Glycine, N,N'-1,2-ethanediylbis-, reaction products with formaldehyde, iron chloride (FeCl3) and phenol, potassium salts

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Remarks:

extended OECD 422; investigation of effects on repro and development combined with a full 90-day study.

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2021 - May 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Because the number of animals in OECD 422 is at least 10 animals/sex/group, this extended OECD 422 is carried out with a 10-wk pre-mating period so that animals are exposed for at least 13 weeks, and all 90-day study parameters are included.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

Extended OECD 422: thus with a 10-week pre-mating period. Because animals (at least 10 rats/sex/group) are at least 13 weeks on study, all 90-day parameters/measurements have been included with the required number of animals which means it is also equal to a full OECD 408 study.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

potassium 2-({2-[(carboxymethyl)[(2-hydroxyphenyl)methyl]amino]ethyl}[(2-hydroxyphenyl)methyl]amino)acetic acid iron potassium hydride chloride

EC Number:

938-828-8

Cas Number:

1463474-95-4

Molecular formula:

Not applicable for the reaction product (UVCB)

IUPAC Name:

potassium 2-({2-[(carboxymethyl)[(2-hydroxyphenyl)methyl]amino]ethyl}[(2-hydroxyphenyl)methyl]amino)acetic acid iron potassium hydride chloride

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Commonly used species and strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France.
Number of F0-males: 40 (plus 2 alternates).
Number of F0-females: 54 (nulliparous and non-pregnant, plus 2 alternates); see also section 7.8.1.
Target Age at the Initiation of Dosing (F0-generation): At least 6 weeks.
The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days prior to start of the pre-treatment period.

Caging: On arrival, during pretreatment and prior to mating, F0-animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm or Makrolon type 2000P, height 21.5 cm).
During the mating phase, males and females of the F0-generation (both Cohorts) will be cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, F0-males will be housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. F0-females will be individually housed in Makrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, F0-females will be housed in Makrolon plastic cages (MIII type, height 18 cm), except during locomotor activity monitoring of the dams.
During locomotor activity monitoring, F0-animals will be housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
Animals are socially housed for psychological/environmental enrichment and provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.

The target conditions for animal room environment are as follows:
Temperature: 18 to 24°C.
Humidity: 40 to 70%.
Light Cycle: 12-hours light and 12-hours dark (may be interrupted for designated procedures).
Ventilation: At least 10 air changes per hour.

Administration / exposure

Route of administration:

oral: gavage

Details on oral exposure:

The first day of dosing was designated as Day 1. The dose volume for each animal was based on the most recent body weight measurement. The dose formulations were stirred continuously during dose administration and doses are given using a plastic feeding tube.
A dose control system via DCS was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 85-115% of target concentration).
No test material was detected in the Group 1 formulations.
The formulations checked in Group 2 and Group 4 formulations were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

Males: 7 days a week for a minimum of 90 days, including at least 10 weeks of treatment prior to first mating, during and between the mating periods, up to and including the day before scheduled necropsy.
Females: 7 days a week for at least 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females will not be dosed during littering.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males and 12 females

Control animals:

yes, concurrent vehicle

Positive control:

Not required

Examinations

Observations and examinations performed and frequency:

Mortality: At least twice daily beginning upon arrival through termination/release. Except on days of receipt and necropsy where frequency is at least once daily.

Clinical Observations: At least once daily from the Pretreatment Period onwards, up to the day prior to necropsy. On dosing days, these clinical observations are at least conducted at no specific time point, but within a similar time period after dosing for the respective animals.

Arena Observations: Once before the first administration of the test item and weekly during the Treatment Period.

Individual Body Weights: Weekly from the Pretreatment Period onwards. Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post coitum and during lactation on PND 1, 4, 7, 13 and 20.

Food Consumption: Weekly, except for males and females which are housed together for mating and for females without evidence of mating. Mated females: on Days 0, 4, 7, 11, 14, 17, and 20 post coitum and during lactation on PND 1, 4, 7, 13 and 20.

Water Consumption: Regular basis throughout the study. Water consumption is monitored by visual inspection of the water bottles. If inter group differences are noted, consumption may be assessed by weight.

