pentapotassium 2,2’,2’’,2’’’,2’’’’-(ethane-1,2-diylnitrilo)pentaacetate

Endpoint summary

Description of key information

One 28-day oral gavage study in rats using pentapotassium DTPA and one 28-day oral drinking water study using pentasodium DTPA, one 90-day and one 5-day inhalation study using disodium EDTA. Additional information comes from published literature on the salts of DTPA such as zinc and calcium.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

April-May 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No data on batch number and composition; basic data given, comparable to guidelines/standards

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

study carried out according to previous version of OECD 407

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., UK
- Age at study initiation: 28 ± 1 days
- Weight at study initiation: 66-77 g
- Fasting period before study: no
- Housing: groups of 5 per sex
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.6-24.8
- Humidity (%): mean 68.4
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 9 April To: 7 May 1987

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: freshly each day

VEHICLE
- Concentration in vehicle: 13.3% w/v solution in distilled water (13.3% active ingredient). The intermediate (3.325%) and low
(0.831%) dosage concentrations were prepared by dilution of the high dosage level with the vehicle.
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aliquots (25 mL) of each of the test substance formulation and the vehicle were retained on days 1 and 27. These samples were consiged to the sponsor for concentration analyses; results were not included in the report.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 83, 333, 1330 mg/kg bw/day
Basis:
nominal in water

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: doses were selected on the basis of an acute oral toxicity study (oral LD50 > 5.0 g/kg bw)
- Rationale for animal assignment (if not random): allocation 2 days before start study based on BW, using a computer programme
- Section schedule rationale (if not random): at random

Positive control:

Not required.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes (in g per day)
- Mean daily diet consumption calculated as g food/kg body weight/day: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption
and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in week 4
- Anaesthetic used for blood collection: light ether anaesthesia
- Animals fasted: Yes
- How many animals: all (control, low and mid dose groep) 4/5 females (high concentration group)
- Parameters: PCV, Hb, RBC, Platelets, MCHC, MCV, WBC, absolute WBC differentials, thrombotest

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in week 4
- Animals fasted: Yes
- How many animals: all (control, low and mid dose groep) 1/5 male and 4/5 females (high concentration group)
- Parameters: Glucose, ALP, ALAT, ASAT, total bilirubin, cholesterol, urea nitrogen, total protein, albumin, globulin (= total protein -
albumin), albumin/globulin ratio, Na, K, Ca, Cl, inorganic P, creatinine.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals
HISTOPATHOLOGY: adrenals, heart, kidneys, liver, spleen, macroscopical abnormal tissue (control and high dose group animals,
and all deceased animals)

Other examinations:

Organ weights:adrenals, kidneys, liver, ovaries, testes (with epidydimides)

Statistics:

Bodyweight data were analysed using weight gains.

The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology data:
(i) If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of
values different from the mode was analysed by appropriate methods. Otherwise:
(ii) Bartlett's test was applied to test for heterogeneity of variance between treatments. Where significant (at the 1% level)
heterogeneity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained.
(iii) If no significant heterogeneity was detected (or if a satisfactory transformation was found), a one-way analysis of variance
was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-
Wallis analysis of ranks was used.
(iv) Analyses of variance were followed by Student's 't' test and Williams' test for a dose-related response, although only the
one thought more appropriate for the response pattern observed is reported. The Kruskal-Wallis analyses were followed by the
non-parametric equivalents of the 't' test and Williams' test (Shirleys' test).
For organ weight data, where appropriate, analysis of covariance was used in place of analysis of variance in the above
sequence. The final bodyweight was used as covariate in an attempt to allow for differences in bodyweight which might influence
the organ weights.

References
Bartlett, M.S., (1937), Proc. Roy. Soc. A., 160: 268-282.
Kruskal, W.H. and Wallis, W.A., (1952/3), J. Amer. Statist. Ass., 47: 583-621 and 48: 907-912.
Shirley, E., (1977), Biometrics, 33: 386-389.
Williams, D.A., (1971/2), 8iometrics 27 :103-117 and 28: 519-531.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
4/5 males of the high concentration group were found dead at the end of the study period; 1/5 females of that group was killed at
the same time due to a poor condition. Clinical signs consisted of hunched posture, abnormal gait, pilo-erection, ptosis, decreased
respiratory rate and diarrhoea (see Table 1 below). Pallor was seen in all groups at the end of the study period and was
considered to have probably resulted from the blood sampling procedure.

BODY WEIGHT AND WEIGHT GAIN
Statistically significant BW loss observed in female animals of the high dose group during week 4; tendency to reduced BW gain in males of the mid and high dose group during weeks 3 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Reduced food intake in week 4 in males and females of the high dose group, and in males of the mid dose group

HAEMATOLOGY
No statistically significant changes observed.

CLINICAL CHEMISTRY
See Table 2 (below).

NEUROBEHAVIOUR
No specific test performed. However, abnormal gait was observed towards the end of the stduy period (see Table 1 below).

ORGAN WEIGHTS
Reduced absolute liver weight in males and females of the high dose group and in males of the mid dose group (statistically
significant). See Table 2 (below).

GROSS PATHOLOGY
Watery contents in caecum in 3/4 surviving female rats of the high dose group at necropsy (see Table 3 below).

HISTOPATHOLOGY: NON-NEOPLASTIC
A contracted spleen was found in 4 male and 1 female rat of the high dose group that did not survive (see Table 3 below).

