trans-cyclohexane-1,2-dinitrilotetraacetic acid

Endpoint summary

Description of key information

The acute toxicity of CDTA HHQ was investigated following a single oral administration (10mL/kg in 0.5% aqueous solution of carboxymethylcellulose) to the Sprague Dawley rat followed by a 14-day observation period. No mortality occurred and no signs of toxicity were observed in the 6 animals following dosing at 2000mg/kg.These results indicate that the test item CDTA HHQ did not induce toxic effects in the rat following oral administration of a single dose at a level of 2000mg/kg. The lack of mortality demonstrates the acute toxicity estimate (ATE) to be greater than 2000mg/kg body weight.

In a repeated dose range finding study rats were exposed to aerosol of the test substance.

6 death occured in the high dose group. No deaths occured in the middle dose group (300 mg/m³).

Taking into account that the exposure duration was significantly extended compared to an acute toxicity study it is reasonable that less than 50% of the animals of the highest dose group would die when exposed to the substance once for 4 hours.

Thus the LC50 can be stated as:

LC50 > 1000 mg/m³ (nominal)

LC50 > 1103 mg/m³ (actual)

This leads to the following classification according to CLP:

acute oral toxicity: not classified

acute dermal toxicity: not data available

acute inhalative toxicity: classified as acute toxic category 4 (H332)

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 - 03. 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

GLP compliance:

yes

Test type:

acute toxic class method

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

Age at order : 6-7 weeks old
Weight range at arrival : 158.4-161.3 grams (The body weight of each individual was within 20% of the group’s mean)
Acclimatisation period : At least 5 days
Veterinary health check : During acclimatisation period
Animals per cage 3 during the study: up to 5 during acclimatisation
Housing : Polisulfone solid bottomed cages measuring 59.5×38×20 cm with nesting material provided into suitable bedding bags
Cage control : Daily inspected and changed as necessary (at least 3 times/week)
Water : Drinking water supplied to each cage via a water bottle
Water supply : ad libitum
Diet : 4 RF 18 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy)
Diet supply : ad libitum throughout the study except for the dosing procedure
Room lighting : Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours
Air changes : Approximately 15 to 20 air changes per hour
Temperature range : 22°C±2°C
Relative humidity range : 55%±15%

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous solution of carboxymethylcellulose

Details on oral exposure:

Test item was administered, by gavage, at a dose volume of 10mL/kg using a plastic feeding tube attached to a graded syringe.

Doses:

On the day of dosing (Day 1), the amount of the formulated test item to be administered was calculated for each fasted animal according to body weight. Animals were dosed once only on Day 1.

No. of animals per sex per dose:

3 female animals/group

Details on study design:

Food was removed fromthe cages overnight prior to dosing (Day -1) and was made available approximately 4 hours after dosing.
Animals were observed for clinical signs as indicated below:
– Day of dosing
· Session 1: on dosing
· Session 2: approximately 0.5 hour after dosing
· Session 3: approximately 2 hours after dosing
· Session 4: approximately 4 hours after dosing
– Daily thereafter for a total of 14 days (Session 1).

Key result

Sex:

female

Dose descriptor:

other: acute toxicity estimate (ATE)

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

A first group of 3 female animals was initially dosed at 300mg/kg (Group 1, Step 1). No mortality occurred and no clinical signs were observed. A second group of 3 female animals was then dosed at the same dose level (Group 2, Step 2). No death occurred and no clinical signs were noted. A third group, similarly composed, was dosed at 2000mg/kg (Group 3, Step 3). No death occurred and no clinical signs were seen. A fourth group of 3 female
animals was administered at the same dose level (Group 4, Step 4). No mortality occurred and no clinical signs were observed.

Body weight:

Changes in body weight observed during the study were within the expected range for this strain and age of animals.

Other findings:

No abnormalities were observed at necropsy examination performed on all animals dosed at 300 and 2000mg/kg (Groups 1, 2, 3 and 4) at the end of the observation period.

