Sodium [N-[2-[bis(carboxylatomethyl)amino]ethyl]-N-(2-hydroxyethyl)glycinato(3-)]zincate(1-)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
See the documentation in support for the category approach.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
See the documentation in support for the category approach.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetate;manganese, potassium;sodium;2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetate;manganese and potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetate;manganese

IUPAC Name:

Reaction mass of potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetate;manganese, potassium;sodium;2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetate;manganese and potassium;sodium;2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetate;manganese

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Taconic Biosciences, Inc., USA, through its representative Vivo Bio Tech Ltd., Telangana, INDIA, a vendor approved by the Test Facility Management.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
11-12 weeks
- Weight at study initiation:
mal =: 302-372 g, female: = 204-249 g
- Fasting period before study:
no
- Housing:
Animals were housed in Room No. AR-47 of the experimental animal facility of INTOX. Animals were housed maximum three per sex / cage in solid [Sized:42 cm (L) x 29 cm (W) x 19 cm (H)] bottom polypropylene cages with stainless steel grill tops, facilities for food and water bottle, and with bedding of clean and sterilized corn cob. The cages were suspended on stainless steel racks. Cages were suspended on movable stainless steel racks arranged in such a way that every cage would receive almost same amount of light. For this purpose, cages arranged on the first row were shifted to the last row, those on the second row were shifted to first row. Likewise all the cages were rotated. This kind of cage rotation was carried out every week throughout the treatment and observation period. Animals were housed in groups or singly, as below.
Pre-mating period: males and females were housed in groups of maximum two or three per cage per sex.
During mating (co-habitation): one male : one female, per cage.
Post-mating: females were housed individually once they were successfully mated. Males were housed in groups of maximum three per cage. During the post-natal (lactation) period the dam was housed individually.
- Diet (e.g. ad libitum):
'Altromin' brand extruded pelleted rat feed manufactured by M/s Altromin Spezialfutter GmbH & Co. KG, Germany, was provided ad libitum.
- Water (e.g. ad libitum): Potable water, passed through ‘Aquaguard’ water filter, and subjected to ultra violet irradiation, was provided ad libitum in sterilized glass bottles.
- Acclimation period:

The animals were acclimatised for a period of at least five days to the experimental room.

DETAILS OF FOOD AND WATER QUALITY:
The diet has been tested and certified to be free from undesired levels of environmental contaminants. Results of feed analysis were provided by the manufacturer for each batch of diet.
Water is analysed quarterly for potability and once in a year for various environmental contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
19-25°
- Humidity (%):
30-70%
- Air changes (per hr):
10-15/h
- Photoperiod (hrs dark / hrs light):
12 hours dark / 12 hours light

IN-LIFE DATES: From: 17 November 2017 To: 07 February 2018

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared freshly each day before dosing. Analytical grade water was used as vehicle control item. Formulations for different doses were vary in concentrations to allow a constant dosage volume of 5 ml/kg body weight.
Required amount of test item was weighed on analytical balance, then analytical grade water was added gradually to achieve appropriate concentrations i.e. 50, 100 and 200 mg/ml to meet dosage level requirements.


- VEHICLE
Test item was completely soluble in analytical grade water, hence analytical grade water was chosen as a vehicle for the dosing formulation.

Details on mating procedure:

- M/F ratio per cage: One male to one female (1 : 1) mating
- Length of cohabitation: The female was placed with the same male until pregnancy occurs or one week have elapsed.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
In case pairing was unsuccessful, re-mating of females with proven males of the same group was considered.
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged (how):
Post-mating: females were housed individually once they were successfully mated. Males were housed in groups of maximum three per cage. During the post-natal (lactation) period the dam was housed individually.
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test item formulations (for all concentrations) were subjected for verification of concentration twice (i.e. in first week after initiation of treatment and in last week at termination of treatment) during the study.

The following conditions were used:
Element-Matrix: Manganese (Mn)
Instrument type: Flame -AAS
Lamp current: 10 mA
Wavelength: 279.5 nm
Slit width: 0.2 nm
Flame Type: Air/Acetylene
Air Flow: 10 L/minute
Acetylene Flow: 2 L/minute
Diluent: Analytical grade water
Instrument mode: Absorbance
Calibration mode: Concentration
Measurement mode: Integrate

The analytical method was validated by determining the parameters viz., Linearity, Accuracy (% Recovery), Precision (Repeatability), Limit of detection (LOD) and Limit of quantification (LOQ) using Atomic Absorption Spectrometer.