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
Not applicable

Dose descriptor:

NOAEL

Effect level:

83 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Changes in clinical signs, clinical chemistry and decreased liver weights were observed at 333 mg/kg bw/day

Critical effects observed:

not specified

Table 1

No. of animals           Examined	Males (mg/kg bw day)				Females (mg/kg bw/day)
	0	83	333	1330	0	83	333	1330
	5	5	5	5	5	5	5	5
Mortality Found dead     Killed	0	0	0	4 0	0	0	0	0 1
Clinical findings
Hunched posture Abnormal gait Decreased respiratory rate Diarrhoea Pilo-erection Ptosis Pallor	0 0 0 0 0 0 5	0 0 0 0 0 0 5	1 1 0 0 0 0 5	4 4 2 3 3 2 2	0 0 0 0 0 0 5	0 0 0 0 0 0 5	0 0 0 0 0 0 5	5 5 0 5 5 0 5

Table 2

	Males (mg/kg bw day)				Females (mg/kg bw/day)
	0	83	333	1330	0	83	333	1330
BW (g) week 4	332	320	298	(329)	216	209	223	180**
Glucose (mg/dl)	91	99*	98*	(119)	88	86	81	88
Total protein (g/dl)	6.4	6.6	6.5	(6.5)	6.9	6.9	6.4**	6.7**
Globuline (g/dl)	2.7	2.8	2.8	(2.7)	2.9	2.9	2.6**	2.6**
A/G	1.38	1.33	1.36	(1.41)	1.39	1.39	1.44	1.61**
Urea N (mg/dl)	13	12	15*	(13)	15	15	14	14
ALP (mU/ml)	386	324	383	(112)	199	224	186	69**
ALAT (mU/ml)	22 #	27 #	22 #	(22) #	18	18	19	23*
Na (mEq/L)	141	142	144*	(144)	143	143	143	144
K (mEq/L)	4.2	4.5	4.4	(4.3)	3.8	4.0	4.2*	4.1*
Cl (mEq/L)	102	101	100	(100)	102	103	103	104*
Liver weight (g)	16.8	15.9	13.7**	(12.7)	10.2	10.4	9.9	8.4

( ) value for one animal only; # log transformed

* p0.05, ** p0.01

Table 3

No. of animals           Examined	Males (mg/kg bw day)				Females (mg/kg bw/day)
	0	83	333	1330	0	83	333	1330
	5	5	5	1	5	5	5	4
Macroscopy Watery contents in caecum	0	0	0	0	0	0	0	3
No. of animals          Examined	5	0	0	5	5	0	0	5
Microscopy Spleen contracted	0	-	-	4	0	-	-	2

Conclusions:

Based on clinical signs, slight changes in clinical chemistry, and decreased absolute liver weight in animals dosed with 333 mg DTPA-K5 per kg bw, the next lower level tested, viz. 83 mg DTPA-K5 per kg bw was considered a NOAEL.

Executive summary:

Glycine N-N bis[2-[bis(carboxymethyl)amine]ethyi]-pentapotassium salt (hereafter named Glycine derivative) was administered to rats daily, for twenty-eight consecutive days by intragastric intubation. The test substance, formulated with distilled water, was administered at dosage levels of 83, 333 or 1330 mg DTPA-K5 per kg bw per day. Control animals similarly received distilled water.

No abnormal clinical signs were recorded for rats receiving Glycine derivative, 83 or 333 mg/kg/day that were considered to be related to treatment. On Days 26 or 27 until termination, a number of clinical signs were noted for the surviving male rat and four surviving female rats receiving Glycine derivative, 1330 mg/kg/day. These clinical findings included pilo-erection, hunched posture, abnormal gait (waddling), pallor and diarrhoea (with or without mucus).

Four male rats receiving Glycine derivative, 1330 mg/kg/day were found dead during the fourth week of treatment. Additionally, one female rat in this high dosage group was killed during Week 4 due to its poor health status. The spleen was found on microscopic examination to be contracted in all mortalities; this finding may possibly, therefore, be related to treatment. However, the severity of the change was not considered sufficient to be a factor contributory to death and the aetiology of this change remains obscure.

During Week 4, bodyweight losses were recorded for the four surviving female rats receiving Glycine derivative, 1330 mg/kg/day, while lower bodyweight gains were recorded during Weeks 3 and 4 for male rats receiving Glycine derivative, 333 or 1330 mg/kg/day in comparison with controls. The changes in male bodyweights did not, however, achieve statistical significance. In all other instances, bodyweight gains for rats receiving Glycine derivative were similar to those of the controls receiving distilled water.

Lower food consumption was recorded during Week 4 for male and female rats receiving Glycine derivative, 1330 mg/kg/day in comparison with controls. Reduced food consumption was similarly apparent during Weeks 3 and 4 for male rats receiving Glycine derivative, 333 mg/kg/day in comparison with those receiving distilled water. No other changes in food consumption were recorded that were considered to be related to treatment with Glycine derivative.

No treatment-related haematological changes were observed during Week 4 for treated female rats or for male rats receiving Glycine

derivative, 83 or 333 mg/kg/day. No haematological data was recorded for male rats receiving Glycine derivative, 1330 mg/kg/day.