Interpretation of results:

GHS criteria not met

Conclusions:

The acute toxicity of CDTA HHQ was investigated following a single oral administration (10mL/kg in 0.5% aqueous solution of carboxymethylcellulose) to the Sprague Dawley rat followed by a 14-day observation period. No mortality occurred and no signs of toxicity were observed in the 6 animals following dosing at 2000mg/kg.These results indicate that the test item CDTA HHQ did not induce toxic effects in the rat following oral administration of a single dose at a level of 2000mg/kg. The lack of mortality demonstrates the acute toxicity estimate (ATE) to be greater than 2000mg/kg body weight. European Directives concerning the classification, packaging and labelling of dangerous substances (Council Regulation (EC) No. 1272/2008 and subsequent revisions) would indicate the following:
Classification : No category
Signal word : No signal word required
Hazard statement : No hazard statement required

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 412, dose range finding study

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.: of test material: 06088797V0

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Age: 7 weeks (approx)
Identification: Tattooing of ears
All animals free of disease and clinical signs
Rats housed together (5 animals per cage) in Polysulfon cages
Bedding: Type Lignocel fibres, dust free bedding
Woodne gnawing blocks for enrichment
Rooms: Fully ariconditioned, temperature range 20 to 24 degrees celcius, 30 to 70% humidity
Light/dark cycle of 12 hours (6 am to 6pm light, 6pm to 6 am dark)
Food, drinking water and bedding/enrichment materials were analysed for chemical and microbiological contaminants.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Remark on MMAD/GSD:

MMAD / GSD: MMAD : 2.0 - 2.7 µm
GSD: 2.0 - 2.22

Details on inhalation exposure:

A dust aerosol was generated using a dust generator and compressed air inside a mixing stage mixed with conditioned dilution air and passed into the inhalation system. The test substance was mixed with Aerosil R972 prior to facilitate aerosol generation.

The inhalation atmosphere was maintained inside aerodynamic exposure systems consisting of cylindrical inhalation chamber made of stainless steel sheeting and cone shaped outlets and inlets. The rats were restrained in glass exposure tubes with their snouts projecting into the inhalation chamber.
The animals did not ave access to feed or water during the exposure period.

Analytical verification of test atmosphere concentrations:

yes

Remarks on duration:

Exposures: 6 hours per day High dose group (1000 mg/m³) was exposued for 1 day only All other groups were exposed for 5 consecutive days

Concentrations:

Concentrations of the inhalation atmospheres were analyzed using gravimetry. Daily means were calculated based on 2 measured samples per concentration and exposure. From the Daily mean values of each concentration, mean concentrations and standard deviations were derived.
Consistency of concentrations in each inhalation system was continuously monitored using scattered light photometry.
Particle size analysis was conducted using a cascade impactor.
The concentrations were [mg/m³]:
- dose group one: 30 (nominal), 33.3 +- 2.3 (actual)
- dose group one: 300 (nominal), 320 +- 27 (actual)
- dose group one: 1000 (nominal), 1103 +- 52 (actual)

No. of animals per sex per dose:

10 animals per dose group
An additional 10 animals for the high dose group and control

Control animals:

yes

Details on study design:

The animals were exposed to a respirable dust aerosol for 6 hours per day for 5 consecutive days. The exception was the high dose group (1000mg/m³) where exposure was for one day only due to mortality observed.
In the control, low and mid dose groups, 5 animals were sacrificed on the day after the last exposure period and 5 were sacrificed 17 days after the last exposure.
10 additional control animals and the 14 surviving high dose group animals were sacrificed on day 14 of the study (14 days after first exposure).

Key result

Sex:

male

Dose descriptor:

LC50

Effect level:

>= 1 103 mg/m³ air (analytical)

95% CL:

>= 1 051 - <= 1 155

Exp. duration:

6 h

Mortality:

6 deaths in the high dose group on days 0 and 1. Accelerated respiration, respiration sounds, piloer
ection, red encrusted nose, hunched position; Mid dose group - accelerated respiration, respiration
sounds, piloerection, reduced fur care

Detials on Results

Histopathology results:

High dose: Multifocal hemorraghes in the lungs; Inflammatory cell infiltrates

Mid dose:

Larynx: laryngeal, epithelial necrosis, multifocal, in various levels of the larynx

Inflammatory cell infiltrates in various levels of the larynx

laryngeal squamous metaplasia, multifocal, in various levels of the larynx

Regenerative hyperplasia of the laryngeal epithelium, multifocal, in various levels of the larynx

Lungs: Regenerative hyperplasia of bronchiolar epithelium (predominantly: medium bronchi, terminal bronchioles)

Mucous cell hyperplasia in large bronchi

interstitial infiltration of eosinophylic granulocytic cells

Low dose:

Larynx: Laryngeal, epithelial necrosis, multifocal, at the base of the epiglittis (level 1)

Inflammatory cell infiltrates at the base of the epiglottis (level 1)

Lungs: Regenerative hyperplasia of the bronchiolar epithelium (predominantly medium bronchi and terminal bronchioles)

Mucous cell hyperplasia in large bronchi

interstitial infiltration of eosinophylic granulocytic cells.

