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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

extended OECD 422; investigation of effects on repro and development combined with a 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:

Investigation of effects on repro and development combined with a 90-day study; because the number of animals in OECD 422 is at least 10 animals/sex/group, the extended OECD 422 is carried out with a 10-wk pre-mating period so that animals will be exposed for at least 13 weeks (conform a 90-day study), and all repro-screening parameters are included.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Extended OECD 422: thus with a 10-wk pre-mating period.
The study also includes all required parameters for a 90-day study (OECD 408). See also at section 7.5.1.

In addition, pups from three additional females/group (using a pre-mating period of 7 weeks) in the mid and high dose groups were dosed for at least 15 days after weaning, to evaluate systemic toxicity in these pups (named 'extra cohort').

GLP compliance:

yes

Limit test:

no

Justification for study design:

Because for an OECD 422 study at least 10 animals/sex/group are required, and the pre-mating period should at least be 2 weeks, the OECD 422 provides the possibility to extend the pre-mating period from 2 to 10 weeks. In addition, this set up also results in and males females being at least 13 weeks on study so that all 90-day study parameters can be included.

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:

Preferred testing species

Sex:

male/female

Details on test animals or test system and environmental conditions:

On 10 Nov 2021, Crl: WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. At initiation of dosing, the animals were 6-7 weeks old; males weighed between 148 and 228 g and females weighed between 126 and 155 g. Female rats were non-pregnant and nulliparous.
The animals were allowed to acclimate to the Test Facility accommodation for 6 days prior to start of the pretreatment period.

The conditions for animal room environment were as follows:
Temperature: targeted: 18 to 24°C, actual mean: 20 to 21°C.
Humidity: targeted: 40 to 70%, actual mean: 34 to 63%.
Light Cycle: 12 hours light and 12 hours dark (except during designated procedures)
Ventilation: Ten or more air changes per hour
The values that were outside the targeted humidity range occurred for 26 out of 125 days with a minimum of 34% and were without a noticeable effect on the clinical condition of the animals and were not considered to have had any impact on the outcome or integrity of the study.

For psychological/environmental enrichment and nesting material, animals were provided with materials such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities. Results of analysis for contaminants were provided by the supplier and are on file at the Test Facility. It was checked there were no known contaminants that would interfere with the objectives of the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Elix

Details on 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 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 (Reference Nos. 523072 (F0-animals) and 523074 (F1 animals) are used for DCS).

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 of Group 2 and Group 4 were homogeneous (i.e., coefficient of variation ≤ 10%).

Duration of treatment / exposure:

F0-males: 7 days a week for a minimum of 90 days, including at least 7 weeks of treatment prior to first mating, during and between the mating periods, up to and including the day before scheduled necropsy.
F0-females: 7 days a week for at least 7 weeks prior to mating (Cohort A) or 10 weeks prior to mating (Cohort B), the variable time to conception, the duration of pregnancy and at least 13 (Cohort B) or 20 (Cohort A) days after delivery, up to and including the day before scheduled necropsy. Females will not be dosed during littering.
Pups will not be treated directly but could potentially be exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.
From weaning onwards (PND 21), F1-animals from Cohort A will be dosed once daily for at least 15 days, up to and including the day before scheduled necropsy (PND 36-38 ).

Frequency of treatment:

Daily

Details on study schedule:

See above

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 per group; control,low, mid and high dose

Control animals:

yes, concurrent vehicle

Positive control:

Not required

Examinations

Parental animals: Observations and examinations:

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 (all F0 animals): At least once daily from the Pretreatment Period onwards, up to the day prior to necropsy.
Arena observations (all F0 animals): Once before the first administration of the test item and weekly during the Treatment Period.
Body weights (all F0 animals): 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.
Food intake (all F0 animals): Weekly, except for males and females which were 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.
Water intake (all F0 animals): Regular basis throughout the study. Water consumption is monitored by visual inspection of the water bottles.
Ophthalmic Examinations: Pre-treatment Period – All animals, including spares. End of Treatment – All Group 1 and 4 animals in Week 13.
Functional Tests (F0-Generation). Premating period: All F0-females were tested once before mating in Week 10, prior to mating. End of Treatment: All F0 males during Week 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: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity (total movements and ambulations).