Changes in several biochemical parameters were observed during Week 4 that were considered to be related to treatment with Glycine derivative. Glutamic-pyruvic transaminase (GPT or ALAT) levels for female rats receiving Glycine derivative, 1330 mg/kg/day were statistically significantly higher (p<0.05) than those of the controls while alkaline phosphatase (ALP) levels for female rats in this dosage group were statistically significantly lower (p<0.01) than those receiving distilled water. Individual ALP levels for 4/5 female rats and also for the surviving male rat receiving Glycine derivative, 1330 mg/kg/day were below the lower limits of the expected parameter ranges. Similarly, lower globulin levels, resulting in lower total protein levels and higher albumin/globulin ratios were recorded for female rats receiving Glycine derivative, 333 or 1330 mg/kg/day with statistical significance (P<0.01) being achieved in most instances. Sodium levels for male rats receiving Giycine derivative, 333 mg/kg/day were statisticaliy significantly higher (p<0.05) than those of the controls. The sodium level for the surviving male rat receiving Glycine derivative, 1330 mg/kg/day was similarly higher than those of rats receiving distilled water. Potassium and chloride levels for female rats receiving Glycine

derivative were also higher than those of the controls with statistical significance being achieved for female rats in the intermediate (potassium levels) and high (potassium and chloride levels) dosage groups. The above changes in female GPT, globulin, potassium and chloride levels and in male sodium levels were not observed for rats of the opposite sex. No other changes in biochemical parameters were observed that were considered to be related to treatment with Glycine derivative.

In comparison with rats receiving distilled water, lower liver weights were recorded for male rats receiving Glycine derivative, 83 or

333 mg/kg/day with statistical significance being achieved for male rats in the intermediate dosage group. The liver weight for the

surviving male rat in the high dosage group was also lower than those of the controls. Adjusted liver weights for treated female rats were similar to those of the controls.

Watery contents in the caecum were noted for 3/4 female rats treated with Glycine derivative, 1330 mg/kg/day. This macroscopic finding was considered to be treatment-related.

The spleen was found to be contracted in four male rats and one female rat which died during treatment with Glycine derivative,

1330 mg/kg/day (see Mortalities). Contraction of the spleen was also present in one female rat from this treatment group which was killed at termination. These findings, the aetialogy of which remain obscure, may possibly be related to treatment.

No other changes were apparent that were considered to be of toxicological importance. Based on clinical signs, slight changes in clinical chemistry, and decreased absolute liver weight in animals dosed with 333 mg/kg bw, the next lower level tested, viz. 83 mg/kg bw was considered a NOAEL.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

83 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Two studies available.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Study period:

2013-2014

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 7 weeks
- Housing: up to 5 animals per cage in Polysulfon cages (H-Temp [PSU])
- Diet (e.g. ad libitum): 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): A light/dark rhythm of 12 hours was maintained: 06.00 a.m. 06.00 p.m. light, 06.00 p.m. 06.00 a.m. dark

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: Test group 1 (0.5 mg/m3): MMAD (µm) 2.3-2.8; Geometric standard deviation 1.7-2.2
Test group 2 (3 mg/m3): MMAD (µm) 2.0-2.4; Geometric standard deviation 1.8-2.0
Test group 3 (15 mg(m3): MMAD (µm) 2.3-2.5; Geometric standard deviation 1.8-2.1

Details on inhalation exposure:

For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage, mixed with conditioned dilution air and passed into the inhalation system. To achieve stable concentration in the atmosphere, a part of generated atmosphere was replaced by fresh conditioned air.
The inhalation atmosphere was maintained inside aerodynamic exposure systems, consisting of a cylindrical inhalation chamber made of stainless
steel sheeting and cone-shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol. The exposure systems were located in exhaust hoods in an air conditioned room. All test groups were exposed for 6 hours on each workday over a time period suitable to reach 65 exposures. The animals did not have access to water or feed during the exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the inhalation atmospheres were analyzed by gravimetry in all test groups including. This analytical method is judged to be valid because the test substance does not possess an appreciable vapor pressure. Daily means were calculated based on 2 measured samples in test group 1 and 3 measured samples per concentration and exposure in test groups 2 and 3. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived.
Scattered light photometry was used to continuously monitor the constancy of concentrations of test substance aerosols in the inhalation systems. To this end the inhalation atmosphere was continuously sampled by the measuring devices.
The particle size analysis was carried out with a cascade impactor.

Duration of treatment / exposure:

Exposures: 6 hours per day

Frequency of treatment:

daily (5 consecutive days/week), 13 weeks, 65 exposures in total

Remarks:

Doses / Concentrations:
0, 0.5, 3, 15 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were examined for evident signs of toxicity or mortality twice a day on working days and once a day on Saturdays, Sundays and public holidays

CLINICAL OBSERVATIONS: Yes
The clinical condition of the test animals was recorded once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days. During exposure only a group wise examination was possible.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: days 0, 42, 84

BODY WEIGHT: Yes
- Time schedule for examinations: at start of the pre-exposure, at start of the exposure, then twice a week (Monday, Friday), and prior to gross necropsy

FOOD CONSUMPTION:
- Food consumption was determined cage-wise weekly (Monday-Friday) and calculated as g food/animal/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before the start of the exposure period (day -1/ -2) the eyes of all animals, and at the end of the study (day 82/83) the eyes of the animals of test group 0 (control group) and test group 3 (high concentration) were examined for any changes in the refracting media with an ophthalmoscope after administration of a mydriatic.