There were no histopathological findings in any of the recovery group animals. Thus all pathology was reversible within the recovery period.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

In a repeated dose range finding study rats were exposed to aerosol of the test substance.
6 death occured in the high dose group. No deaths occured in the middle dose group (300 mg/m³).
Taking into account that the exposure duration was significantly extended compared to an acute toxicity study it is reasonable that less than 50% of the animals of the highest dose group would die when exposed to the substance once for 4 hours.
Thus the LC50 can be stated as:
LC50 > 1000 mg/m³ (nominal)
LC50 > 1103 mg/m³ (actual)

Executive summary:

Inhalation exposure to 1000 mg/m3 disodium EDTA for 6 hours caused lethality in 6 out of 20 male rats. Histological examination of the lung of the dead rats revealed congestion, edema, multifocal hemorraghes and inflammatory cell infiltrates. Inhalation exposure of rats to disodium EDTA for 6 hours per day, 5 consecutive days cause concentration dependant lesions inthe larynx and lungs that were fully reversible within 14 days. Due to histopahological changes in the low dosegroup a no observed effect level could not be determined.

In a subacute repeated dose toxicity study (BASF, 2009) 10 male Wistar rats per dose were exposed to a respirable dust aerosol of Na2H2EDTA for 6 hours per day for 5 consecutive days at concentrations of 0, 30, 300, 1000 mg/m³ air (also see capter 7.5).

Exposure in the high dose group (1000 mg/m3) was for one day only due to mortality observed. Inhalation exposure to 1000 mg/m³ disodium EDTA for 6 hours caused lethality in 6 out of 20 male rats. Histological examination of the lung of the dead rats revealed congestion, edema, multifocal hemorraghes and inflammatory cell infiltrates.

Inhalation exposure of rats to disodium EDTA for 6 hours per day, 5 consecutive days cause concentration dependant lesions in the larynx and lungs that were fully reversible within 14 days. Due to histopahological changes in the low dose group a no observed effect level could not be determined.

The LOAEC was considered to be 30 mg/m³ air.

Reference 2

Endpoint:

acute toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2009-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The substance Disodium dihydrogen ethylenediaminetetraacetate (CAS 139-33-3) is structurally similar to the substance trans-cyclohexane-1,2-dinitrilotetraacetic acid (CAS 13291-61-7), therefore used for the read-across.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 412, dose range finding study

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.: of test material: 06088797V0

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Age: 7 weeks (approx)
Identification: Tattooing of ears
All animals free of disease and clinical signs
Rats housed together (5 animals per cage) in Polysulfon cages
Bedding: Type Lignocel fibres, dust free bedding
Woodne gnawing blocks for enrichment
Rooms: Fully ariconditioned, temperature range 20 to 24 degrees celcius, 30 to 70% humidity
Light/dark cycle of 12 hours (6 am to 6pm light, 6pm to 6 am dark)
Food, drinking water and bedding/enrichment materials were analysed for chemical and microbiological contaminants.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Remark on MMAD/GSD:

MMAD / GSD: MMAD : 2.0 - 2.7 µm
GSD: 2.0 - 2.22

Details on inhalation exposure:

A dust aerosol was generated using a dust generator and compressed air inside a mixing stage mixed with conditioned dilution air and passed into the inhalation system. The test substance was mixed with Aerosil R972 prior to facilitate aerosol generation.

The inhalation atmosphere was maintained inside aerodynamic exposure systems consisting of cylindrical inhalation chamber made of stainless steel sheeting and cone shaped outlets and inlets. The rats were restrained in glass exposure tubes with their snouts projecting into the inhalation chamber.
The animals did not ave access to feed or water during the exposure period.