F0 females (week 10): haematology, coagulation, clinical chemistry, thyroid hormone analysis total (T3, T4 and TSH), urinalysis
F0 females (scheduled necropsy): haematology, coagulation, clinical chemistry, thyroid hormone analysis (total T3, T4 and TSH)
F0 males (scheduled necropsy): haematology, coagulation, clinical chemistry, thyroid hormone analysis total (T3, T4 and TSH), urinalysis

General reproduction data: Daily from the mating period onwards. Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. The females were allowed to litter. Postnatal day (PND) was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 was considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females was examined for evidence of premature delivery. Signs of difficult or prolonged parturition were recorded, if applicable. Deficiencies in maternal care, such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding, were recorded, if applicable.

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed for all females beginning 14 days prior to mating 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. At the end of treatment, on the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

Sperm parameters (parental animals):

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. In the case of any abnormalities in the right epididymis, the right side organ(s) was fixed in modified Davidson's solution, and the left side organ was used for evaluation of sperm numbers.

Litter observations:

For F1 animals:
Mortality (all pups): The number of live and dead pups was determined on PND 1 and daily thereafter.
Clinical Observations (all pups): At least once daily, up to the day prior to necropsy.
Body Weights (all pups): on PND 1, 4, 7, and 13.
Sex (all pups): on PND 1 and 4.
Anogenital Distance (all pups): on PND 1. Anogenital distance (AGD) was measured for all live pups. The AGD was normalized to the cube root of body weight.
Areola/Nipple Retention (all male pups in each litter): on PND 13.
Culling (all litters): on PND 4. To reduce variability among the litters, eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) was considered acceptable.

2 pups/litter: thyroid hormone analysis (possible total T4) on PND 4 at culling
2 pups/litter: thyroid hormone analysis (total T4 and possible TSH) on PND 14-16

For F1 animals (extra cohort):
Mortality (all animals): At least twice daily throughout the study.
Clinical Observations (all animals): At least once daily, up to the day prior to necropsy. These clinical observations were at least conducted at no specific time point, but within a similar time period after dosing for the respective animals.
Body Weights (all animals): Day 1, 4, 8, 11 and 15 (equal to PND 21, 24, 28, 31 and 35).

Postmortem examinations (parental animals):

A necropsy was conducted on the animal that was found dead; specified tissues were saved, but not weighed.

All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or another qualified person, was available.
The numbers of former implantation sites were recorded for all paired females. The number of corpora lutea was recorded in all pregnant and formerly pregnant females (i.e. all females with implantation sites).

Scheduled necropsies are summarized below:
Males (which sire or fail to sire): Following completion of the mating period (a minimum of 28 days of administration).
Cohort B Females which deliver: LD 14-16.
Cohort A Females which deliver: LD 21-23.
Females which fail to deliver: With evidence of mating: Post-coitum Days 25-27.
Without evidence of mating: Approximately 24-26 days after the last day of the mating period.
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 organs (as listed in OECD 422/408) were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

Representative samples of the tissues identified in the table below were collected from all Cohort B animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated. For females which fail to deliver a complete litter, uterine contents (i.e. any fetuses, placenta and implantation sites) will be fixed (if applicable).

The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
Group 1 and 4: Tissues identified in OECD 422/408
Group 2 and 3: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus, vagina and gross lesions/masses.

All tissues were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.

Postmortem examinations (offspring):

Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND 7-16), except the pups selected for blood collection, were euthanized by an intraperitoneal injection of sodium pentobarbital.
The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane and subsequently exsanguinated.
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
On PND 4, the surplus pups were euthanized by decapitation. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. From two surplus pups per litter, blood was collected, if possible.
All remaining pups were euthanized on PND 14-16.
Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, the thyroid was collected from two pups per litter (if possible, from one male and one female pup and preferably from the same pups as selected for blood collection), and were preserved in 10% buffered formalin.

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
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.
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
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.

Reproductive indices:

Mating index (%): Number of females mated x 100 / Number of females paired;
Precoital time: Number of days between initiation of cohabitation and confirmation of mating;
Fertility index (%): Number of pregnant females x 100 / Number of females mated;
Gestation index (%): Number of females with living pups on Day 1 x 100 / Number of pregnant females;
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition;
Post-implantation survival index (%): Total number of offspring born x 100 / Total number of uterine implantation sites
(Post-implantation survival index will be expressed as 100% when the number of offspring exceeds the number of implantation sites recorded);
Live birth index (%): Number of live offspring on Day 1 after littering x 100 / Total number of offspring born;
Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check x 100 / Number of live pups at First Litter Check;
Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check x 100 / Number of live pups at First Litter Check

Offspring viability indices:

Viability index (%): Number of live offspring on Day 4 before culling x 100 / Number live offspring on Day 1 after littering;
Lactation index (%): Number of live offspring on Day 13 after littering x 100 / Number live offspring on Day 4 (after culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 (see further section 7.5.1).