Ophthalmic Examinations: Frequency: Pretreatment Period, all animals, including spares. End of Treatment – All Group 1 and 4 animals in Week 13. If treatment-related findings are noted, all males and selected females of the intermediate groups will also be examined.

Females (week 10): haematology, coagulation, clinical chemistry, thyroid hormone analysis (total T3, T4 and TSH), urinalysis.
Females (scheduled necropsy): haematology, coagulation, clinical chemistry, thyroid hormone analysis (total T3, T4 and TSH).
Males (scheduled necropsy): haematology, coagulation, clinical chemistry, thyroid hormone analysis (total T3, T4 and TSH), urinalysis.

Haematology: White blood cells (WBC), Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Large unstained cells (LUC) (absolute), Red blood cells (RBC), Reticulocytes (absolute), Red blood cell distribution (RDW),
Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH),
Mean corpuscular hemoglobin concentration (MCHC), Platelets.

Coagulation: Prothrombin Time (PT), Activated partial thromboplastin time (APTT).

Urinalysis: Volume, Specific gravity, Clarity, Colour, pH, Blood, Leukocyte esterase, Bilirubin, Protein
Ketones, Glucose. In Sediment: White blood cells (WBC-sed.), Red blood cells (RBC-sed.), Casts,
Epithelial cells, Crystals, Bacteria, Other.

Sacrifice and pathology:

A necropsy was conducted for the animal that died on study, and specified tissues were collected and kept.
Animals surviving until scheduled euthanasia were weighed, and deeply anesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.

Scheduled necropsies were conducted on the following days:
Males (which sired or failed to sire): Following completion of the mating period (a minimum of 90 days of administration).
Females which delivered: LD 14-16.
Females which failed to deliver (Nos. 57, 62, 63, 94, 102 and 104): With evidence of mating: Post-coitum Day 26-27
All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available. Females were not fasted overnight.

The following organs/tissues were collected: Artery, aorta; Body cavity; nasopharynx; Bone marrow, sternum; Bone, femur; Bone, sternum; Brain; Epididymis; Esophagus; Eye; Gland, adrenal; Gland, clitoral; Gland, harderian; Gland, lacrimal; Gland, mammary; Gland, parathyroid; Gland, pituitary; Gland, preputial; Gland, prostate; Gland, salivary, submandibular; Gland, salivary, sublingual; Gland, salivary, parotid; Gland, seminal vesicle including coagulation gland and fluid;
Gland, thyroid; Gut-associated lymphoid tissue; Heart; Kidney; Large intestine, cecum; Large intestine, colon; Large intestine, rectum; Larynx; Liver; Lung; Lymph node, mandibular; Lymph node, mesenteric; Muscle, skeletal; Nerve, optic; Nerve, sciatic; Nerve, tibial; Ovaries; Pancreas; Skin; Small intestine, duodenum; Small intestine, ileum; Small intestine, jejunum; Spinal cord;
Spleen; Stomach; Testes; Thymus; Tongue; Trachea; Urinary bladder; Uterus/Cervix; Vagina.

Organ weights: Brain; Epididymis; Gland, adrenal; Gland, parathyroid; Gland, pituitary; Gland, prostate; Gland, seminal vesicle including coagulation gland and fluid; Gland, thyroid; Heart; Kidneys; Liver; Ovaries; Spleen; Testes; Thymus; Uterus/cervix.

The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
Group 1 and 4: Tissues as listed in OECD 408 (except animal identification, nasopharynx, clitoral gland, harderian gland, lacrimal gland, preputial gland, parotid salivary gland, Tibial nerve, larynx and tongue).
Group 2 and 3: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus, vagina and gross lesions/masses.

These tissues were evaluated histopathologically by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.

Other examinations:

Neurobehavioral assessments:
Premating period: All F0-females were tested once before mating in Week 10, prior to mating.
End of Treatment: All F0 males during Week 12 13 and all F0 females during the last week of lactation (i.e. PND 6-13).
These tests were performed after clinical observations (including arena observation, if applicable).
The following tests were performed (abbreviations mentioned in the respective tables are indicated between brackets):
• Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
• Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous. This was done for all females.