HAEMATOLOGY: Yes
All animals per test group and sex at the end of the administration period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
Parameters: WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, DBC, RET

CLINICAL CHEMISTRY: Yes
All animals per test group and sex at the end of the administration period
- Animals fasted: Yes
Parameters. ALT, AST, ALP, GGT, NA, K, CL, INP, CA, UREA, CREA, GLUC, TBIL, TPROT, ALB, GLOB, TRIG, CHOL

URINALYSIS: Yes
All animals per test group and sex at the end of the administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity / grip strength / motor activity / Open filed observations

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals assigned for light microscopic examination were sacrificed under pentobarbital anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights: all animals sacrificed on schedule
Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lung, Ovaries, Spleen, Testes, Thymus, Thyroid glands, Uterus

Organ/Tissue Fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution:
All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain with olfactory bulb, Cecum, Colon, Duodenum, Epididymides, Esophagus, Extraorbital lacrimal gland, Eyes with optic nerve and eyelid (modified Davidson’s solution), Femur with knee joint, Harderian glands, Heart, Ileum, Jejunum, Kidneys, Larynx, Liver, Lung, Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes), Mammary gland (male + female), Nose (nasal cavity), Ovaries, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Teeth, Testes, Thymus, Thyroid glands, Tongue, Trachea, Ureter, Urethra, Urinary bladder, Uterus

HISTOPATHOLOGY: Yes
List of organs and tissues of histological examinations: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Colon, Duodenum, Esophagus, Femur with knee joint, Heart, Ileum, Jejunum, Kidneys, Larynx (3 levels), Liver, Lung, Lymph nodes (tracheobronchial, mediastinal and mesenteric), Mammary gland (female), Nasal cavity (4 levels), Ovaries, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Teeth, Testes, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus

Statistics:

Body weight, body weight change - comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the
hypothesis of equal means
Feces, rearing, grip, strength length, forelimbs, grip, strength length, hindlimbs, footsplay, test, motor activity - Non-parametric one-way analysis
using KRUSKAL-WALLIS test (twosided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians
Blood parameters - For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians
Weight parameters - Non-parametric one-way analysis using KRUSKAL-WALLIS test (twosided).If the resulting p-value was equal or less than 0.05, a pairwise
comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians

Clinical signs:

no effects observed

Description (incidence and severity):

One male rat (No. 33) of the high concentration group showed discoloration (orange) of feces and smeared fur (red) in anogenital region. The effects were observed in the morning before exposure on study day one. Therefore, the animal was sacrificed after the first exposure on study day one. As no other animals of this group showed similar clinical signs of toxicity during the whole exposure period of 90 days (65 exposures), the findings in animal No. 33 were considered to be incidental and not substance-related. No further deaths were recorded in the study.

Mortality:

no mortality observed

Description (incidence):

One male rat (No. 33) of the high concentration group showed discoloration (orange) of feces and smeared fur (red) in anogenital region. The effects were observed in the morning before exposure on study day one. Therefore, the animal was sacrificed after the first exposure on study day one. As no other animals of this group showed similar clinical signs of toxicity during the whole exposure period of 90 days (65 exposures), the findings in animal No. 33 were considered to be incidental and not substance-related. No further deaths were recorded in the study.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Dose descriptor:

NOAEC

Effect level:

3 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Organ weights:

Relative changes of absolute liver and lung weights in comparison to the control

	Male animals			Female animals
Test group (mg/m³)	1 (0.5)	2 (3)	3 (15)	1 (0.5)	2 (3)	3 (15)
Liver				107%	102%	109%*
Lungs	99%	93%	108%*	107%*	112%**	124%**

* : p <= 0.05, **: p <= 0.01

 

All other mean absolute weight parameters did not show significant differences when

compared to the control group 0.

Relative organ weights

When compared with control group 0 (=100%), the following mean relative organ weights

were significantly changed (statistically significant changes printed in bold):

	Female animals
Test group (mg/m³)	1 (0.5)	2 (3)	3 (15)
Lungs	104%	112%**	120%**

* : p <= 0.05, **: p <= 0.01

 

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

The increased lung weights in males of test group 3 (15 mg/m³) and females of all test groups might be related to the treatment. No histopathologic finding could explain the weight increase. In males, the relative lung weight was not significantly changed. Furthermore was the lung weight of female animals within the range of historical control data. Therefore the lung weight increase was regarded to be not adverse.

Treatment-related findings were observed in male and females with incidences and grading according to the table below

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (0.5)	2 (3)	3 (15)	0 (0)	1 (0.5)	2 (3)	3 (15)
No.of animals	10	10	10	10	10	10	10	10
Hyperplasia (m)focal	0	0	0	0	0	0	0	1
Metaplasia, squamous	0	0	1	6	0	0	1	3
·                  Grade1			1	2			1	1
·                  Grade2				4				2
Epithelial alteration	2	4	6	4	1	7	8	6
·                  Grade1	2	4	5	1	1	7	8	2
·                  Grade2			1	3				4
Infiltrates, granulocytic								2
·                 Grade2								2

Whenever no grading was given the finding was recorded to be present

Animals of all test groups as well as single control animals revealed minimal to slight epithelial alteration at the base of the epiglottis. The term was used, if at the base of the epiglottis a small focal area was covered by flattened epithelium, which differed from the normal cuboidal to columnar laryngeal epithelium. Secondary, some animals of test group 3 (15 mg/m³) and 2 (3 mg/m³) showed a minimal to slight focal squamous metaplasia in this region. These findings were regarded to be test substance related and adaptive. One female of test group 3 (15 mg/m³) revealed a small focal hyperplasia of the laryngeal epithelium at the base of the epiglottis and in addition slight granulocytic cell infiltrates. These infiltrates were also observed in a second female of this test group. These findings were regarded to be treatment related and adverse.