Analytical verification of test atmosphere concentrations:

yes

Remarks on duration:

Exposures: 6 hours per day High dose group (1000 mg/m³) was exposued for 1 day only All other groups were exposed for 5 consecutive days

Concentrations:

Concentrations of the inhalation atmospheres were analyzed using gravimetry. Daily means were calculated based on 2 measured samples per concentration and exposure. From the Daily mean values of each concentration, mean concentrations and standard deviations were derived.
Consistency of concentrations in each inhalation system was continuously monitored using scattered light photometry.
Particle size analysis was conducted using a cascade impactor.
The concentrations were [mg/m³]:
- dose group one: 30 (nominal), 33.3 +- 2.3 (actual)
- dose group one: 300 (nominal), 320 +- 27 (actual)
- dose group one: 1000 (nominal), 1103 +- 52 (actual)

No. of animals per sex per dose:

10 animals per dose group
An additional 10 animals for the high dose group and control

Control animals:

yes

Details on study design:

The animals were exposed to a respirable dust aerosol for 6 hours per day for 5 consecutive days. The exception was the high dose group (1000mg/m³) where exposure was for one day only due to mortality observed.
In the control, low and mid dose groups, 5 animals were sacrificed on the day after the last exposure period and 5 were sacrificed 17 days after the last exposure.
10 additional control animals and the 14 surviving high dose group animals were sacrificed on day 14 of the study (14 days after first exposure).

Key result

Sex:

male

Dose descriptor:

LC50

Effect level:

>= 1 103 mg/m³ air (analytical)

95% CL:

>= 1 051 - <= 1 155

Exp. duration:

6 h

Mortality:

6 deaths in the high dose group on days 0 and 1. Accelerated respiration, respiration sounds, piloer
ection, red encrusted nose, hunched position; Mid dose group - accelerated respiration, respiration
sounds, piloerection, reduced fur care

Detials on Results

Histopathology results:

High dose: Multifocal hemorraghes in the lungs; Inflammatory cell infiltrates

Mid dose:

Larynx: laryngeal, epithelial necrosis, multifocal, in various levels of the larynx

Inflammatory cell infiltrates in various levels of the larynx

laryngeal squamous metaplasia, multifocal, in various levels of the larynx

Regenerative hyperplasia of the laryngeal epithelium, multifocal, in various levels of the larynx

Lungs: Regenerative hyperplasia of bronchiolar epithelium (predominantly: medium bronchi, terminal bronchioles)

Mucous cell hyperplasia in large bronchi

interstitial infiltration of eosinophylic granulocytic cells

Low dose:

Larynx: Laryngeal, epithelial necrosis, multifocal, at the base of the epiglittis (level 1)

Inflammatory cell infiltrates at the base of the epiglottis (level 1)

Lungs: Regenerative hyperplasia of the bronchiolar epithelium (predominantly medium bronchi and terminal bronchioles)

Mucous cell hyperplasia in large bronchi

interstitial infiltration of eosinophylic granulocytic cells.

There were no histopathological findings in any of the recovery group animals. Thus all pathology was reversible within the recovery period.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

In a repeated dose range finding study rats were exposed to aerosol of the test substance.
6 death occured in the high dose group. No deaths occured in the middle dose group (300 mg/m³).
Taking into account that the exposure duration was significantly extended compared to an acute toxicity study it is reasonable that less than 50% of the animals of the highest dose group would die when exposed to the substance once for 4 hours.
Thus the LC50 can be stated as:
LC50 > 1000 mg/m³ (nominal)
LC50 > 1103 mg/m³ (actual)

Executive summary:

Inhalation exposure to 1000 mg/m3 disodium EDTA for 6 hours caused lethality in 6 out of 20 male rats. Histological examination of the lung of the dead rats revealed congestion, edema, multifocal hemorraghes and inflammatory cell infiltrates. Inhalation exposure of rats to disodium EDTA for 6 hours per day, 5 consecutive days cause concentration dependant lesions inthe larynx and lungs that were fully reversible within 14 days. Due to histopahological changes in the low dosegroup a no observed effect level could not be determined.

In a subacute repeated dose toxicity study (BASF, 2009) 10 male Wistar rats per dose were exposed to a respirable dust aerosol of Na2H2EDTA for 6 hours per day for 5 consecutive days at concentrations of 0, 30, 300, 1000 mg/m³ air (also see capter 7.5).

Exposure in the high dose group (1000 mg/m3) was for one day only due to mortality observed. Inhalation exposure to 1000 mg/m³ disodium EDTA for 6 hours caused lethality in 6 out of 20 male rats. Histological examination of the lung of the dead rats revealed congestion, edema, multifocal hemorraghes and inflammatory cell infiltrates.

Inhalation exposure of rats to disodium EDTA for 6 hours per day, 5 consecutive days cause concentration dependant lesions in the larynx and lungs that were fully reversible within 14 days. Due to histopahological changes in the low dose group a no observed effect level could not be determined.

The LOAEC was considered to be 30 mg/m³ air.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

1 103 mg/m³