For all males, sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with hematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal were recorded. Evaluation was performed for all samples.
One epididymis (right) was removed, placed in labeled bags, and kept in the freezer set to maintain 20°C. After thawing, the right epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
Non-Parametric: Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Discolored feces were noted from 100 mg/kg/day onwards in both sexes, starting from Week 2 of treatment at 1000 mg/kg/day and from Week 13 of treatment at 100 and 300 mg/kg/day. At 100 and 300 mg/kg/day, red or blue discoloration of the feces was noted in all cages from Week 13 of treatment onwards until necropsy. At 1000 mg/kg/day, dark or red discoloration of the feces was noted from Week 2 or 3 of treatment onwards in all cages throughout the Treatment Period, until necropsy.
Purple discoloration of the tail was noted at 1000 mg/kg/day for both sexes and in all animals from Week 8 or 9 of treatment onwards, until the end of the Treatment Period.
No findings were noted during the weekly arena observations in this study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period up to 300 mg/kg/day.
Female No 98 (1000 mg/kg/day) was found dead on Day 50 of treatment before dosing. No relevant clinical observations were noted for this animal prior to death. At necropsy, main findings included pale discoloration of the lungs, a black-brown and soft nodule on the liver papillary process (12x17 mm), diaphragmatic hernia of the liver, and watery-cloudy, red brown fluid in the thoracic cavity. At microscopic examination, the main finding was lobar necrosis of the liver with moderate hemorrhage in the abdominal cavity (correlating to the black-brown, soft nodule on the papillary process), which likely attributed to the death of this female. Lobar necrosis can be seen as a spontaneous finding, and this alteration in a single female at 1000 mg/kg/day was regarded spontaneous in nature and unrelated to the treatment with the test material.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the Treatment Period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before and after correction for body weight of treated animals remained in the same range as controls over the Treatment Period.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The nature and incidence of ophthalmology findings noted during the Pretreatment Period and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test material.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

F0-females were nulliparous in Week 10 and lactating at end of treatment.
Hematological parameters of treated animals remained in the same range as controls over the Treatment Period up to 300 mg/kg/day.
In Week 10 of the Treatment Period (pre-mating), a lower red blood cell count (0.96x of control), a higher reticulocyte count (1.20x of control) and consequently a higher red blood cell distribution width (1.05x of control) were noted in females treated at 1000 mg/kg/day when compared to controls (see Table attached).
Any other findings in hematology parameters, regardless of statistical significance, were considered unrelated to treatment with the test material as these occurred in the absence of a dose-related trend.
No relevant differences were noted in males or in lactating females at the end of the Treatment Period.
Coagulation parameters generally remained in the same range as controls over the Treatment Period for males and females at the end of treatment.
In Week 10 of the Treatment Period (pre-mating), a longer activated partial thromboplastin time (APTT) was noted for females treated at 1000 mg/kg/day (1.10x of control) (see Table attached).
While a few other statistically significant findings changes were noted, the findings in coagulation parameters were considered unrelated to administration of the test material due to the minimal magnitude of the difference with the control group and the large overlap of individual values between the groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical chemistry parameters of treated animals remained in the same range as controls over the Treatment Period in females and up to 300 mg/kg/day in males.
At the end of the Treatment Period, lower albumin and total protein levels (both 0.95x of control) and higher triglyceride levels (1.52x of control) were noted in males treated at 1000 mg/kg/day.
The higher calcium levels in (lactating) females treated at 300 and 1000 mg/kg/day at the end of the Treatment Period were considered due to a relatively low concurrent control mean when compared to historical control data and was therefore regarded as unrelated to treatment with the test material (see Table attached).
While a few other differences were noted between control and treated groups, these findings in clinical chemistry parameters were considered unrelated to administration of the test material due to the minimal magnitude of the difference with the control group, the absence of a dose response and/or general overlap between the groups.