Microscopic findings in lung and their grading

	Maleanimals				Femaleanimals
Test group (mg/m³)	0 (0)	1 (0.5)	2 (3)	3 (15)	0 (0)	1 (0.5)	2 (3)	3 (15)
No.ofanimals	10	10	10	10	10	10	10	10
Histiocytosisalveolar,d	0	0	6	10	0	0	5	10
·                  Grade1			6	4			5	6
·                  Grade2				6				4
Metaplasiamucouscells	0	0	0	4	0	0	0	5
·                  Grade1				4				5

Animals of test group 2 and 3 (3 and 15 mg/m³) revealed minimal to slight increased numbers of alveolar histiocytes. These histiocytes showed an eosinophilic cytoplasm, occasionally with clear vacuoles and were located as single cells within the alveoli all over the lung lobes. When compared to the control animals, males and females of test group 3 (15 mg/m³) showed a minimal to slight increase in number of mucous cells in the large bronchi. These findings were regarded to be treatment related effects and adaptive.

Testes

Tubular degeneration (up to moderate) was observed in control (8 animals affected) and test group 3 animals (7 animals affected) in similar incidences. This finding was characterized by randomly affected (not stage specific) tubules with sloughed spermatogenic cells, vacuolation of the spermatogenic epithelium or missing germ cell layers. This effect in the testis is likely due to the technical exposure scenario (heat [e.g. Brock WJ et al., 1996] and possibly evading movements by the animals leading to pressing backwards in tubes), rather than being a direct effect of the test substance as this finding is also found in similar incidences in control animals.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Conclusions:

Under the current test conditions, the No Observed Adverse Effect Concentration (NOAEC) for local effects in the larynx is 3 mg/m3, the NOEC for systemic effects is 15 mg/m³.

Executive summary:

Inhalation exposure of rats to Trilon BD for 90 day (65 exposures) did not lead to any substance-related clinical signs of toxicity. Nor were there any effect in clinical chemistry, hematology. Histological examination revealed some effects in larynx at the highest tested concentration of 15 mg/m³ in female animals. No signs of systemic toxicity were observed up to a concentration of 15 mg/m³. Signs of local toxicity were observed only at the high concentration of 15 mg/m³ in female animals.

Under the current test conditions, the No Observed Adverse Effect Concentration (NOAEC) for local effects in the larynx is 3 mg/m3, the NOEC for systemic effects is 15 mg/m³.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

15 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

See read across argument in section 7 (DNEL setting) and read across document in section 13.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Study period:

2013-2014

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 7 weeks
- Housing: up to 5 animals per cage in Polysulfon cages (H-Temp [PSU])
- Diet (e.g. ad libitum): 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): A light/dark rhythm of 12 hours was maintained: 06.00 a.m. 06.00 p.m. light, 06.00 p.m. 06.00 a.m. dark

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: Test group 1 (0.5 mg/m3): MMAD (µm) 2.3-2.8; Geometric standard deviation 1.7-2.2
Test group 2 (3 mg/m3): MMAD (µm) 2.0-2.4; Geometric standard deviation 1.8-2.0
Test group 3 (15 mg(m3): MMAD (µm) 2.3-2.5; Geometric standard deviation 1.8-2.1

Details on inhalation exposure:

For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage, mixed with conditioned dilution air and passed into the inhalation system. To achieve stable concentration in the atmosphere, a part of generated atmosphere was replaced by fresh conditioned air.
The inhalation atmosphere was maintained inside aerodynamic exposure systems, consisting of a cylindrical inhalation chamber made of stainless
steel sheeting and cone-shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol. The exposure systems were located in exhaust hoods in an air conditioned room. All test groups were exposed for 6 hours on each workday over a time period suitable to reach 65 exposures. The animals did not have access to water or feed during the exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the inhalation atmospheres were analyzed by gravimetry in all test groups including. This analytical method is judged to be valid because the test substance does not possess an appreciable vapor pressure. Daily means were calculated based on 2 measured samples in test group 1 and 3 measured samples per concentration and exposure in test groups 2 and 3. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived.
Scattered light photometry was used to continuously monitor the constancy of concentrations of test substance aerosols in the inhalation systems. To this end the inhalation atmosphere was continuously sampled by the measuring devices.
The particle size analysis was carried out with a cascade impactor.