For stability study, formulations at different doses were prepared in analytical grade water and analysed for determination of test item concentration after 4 hours. Data on test item concentrations after 4 hours at specified storage condition were compared with the data obtained before storage. On the basis of the calculated % deviation of test item concentration, stability of Manganese concentration in Reaction mixture of MnEDTA, MnDTPA and MnHEEDTA prepared in analytical grade water was determined. Based on the results of stability study, it is concluded that, Reaction mixture of MnEDTA, MnDTPA and MnHEEDTA is Stable in analytical grade water after specified storage conditions.

Duration of treatment / exposure:

Males were dosed for a minimum of four weeks, up to and including the day before scheduled sacrifice (this included a minimum of two weeks prior to mating, during the mating period and, approximately, two weeks post mating).
Females were dosed throughout the study period. This included two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and thirteen days after delivery, up to and including the day before scheduled sacrifice.

Frequency of treatment:

Daily dosing

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 15 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Repeated Dose 14 Days Oral Toxicity Study of Reaction Mixture of MnEDTA, MnDTPA and MnHEEDTA in Wistar Rat (Dose Range Finding Study) was performed in compliance with the Study Plan (No. P/17189/SOR-14-DRF/17). Groups of three male and three female Wistar rats were administered with test item, by oral gavage daily at the doses of 125, 250, 500 and 1000 mg/kg body weight for 14 days and were sacrificed on day 15 to evaluate its toxicity. Concurrent vehicle control group receiving analytical grade water at the dose of 5 ml/kg was also maintained. The test item did not induce any mortality and treatment related clinical abnormalities in rats treated at and up to the dose of 1000 mg/kg body weight. No mortality or abnormal clinical signs were observed in vehicle control group animals.
Body weight gain and food consumption was not affected at and up to the dose of 1000 mg/kg body weight. The values of absolute and relative organ weights of male and female rats treated with test item, at and up to the dose of 1000 mg/kg were found to be comparable with those of the control rats at termination. No gross pathological alterations were encountered in the rats sacrificed at termination of the study.
Based on these findings, the doses selected for the "Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test of Reaction Mixture of MnEDTA, MnDTPA and MnHEEDTA in Wistar Rat" are 250, 500 and 1000 mg/kg body weight.
- Rationale for animal assignment (if not random): random assignment

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:

All signs of ill health, together with any behavioural changes or reaction to treatment were observed (cage side observation) once a day for individual animals. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets.
Throughout the study, all animals were checked early on each working day and again in the afternoon to look for dead or moribund animals to allow necropsy examination to be carried out during the working hours of that day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Once before the first exposure (to allow for within-subject comparisons), and once a week thereafter, detailed clinical observations were made in all parental animals. The rats were subjected to detailed clinical examinations before initiation of the treatment (to allow for within-subject comparisons) and weekly thereafter during the treatment period.
These observations were made outside the home cage in a standard arena and preferably at the same time. Signs noted included, changes in skin, fur, eyes and mucous membranes, occurrence of secretions, excretions and autonomic activity such as lacrimation, piloerection, pupil size and unusual respiratory pattern. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies, difficult or prolonged parturition or bizarre behavior were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males and females were weighed on the first day of dosing, weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and then within 24 hours of parturition (day 1 post-partum), and at day 4 and day 13 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
During pre-mating, food consumption was measured weekly (on days 0, 7 and 14). During pregnancy it was measured on gestation day 0, 7, 14 and 20 and in lactation on days 4 and 13. Food consumption during mating period was not measured.
Food consumption was computed as the amount of food consumed in grams per animal per day.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
After completion of premating period (day 14 from start of treatment) haematological examination was made in five males and five females randomly selected from each group.
- Anaesthetic used for blood collection: Yes (light CO2 anaesthesia)
- Animals fasted: Yes
- How many animals:
five males and five females randomly selected from each group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
At termination (day 29 for males and day 14 of lactation for females)
- Animals fasted: Yes
- How many animals:
five males and five females randomly selected from each group.
- Parameters checked in table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
In males, these functional observations were made towards the end of their dosing period, shortly before scheduled sacrifice but before blood sampling for haematology or clinical chemistry.
In females these functional tests were made during the last week of lactation (e.g., LD 6-13), shortly before scheduled sacrifice.
- Dose groups that were examined:
each group
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: Yes
Based on the haematology

Oestrous cyclicity (parental animals):