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.
Any statistically significant differences in body weight gain were considered to be unrelated to treatment with the test material, since no trend was apparent regarding dose.

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):

not examined

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 (see further at section 7.5.1).

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

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 at 7.5.1).
Any other findings in hematology parameters, regardless of statistical significance, were considered to be 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 females at the end of treatment and for males in Week 10.
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 further at section 7.5.1).
While a few other statistically significant findings changes were noted, the findings alterations in coagulation parameters were considered unrelated to administration of the test material due to the minimal magnitude of the difference with the control group change 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 further at section 7.5.1).
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.

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 were noted from 100 mg/kg/day onwards in both sexes and are summarized in a table (see section 7.5.1). 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 to be 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):

no effects observed

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 (see further at 7.5.1).
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

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

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. 

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

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. Female Nos. 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.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

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.

Reproductive performance:

no effects observed

Description (incidence and severity):

No differences between control and treated animals were noted for mating index. All females showed evidence of mating. The mating indices were therefore 100% for all groups.

No differences between control and treated animals were noted for precoital time. All females showed evidence of mating within 4 days.

No differences were noted between control and treated groups for the number of corpora lutea or implantation sites. Female No. 94 (1000 mg/kg/day) had seven corpora lutea and only one implantation site. As this occurred in a single animal only, the pre-implantation loss of this female was considered to be unrelated to treatment with the test material.

Fertility indices for the control, 100, 300 and 1000 mg/kg/day groups were 100, 75, 100 and 82%, respectively.
At 100 mg/kg/day, 3 out of 12 females and at 1000 mg/kg/day 2 out of 11 females were not pregnant. The two non pregnant females at 1000 mg/kg/day (Female Nos. 102 and 104) had mated with the same male (Male No. 39). In the absence of a dose-related incidence of non-pregnancy, the lower fertility index at 100 and 1000 mg/kg/day was considered not related to treatment with the test material.

Details on results (P0)

Developmental data:
No differences between control and treated groups were noted for gestation length.
Except for one female at 1000 mg/kg/day (No. 94) with a single implantation site only, all pregnant females had live offspring. Gestation indices (females with living pups on Day 1 compared to the number of pregnant females) were therefore 100% for the control, 100 and 300 mg/kg/day groups, and 91% for the 1000 mg/kg/day group. The failed pregnancy of Female No. 94 (1000 mg/kg/day), without related histopathology changes in reproductive organs, was judged to be unrelated to treatment with the test material, due to its incidental occurrence and given the absence of any reproductive/developmental toxicity.

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

No differences were noted between control and treated groups for the total number of offspring born compared to the total number of uterine implantations. Post-implantation survival indices (total number of offspring born as percentage of total number of uterine implantation sites) were 90, 90, 85 and 92% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

No differences changes were noted in litter size between litters of the control group and those of treated animals. Live litter sizes were 11.4, 9.7, 10.9 and 11.6 living pups/litter for the control, 100, 300 and 1000 mg/kg/day groups, respectively. A slightly lower mean number of living pups was recorded at 100 mg/kg/day (9.7 vs. 11.4 in the control group). As a dose-related response was absent, this was regarded as unrelated to treatment with the test material.

No differences were noted between control and treated groups for the number of live offspring on Day 1 after littering compared to the total number of offspring born. The live birth indices were 100, 99, 99 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. One pup of the 100 and 300 mg/kg/day groups each were found dead at first litter check. For one of the pups, absence of milk in the stomach and beginning autolysis were noted. The death of these pups was considered unrelated to treatment with the test material since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

No differences were noted between control and treated groups for the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1. Viability indices were 100, 99, 99 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. One pup of the 100 and 300 mg/kg/day groups each were found missing on PND 2 or 3. No clinical observations were noted for these pups the day(s) before they went missing. Pups missing were most likely cannibalized. These missing pups were considered to be unrelated to treatment with the test material, since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

No differences were noted between control and treated groups for the number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling). The lactation indices were 100, 100, 99 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively. One pup of the 300 mg/kg/day group was in a poor condition following a procedure error and was therefore sacrificed in extremis on PND 6.