Duration of treatment / exposure:

Exposures: 6 hours per day

Frequency of treatment:

daily (5 consecutive days/week), 13 weeks, 65 exposures in total

Remarks:

Doses / Concentrations:
0, 0.5, 3, 15 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were examined for evident signs of toxicity or mortality twice a day on working days and once a day on Saturdays, Sundays and public holidays

CLINICAL OBSERVATIONS: Yes
The clinical condition of the test animals was recorded once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days. During exposure only a group wise examination was possible.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: days 0, 42, 84

BODY WEIGHT: Yes
- Time schedule for examinations: at start of the pre-exposure, at start of the exposure, then twice a week (Monday, Friday), and prior to gross necropsy

FOOD CONSUMPTION:
- Food consumption was determined cage-wise weekly (Monday-Friday) and calculated as g food/animal/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before the start of the exposure period (day -1/ -2) the eyes of all animals, and at the end of the study (day 82/83) the eyes of the animals of test group 0 (control group) and test group 3 (high concentration) were examined for any changes in the refracting media with an ophthalmoscope after administration of a mydriatic.

HAEMATOLOGY: Yes
All animals per test group and sex at the end of the administration period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
Parameters: WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, DBC, RET

CLINICAL CHEMISTRY: Yes
All animals per test group and sex at the end of the administration period
- Animals fasted: Yes
Parameters. ALT, AST, ALP, GGT, NA, K, CL, INP, CA, UREA, CREA, GLUC, TBIL, TPROT, ALB, GLOB, TRIG, CHOL

URINALYSIS: Yes
All animals per test group and sex at the end of the administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity / grip strength / motor activity / Open filed observations

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals assigned for light microscopic examination were sacrificed under pentobarbital anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights: all animals sacrificed on schedule
Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lung, Ovaries, Spleen, Testes, Thymus, Thyroid glands, Uterus

Organ/Tissue Fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution:
All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain with olfactory bulb, Cecum, Colon, Duodenum, Epididymides, Esophagus, Extraorbital lacrimal gland, Eyes with optic nerve and eyelid (modified Davidson’s solution), Femur with knee joint, Harderian glands, Heart, Ileum, Jejunum, Kidneys, Larynx, Liver, Lung, Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes), Mammary gland (male + female), Nose (nasal cavity), Ovaries, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Teeth, Testes, Thymus, Thyroid glands, Tongue, Trachea, Ureter, Urethra, Urinary bladder, Uterus

HISTOPATHOLOGY: Yes
List of organs and tissues of histological examinations: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Colon, Duodenum, Esophagus, Femur with knee joint, Heart, Ileum, Jejunum, Kidneys, Larynx (3 levels), Liver, Lung, Lymph nodes (tracheobronchial, mediastinal and mesenteric), Mammary gland (female), Nasal cavity (4 levels), Ovaries, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Teeth, Testes, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus

Statistics:

Body weight, body weight change - comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the
hypothesis of equal means
Feces, rearing, grip, strength length, forelimbs, grip, strength length, hindlimbs, footsplay, test, motor activity - Non-parametric one-way analysis
using KRUSKAL-WALLIS test (twosided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians
Blood parameters - For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians
Weight parameters - Non-parametric one-way analysis using KRUSKAL-WALLIS test (twosided).If the resulting p-value was equal or less than 0.05, a pairwise
comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians

Clinical signs:

no effects observed

Description (incidence and severity):

One male rat (No. 33) of the high concentration group showed discoloration (orange) of feces and smeared fur (red) in anogenital region. The effects were observed in the morning before exposure on study day one. Therefore, the animal was sacrificed after the first exposure on study day one. As no other animals of this group showed similar clinical signs of toxicity during the whole exposure period of 90 days (65 exposures), the findings in animal No. 33 were considered to be incidental and not substance-related. No further deaths were recorded in the study.

Mortality:

no mortality observed

Description (incidence):

One male rat (No. 33) of the high concentration group showed discoloration (orange) of feces and smeared fur (red) in anogenital region. The effects were observed in the morning before exposure on study day one. Therefore, the animal was sacrificed after the first exposure on study day one. As no other animals of this group showed similar clinical signs of toxicity during the whole exposure period of 90 days (65 exposures), the findings in animal No. 33 were considered to be incidental and not substance-related. No further deaths were recorded in the study.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see details on results

Dose descriptor:

NOAEC

Effect level:

3 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Organ weights:

Relative changes of absolute liver and lung weights in comparison to the control

	Male animals			Female animals
Test group (mg/m³)	1 (0.5)	2 (3)	3 (15)	1 (0.5)	2 (3)	3 (15)
Liver				107%	102%	109%*
Lungs	99%	93%	108%*	107%*	112%**	124%**

* : p <= 0.05, **: p <= 0.01

 

All other mean absolute weight parameters did not show significant differences when

compared to the control group 0.

Relative organ weights

When compared with control group 0 (=100%), the following mean relative organ weights

were significantly changed (statistically significant changes printed in bold):

	Female animals
Test group (mg/m³)	1 (0.5)	2 (3)	3 (15)
Lungs	104%	112%**	120%**

* : p <= 0.05, **: p <= 0.01

 

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

The increased lung weights in males of test group 3 (15 mg/m³) and females of all test groups might be related to the treatment. No histopathologic finding could explain the weight increase. In males, the relative lung weight was not significantly changed. Furthermore was the lung weight of female animals within the range of historical control data. Therefore the lung weight increase was regarded to be not adverse.

