortality upto a dose of 800 mg/kg body weight when dermal application was done for 28 days. Further, no clinical signs or symptoms were observed in any animals of the study. While feed consumption remained comparable across groups through the course of the study, a difference in the percent change in body weights was noted between females of G1 and G3 groups and in G1R and G4R male, which, nevertheless, was found unrelated to test-item administration.

Ophtalmological profile was also found normal in the control and high dose groups in both the sex.

Clinical chemistry parameters tested herein were inclusive of serum biochemistry, electrolytes and hematology. The values largely remained unperturbed due to test-item application. The observed variation in different hematological and biochemical parameters at the end of treatment and recovery period, did not show dose dependency and further these variations are noted in either male or female animals. The observed values which varied significantly are well within biological range. Further, no abnormality of pathological significance is observed at histopathological observations which are consistent with these noted variations.

Examination of external features and gross pathology during necropsy revealed no noteworthy observations attributable to test-item administration.

Histopathological observations were made for G1 (vehicle control) and G4 (high-dose) animals and no treatment related changes showed up in any of the observed tissues at the tested dose (800 mg/kg body weight) in either sex.