Effect levels (P0)

1

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: Absence of parental toxicity

2

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: Absence of effects on fertility

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The nature and incidence of clinical signs noted in treated pups remained within the range of controls and were considered normal for pups of this age. One pup from Litter No. 101 (1000 mg/kg/day, female pup 12) was noted to be generally discolored yellow from PND 2 until culling on PND 4. At the incidence observed, this was considered unrelated to treatment with the test material.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

One pup of the 100 and 300 mg/kg/day groups each were found dead at first litter check. For one of the pups, absence of milk in the stomach and beginning autolysis were noted. In addition, one pup of the 100 and 300 mg/kg/day groups each were found missing on PND 2 or 3. No clinical observations were noted for these pups the day(s) before they went missing. Pups missing were most likely cannibalized. These missing pups were considered to be unrelated to treatment with the test material, since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
One pup of the 300 mg/kg/day group was in a poor condition following a procedure error and was therefore sacrificed in extremis on PND 6.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences were noted between control and treated groups for body weights of pups.
One pup from Litter No. 101 (1000 mg/kg/day, female pup 12) had a relatively low birth weight (4.1 vs. 5.4-6.3 gram for the remaining three female pups in this litter), followed by 22% body weight loss between PND 1 and 4. At the incidence observed, this was considered unrelated to treatment with the test material. This pup was culled on PND 4.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No differences were noted between control and treated groups for T3 and T4 serum levels in PND 4 pups, and for T3, T4 and TSH serum levels in PND 14-16 pups.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No differences were noted between control and treated groups for anogenital distance (absolute and corrected for body weight) in male and female pups.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were observed for any of the examined male pups at PND 13.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings were noted among pups, that were considered related to treatment with the test material, as they also occurred in the control group and/or as the nature and incidence of these macroscopic findings remained within the range considered normal for pups of this age.
One pup from Litter No. 101 (1000 mg/kg/day, female pup 12) was noted to be discolored yellow externally. At the incidence observed, this was considered unrelated to treatment with the test material.

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment with the test material. At 100 mg/kg/day, sex ratio was 36/64 for males/females. As this occurred in the absence of a dose-related response, it was considered to be unrelated to treatment with the test material.

Visceral examinations: Due to the small sample size of pups available for morphological examination results should be interpreted with caution. In addition, it should be noted that evaluation was performed on pups surviving until PND 4, i.e., fetuses and pups not surviving until PND 4 were not part of the evaluation and any variations and malformations, if present, in these fetuses and pups that might be related to their non viability or early death will have been missed. In total, 41, 18, 43 and 30 pups of control, low, mid and high dose, respectively, were evaluated. There were in total two pups with visceral malformations observed in this study, one pup at 100 and 1000 mg/kg/day each: Pup No. 12 in Litter No. 101 (1000 mg/kg/day) had a distended intestine. This pup also had yellow or yellow-green discoloration of the entire body wall (correlating in-life finding: yellow discoloration of the body from PND 2 onwards) and kidneys, and dark red discoloration of the liver. At the incidence observed, this was considered unrelated to treatment with the test material. Pup No. 11 in Litter No. 66 (100 mg/kg/day) had an enlarged kidney with corresponding variations of the urinary tract (absent papilla, convoluted and severely dilatated ureter). This malformation was considered to be spontaneous in origin due to the absence of a dose response.

Extra cohort: No toxicity was observed in the F1-pups dosed at 300 or 1000 mg/kg/day between PND 21 and 35 until necropsy on PND 36.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of developmental toxicity

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

No reproductive and developmental toxicity were observed up to and including the highest dose level tested (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, pups from three additional females/group (using a pre-mating period of 7 weeks) in the mid and high dose groups were dosed for at least 15 days after weaning, to evaluate systemic toxicity in these pups. 

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, sperm analysis and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: estrous cycle, mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone (T4 and T3 in PND 4; T4, T3 and TSH in PND 14-16 pups)) and visceral examination of culled pups on PND 4.

For the post-weaning F₁-generation, the following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight and macroscopic 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, macroscopic examination, organ weights and microscopic examination).

No adverse findings were noted in any of the reproductive parameters investigated in this study (i.e., sperm analysis, mating and fertility indices, precoital time, number of corpora lutea and implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

No adverse findings were noted in any of the developmental parameters investigated in this study (i.e., gestation index and duration, parturition, maternal care, viability and lactation indices, sex ratio, litter size and early postnatal pup development until PND 14-16 consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels, macroscopic examination, and visceral examination of culled PND 4 pups).

No adverse findings were noted in any of the parameters investigated in the weaned pups dosed from PND 21 to 35 (i.e., mortality/moribundity, clinical signs, body weight and macroscopic 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 were established:

Parental NOAEL: at least 1000 mg/kg/day

Reproduction NOAEL: at least 1000 mg/kg/day

Developmental NOAEL: at least 1000 mg/kg/day

In addition, Fe-HBED was considered well-tolerated up to 1000 mg/kg/day in Wistar‑Han rats aged 21 to 35 days.