Treatment-related findings were observed in male and females with incidences and grading according to the table below

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (0.5)	2 (3)	3 (15)	0 (0)	1 (0.5)	2 (3)	3 (15)
No.of animals	10	10	10	10	10	10	10	10
Hyperplasia (m)focal	0	0	0	0	0	0	0	1
Metaplasia, squamous	0	0	1	6	0	0	1	3
·                  Grade1			1	2			1	1
·                  Grade2				4				2
Epithelial alteration	2	4	6	4	1	7	8	6
·                  Grade1	2	4	5	1	1	7	8	2
·                  Grade2			1	3				4
Infiltrates, granulocytic								2
·                 Grade2								2

Whenever no grading was given the finding was recorded to be present

Animals of all test groups as well as single control animals revealed minimal to slight epithelial alteration at the base of the epiglottis. The term was used, if at the base of the epiglottis a small focal area was covered by flattened epithelium, which differed from the normal cuboidal to columnar laryngeal epithelium. Secondary, some animals of test group 3 (15 mg/m³) and 2 (3 mg/m³) showed a minimal to slight focal squamous metaplasia in this region. These findings were regarded to be test substance related and adaptive. One female of test group 3 (15 mg/m³) revealed a small focal hyperplasia of the laryngeal epithelium at the base of the epiglottis and in addition slight granulocytic cell infiltrates. These infiltrates were also observed in a second female of this test group. These findings were regarded to be treatment related and adverse.

Microscopic findings in lung and their grading

	Maleanimals				Femaleanimals
Test group (mg/m³)	0 (0)	1 (0.5)	2 (3)	3 (15)	0 (0)	1 (0.5)	2 (3)	3 (15)
No.ofanimals	10	10	10	10	10	10	10	10
Histiocytosisalveolar,d	0	0	6	10	0	0	5	10
·                  Grade1			6	4			5	6
·                  Grade2				6				4
Metaplasiamucouscells	0	0	0	4	0	0	0	5
·                  Grade1				4				5

Animals of test group 2 and 3 (3 and 15 mg/m³) revealed minimal to slight increased numbers of alveolar histiocytes. These histiocytes showed an eosinophilic cytoplasm, occasionally with clear vacuoles and were located as single cells within the alveoli all over the lung lobes. When compared to the control animals, males and females of test group 3 (15 mg/m³) showed a minimal to slight increase in number of mucous cells in the large bronchi. These findings were regarded to be treatment related effects and adaptive.

Testes

Tubular degeneration (up to moderate) was observed in control (8 animals affected) and test group 3 animals (7 animals affected) in similar incidences. This finding was characterized by randomly affected (not stage specific) tubules with sloughed spermatogenic cells, vacuolation of the spermatogenic epithelium or missing germ cell layers. This effect in the testis is likely due to the technical exposure scenario (heat [e.g. Brock WJ et al., 1996] and possibly evading movements by the animals leading to pressing backwards in tubes), rather than being a direct effect of the test substance as this finding is also found in similar incidences in control animals.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Conclusions:

Under the current test conditions, the No Observed Adverse Effect Concentration (NOAEC) for local effects in the larynx is 3 mg/m3, the NOEC for systemic effects is 15 mg/m³.

Executive summary:

Inhalation exposure of rats to Trilon BD for 90 day (65 exposures) did not lead to any substance-related clinical signs of toxicity. Nor were there any effect in clinical chemistry, hematology. Histological examination revealed some effects in larynx at the highest tested concentration of 15 mg/m³ in female animals. No signs of systemic toxicity were observed up to a concentration of 15 mg/m³. Signs of local toxicity were observed only at the high concentration of 15 mg/m³ in female animals.

Under the current test conditions, the No Observed Adverse Effect Concentration (NOAEC) for local effects in the larynx is 3 mg/m3, the NOEC for systemic effects is 15 mg/m³.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

3 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

See read across argument in section 7 (DNEL setting) and read across document in section 13.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral

There are two standard guideline repeated dose toxicity studies available for DTPA. The studies were conducted using either the potassium or sodium salt. Overall, the two studies are consistent in the adverse effects identified, particularly with respect to the apparent target organs and the no effect levels. The only significant difference between the two studies is the mortality encountered in the study using gavage administration. This increased mortality in the males (4/5) and females (1/5) was probably due to a bolus dose effect rather than a greater toxicity of the potassium salt since the sodium DTPA was administered via the drinking water rather than gavage. With the exception of the high dose group mortality, and the decreased body weight and food consumption in the high and mid dose groups the other adverse effects identified were relatively minimal (changes in clinical chemistry parameters and some alterations in urine parameters (high dose only)). The liver appears to have been a target organ for toxicity, but these effects may have been due to the decreased bodyweight and stress rather than direct compound related toxicity.

The relatively minimal systemic toxicity observed is also consistent with the low degree of absorption following oral administration and its subsequent rapid excretion (half life of approx 2 hours). DTPA is chemically stable and not reactive or metabolized, thus its toxicity is generally associated with the ability to chelate essential metals. DTPA is known to be capable of producing deficiencies in zinc when administered systemically or orally. The effects in the 2 repeated dose studies in the high dose groups are consistent with the development of a nutritional deficiency, such as a zinc deficiency. The reduction in food intake and associated bodyweight decrease are known to be associated with deficiencies of zinc, as the animals reduce their food intake in an effort to trigger catabolism of their tissues to release more zinc. If these studies had continued for a longer period then it is highly likely that more obvious changes in pathology consistent with a zinc deficiency would appear.