Historical control data for Wistar Han rats, F0-females (period 2021-2022): Calcium (mmol/L): mean = 2.492; P2.5 – P97.5 = 2.210 – 2.862 (n = 120)

Endocrine findings:

no effects observed

Description (incidence and severity):

No differences were noted between control and treated groups in T3, T4 and TSH serum levels in females in Week 10 of the Treatment Period (pre-mating) and in males and lactating females at the end of the Treatment Period.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Findings in urine parameters between treated animals and controls were noted from 100 mg/kg/day onwards in both sexes and are summarized in a Table (attached). Differences that were noted between control and treated rats are marked grey.
A dose-dependent discoloration of the urine was noted in all treatment groups which was not considered adverse and most likely representing presence of test material.
Female No. 101 (1000 mg/kg/day) was noted to have a moderate level of white blood cells (score: 3+) in urine sediment. As this was noted in a single animal only, this was considered to be unrelated to treatment with the test material.
Urine being scored more often as cloudy for females treated at 1000 mg/kg/day when compared to control females was considered a chance finding, as this is a finding also commonly observed in untreated rats. For Female No. 99 , the turbidness of the urine was likely caused by the trace of blood noted for this animal. As this was noted in a single animal only, this finding was considered unrelated to treatment with the test material.
Any other findings in urine parameters, including differences in urine sediment parameters, were considered unrelated to treatment with the test material based on the absence of a dose response, general overlap of individual values with the range of control values and/or were considered common findings in rats under similar study conditions.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observation parameters of treated animals remained in the same range as controls over the Treatment Period.
No differences were noted between control and treated groups in hearing ability, pupillary reflex and static righting reflex. Any statistically significant differences in grip strength occurred in the absence of a dose related trend and were therefore considered unrelated to treatment with the test material.
No differences were noted between control and treated groups in motor activity. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the Test Period.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males at 1000 mg/kg/day, higher kidney weights (statistically significant as absolute value) and higher liver weights (statistically significant when expressed relative to body weight) were noted. Differences are presented in a Table attached.
There were no organ weight changes in females.
Other statistically significant findings included higher adrenal gland weights in males at 100 mg/kg/day, higher spleen weights in males at 300 mg/kg/day and higher ovary weights in females at 100 mg/kg/day (all absolute and relative to body weight). These lacked a dose related pattern and were therefore regarded unrelated to the treatment with the test material.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The following macroscopic findings of note were observed:
Cecum:
• Red-brown contents in 1/10 males at 100 mg/kg/day and in 10/10 males at 1000 mg/kg/day.
• Irregular surface in 4/10 males at 1000 mg/kg/day.
Skin (tail):
• Isolated, several or many red-brown foci in 10/10 males and red-brown discoloration in 1/11 females at 1000 mg/kg/day.
The remainder of the recorded macroscopic findings was within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no histologic changes noted between control and treated rats.
There was one finding of note in Female No. 77 (300 mg/kg/day): the stomach showed a chronic inflammatory process in the serosa (correlating to a nodule in the stomach recorded at necropsy), which was attached to the pancreas and contained necrotic liver tissue. This inflammatory process most likely resulted from the gavage procedure.
The remainder of the recorded microscopic findings was within the range of background pathology encountered in rats of this age and strain. There was no test material related alteration in the prevalence, severity, or histologic character of those incidental tissue findings.

Histopathological findings: neoplastic:

no effects observed

Details on results:

A dose-related onset of purple discoloration of the tail as well as red, blue and/or dark feces was noted from 100 mg/kg/day onwards in both sexes. In addition, urine was also noted to be discolored orange or red in a dose-related way upon analysis for females in Week 10 and for males at the end of the Treatment Period. At necropsy, red-brown content of the cecum and red brown foci or discoloration of the tail (without microscopic correlate) were noted. These findings were considered non-adverse and representing discoloration caused by treatment with the test material, which is reddish brown in color.
Clinical pathology including urine analysis was performed in females in Week 10 (pre mating) and in males at the end of the Treatment Period. In addition, clinical pathology (without urine analysis) was performed in lactating females at the end of the Treatment Period.
Hematology findings were noted in females treated at 1000 mg/kg/day in Week 10 only and comprised slight changes in red blood cell parameters, i.e., lower red blood cell count, higher reticulocyte count and consequently higher red blood cell distribution width. These differences were considered related to treatment with the test material, but were regarded as non adverse due to the small magnitude of change and absence of changes at the end of the Treatment Period in these females.
An increase in activated partial thromboplastin time (APTT) was noted for females in Week 10 of the Treatment Period (pre-mating). As this effect was not observed at the end of the Treatment Period anymore, this change was considered non-adverse.
Findings in clinical chemistry were noted in males treated at 1000 mg/kg/day at the end of the Treatment Period only and comprised lower albumin levels with consequently lower total protein levels. In addition, an increase in triglyceride levels was noted in these high dose males. These differences were considered related to treatment with the test material, but were regarded as non adverse due to the small magnitude of change.
Presence of leukocyte esterase and protein was more frequently noted in urine of animals treated at 1000 mg/kg/day compared to controls. Presence of leukocyte esterase could not be correlated with presence of white blood cells in urine sediments. At the low incidence and severity observed, these findings were considered to be related to treatment with the test material but non-adverse.
Higher kidney weights (statistically significant as absolute value) and higher liver weights (statistically significant when expressed relative to body weight) were noted in males at 1000 mg/kg/day. These were regarded likely related to the treatment with the test material. There were no macroscopic or microscopic correlates for these higher organ weights and therefore these minor alterations in weight were regarded non-adverse.
The irregular surface of the cecum in males at 1000 mg/kg/day was without microscopic correlate and therefore regarded non-adverse.