Further support for the toxicity of DTPA being linked to its ability to remove essential metals such as zinc comes from toxicity studies conducted using the zinc salt of DTPA. In a number of comparative studies (predominantly studying developmental toxicity), the zinc salt of DTPA has been significantly less toxic compared to the calcium salt of DTPA.

Due to the nature of DTPA toxicity, it is unlikely that a longer study would identify additional adverse effects or a significantly lower no effect level. There is a finite amount of essential metals in the diet and body. DTPA can only bind to metals during the brief period when it comes into contact (either in the gut or the blood). If a dose of DTPA is insufficient to significantly impact an animal’s intake of essential metals then increasing the number of days of exposure will not have a more severe effect, i.e. there will be a threshold. Thus, rather than leading to a significantly lower no effect level, a longer term study would probably just lead to a greater degree of zinc deficiency thus it would show an increased severity of the effects observed in the shorter study.

There are no longer term guideline studies for DTPA, however there is a longer term study available for another salt of DTPA. In this study Calcium DTPA (approximately 44 mg/DTPA/kg bw) was administered via intraperitoneal injection twice per week to rats for 44 weeks. There were no observed effects on any of the parameters examined (including body weight, clinical signs, pathology, urinalysis, clinical chemistry and hematology). However, by administering DTPA only 2 times per week, there was a recovery period between the doses where the rats would have been able to replace essential nutrients like zinc. Therefore, whilst this study does not indicate any adverse effects following long term exposure to DTPA, if the dose had been administered daily there would probably have been toxicity since it would be equivalent to a daily oral dose of approximately 400 and 800 mg/kg bw oral dose (assuming 5 - 10% absorption in the gut). This study does however illustrate that the toxicity of DTPA is reversible, since there was apparently sufficient time within this study design for the animals to recoup lost essential elements.

Based on the available data it is therefore argued that no further sub-chronic or chronic testing is necessary via the oral route.

Inhalation

To date, two guideline studies have been performed to evaluate the potential toxicity of chelating agents following repeated inhalation exposure.A 5-day inhalation study with a structurally related compound Na2H2EDTA showed histopathological changes in the respiratory tract at a concentration of 30 mg/m3. After a recovery period of 14 days, all changes had disappeared.A 90-day inhalation study with the same substance was carried out according to OECD 413 and EC No 440/2008. Wistar rats, 10 per sex per test group, were exposed (head-nose only), to dust aerosol for 6 hours per day, on 5 consecutive days per week for 13 weeks (65 exposures). The target concentrations were 0.5, 3 and 15 mg/m3. A concurrent control group was exposed to air. On each exposure day a clinical examination was performed before, during and after exposure. Detailed clinical observation was performed at the beginning, midterm and end of the study. Ophthalmology was performed before the beginning of the exposure in all test groups and at the end of the end of the exposure in the control and high concentration group animals. Body weights and food consumption of the animals were determined weekly. At the end of the exposure period, functional observation battery and motor activity tests were performed. On the day after the last exposure, blood was sampled and examined for a range of hematology and clinical chemical parameters as indicated in the guideline. After blood sampling the animals were sacrificed and subject to necropsy (including macroscopic examination of the major internal organs and collection of organ weight data). Selected tissues were processed histopathologically and were evaluated by light microscopy according to the OECD guideline.

When compared with the control group, the following treatment-related adverse findings were noted in Wistar rats after 90 days of inhalation:

 

High concentration (15 mg/m³)

· Focal hyperplasia of the laryngeal epithelium at the base of the epiglottis in one female animal

· Slight granulocytic infiltrates at the base of the epiglottis of the larynx in two female animals

Mid (3 mg/m³) and low concentration (0.5 mg/m³)

No adverse findings.

Conclusion

Inhalation exposure of rats to Na2H2EDTA for 90 days (65 exposures) did not lead to any substance related clinical signs of toxicity. Nor were there any effects in clinical chemistry and hematology. Histological examination revealed some effects in larynx at the highest tested concentration of 15 mg/m³. No signs of systemic toxicity were observed up to a concentration of 15 mg/m³. Signs of local toxicity were observed only at the high concentration of 15 mg/m³. Under the current test conditions, the No Observed Adverse Effect Concentration (NOAEC) for local effects was 3 mg/m3, the NO(A)EC for systemic effects was 15 mg/m³.

Inhalation exposure of rats to Na2H2EDTA for 6 hours per day, 5 consecutive days causes concentration dependent lesions in the larynx and lungsthat were fully reversible within 14 days. Due to histopahological changes in the low-concentration group a no observed effect level could not be determined.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Study available for DTPA-K5; supported by key study (K;imisch 1) with DTPA-Na5 (NOAEL 75 mg/kg bw).

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Well performed 90-day inhalation study with structurally-related substance.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Well performed 90-day inhalation study with structurally-related substance.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Justification for classification or non-classification

Classification for repeated dose toxicity is proposed as the CLP criteria for classification for target organ toxicity have been met. According to the guidance on the application of CLP criteria (Version 4.0, 2013),substances that, on the basis of evidence from studies in experimental animals can be presumed to have the potential to be harmful to human health following repeated exposureshould be classified as STOT RE. For Pentapotassium DTPA, given the available evidence from a structurally related substance, with only slight histopathological effects at the larynx at a concentration of 15 mg/m3, it would be appropriate to apply Category 2, H373, specifically for the inhalation route.