Estrous cycle: No differences between control and treated animals were noted for length and regularity of the estrous cycle. Most females had regular cycles of 4 to 5 days.
Female No. 69 (100 mg/kg/day) had an irregular cycle. For Female Nos. 79 (300 mg/kg/day) and 102 (1000 mg/kg/day), cyclicity could not be determined as these females had only one complete estrous cycle (of 4 or 5 days) during the 14 days observation period. These female 69 and 79 had a normal litter. Female No. 102 was non pregnant. Given their incidental nature, these findings did not indicate a relationship with treatment of the test material.

Sperm analysis: Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present. No adverse findings were noted in epididymal sperm count and sperm morphology parameters between the control and treated groups.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: absence of adverse findings at this dose level

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this extended OECD422 toxicity study with 90-day parameters, the following No Observed Adverse Effect Level (NOAEL) of Fe-HBED for 90-day parental toxicity is at least 1000 mg/kg/day.

Executive summary:

The objectives of this study were to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development and determine the potential toxic effects of Fe-HBED when given orally by gavage for a minimum of 90 days to Wistar Han rats (using a pre-mating period of 10 weeks), and thus also provide information on additional 90‑day parameters. 

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg/day, based on the results of a 14-day Dose Range Finder with oral gavage administration of Fe-HBED in rats (Test Facility Study No. 20291815) attempting to produce graded responses to the test material.

Chemical analyses of formulations were conducted three times during the study to assess accuracy and homogeneity. Formulation analyses confirmed that formulations of test material in Elix water were prepared accurately and homogenously.

For the F₀-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight and food consumption, functional observations, ophthalmoscopy, clinical pathology (hematology, coagulation, clinical chemistry and urinalysis), measurement of thyroid hormones (T4, T3 and TSH), gross necropsy findings, organ weights, oestrous cycle and sperm analysis, and histopathologic examinations.

At 1000 mg/kg/day, a single F₀-female was found dead on Day 50 of treatment before dosing. No abnormalities in clinical appearance or body weight were noted for this animal prior to death. Based on the outcome of macroscopic and microscopic evaluation, lobar necrosis of the liver with hemorrhage in the abdominal cavity was considered the likely cause of the death of this female. Lobar necrosis can be seen as a spontaneous finding, and together with its isolated incidence, it was regarded as unrelated to the treatment with the test material.

A dose-dependent orange-red discoloration of urine and blue/red/dark discoloration of feces was noted in treated animals. A purple discoloration of the tail (without microscopic correlate) was noted in addition. These findings were considered non-adverse and representing discoloration with the test material which is reddish brown in color.

No adverse findings were noted in any of the parameters investigated in this study i.e., mortality, clinical observations, body weight, food consumption, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), ophthalmoscopy, clinical pathology (including other urinalysis parameters), thyroid hormone analysis, estrous cycle and sperm analysis, macroscopic examination, organ weights and microscopic examination.

In conclusion, based on the results of this extended OECD422 toxicity study with 90-day parameters, the following No Observed Adverse Effect Levels (NOAELs) of Fe-HBED was established as Parental 90-day NOAEL: at least 1000 mg/kg/day.