Females were screened for normal oestrous cycles (in two weeks pre-treatment period). Oestrous cycle was monitored before start of treatment to select the females with regular cyclicity.
Females that failed to exhibit typical 4-5 day cycles were not included in the study. Vaginal smears were also monitored daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
Dead pups and pups killed on day 13 post-partum were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
not assessed
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
not assessed

Postmortem examinations (parental animals):

SACRIFICE
All adult animals in the study were humanely sacrificed by exsanguination under deep CO2 anaesthesia.
- Male animals: Males were further dosed after the mating period until the minimum total dosing period of 28 days had been completed. They were then sacrificed.
- Maternal animals: Daily dosing of the parental females were continued throughout pregnancy and up to, and including, day 13 post-partum (the day before sacrifice).

GROSS NECROPSY
All adult animals in the study were humanely sacrificed by exsanguination under deep CO2 anaesthesia and were subjected to a full, detailed gross necropsy which included careful examination of the external surface of the body, all orifices, the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

Dead pups and pups killed on day 13 post-partum were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.

Statistics:

Statistical analysis was performed using IBM SPSS Statistical Software (version 23). For statistical analysis, the litter was used as the basic sampling unit. Following statistical methods were used to analyse the various parameters:
One-way ANOVA followed by Dunnett's test (non-parametric): Live/dead foetuses (%)
Kruskal –Wallis followed by Mann– Whitney test (non-parametric): Number of male/female pups, Sex ratio (M), Number of live/dead foetuses
Chi-Square / Fisher test (non-parametric): Number of pregnant / non-pregnant females, Number of live / dead females
One-Way Anova followed by Dunnett's test (parametric): Foetal body weight, Foetal anogenital distance (AGD), Nipple retentions
The results of these statistical analysis were assessed at 5% level of significance (p = 0.05) and designated as significantly higher (S+) / lower (S-) than control values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Some incidental and spontaneous lesions observed in animals from the control and high dose group (1000 mg/kg) comprised of cortical vacuolation in adrenals; sub-mucosal lymphocytic infiltration in rectum; dilated glands and cystic glands in stomach and ultimobranchial cyst in thyroid. All these changes were of minimal severity and are commonly observed in rats of this age and were not considered to be treatment related.

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The group mean values of the number of oestrous cycles during the two weeks before the mating period was 2.3, 2.2 and 1.8 among the females exposed to test item at the dose levels of 250 mg/kg, 500 mg/kg and 1000 mg/kg respectively. These values were found to be comparable to that (2.8) in the control group.
The length of oestrus cycle was measured in days starting from detection of oestrous stage of the cycle till the oestrous stage of the subsequent cycle. The values of group mean length, in days, of each of the above estrous cycle occurring for two weeks before the mating period were 4.4, 4.5 and 4.2 among the females exposed to test item at the dose levels of 250 mg/kg, 500 mg/kg and 1000 mg/kg respectively and were found to be comparable to that (4.0 days) in the control group. Thus the test item did not have any adverse effect on the oestrous cycle during the treatment period.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The pregnancy rate was 80% in control and 80%, 80%, 66.6% in 250, 500 and 1000 mg/kg dose groups respectively. There was no treatment related adverse effect on pregnancy rate.
A comparison of the incidence of various pregnancy related parameters of the control group rats and those treated with test item did not indicate any remarkable differences indicative of any adverse effects due to the treatment. These parameters included incidence of maternal deaths during pregnancy, pregnancy rate (%), mean gestational length and post implantation loss.

The mean gestational length (duration of pregnancy in days) computed for groups G1 to G4 for all dams was 21.3 ± 0.5 days, 21.9 ± 0.5 days, 21.9 ± 0.6 days and 22.3 ± 0.5 days respectively. The values do not differ from each other significantly (p>0.05).

The gestation index was 100% for 250 and 500 mg/kg dose groups where as its was 94.4% in 1000 mg/kg dose group. The gestation index was 100% for control group. This index measures the female’s ability to maintain pregnancy, based on having delivered at least one live pup. The values do not differ from each other significantly (p>0.05). This indicated that the test item do not influence the gestation index.

The total number of implants in control group dams was 121 whereas it was 152, 140 and 104 in treated group G2, G3 and G4 respectively. The group mean values of the total number of implants per female were 10.1 (Control), 12.7 (G2), 11.7 (G3) and 10.4 in group G4.

There was no any post implantation loss in control as well as any of the treated groups

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

neuropathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Treatment of dams with Reaction Mixture of MnEDTA, MnDTPA and MnHEEDTA did not have any adverse effect on the survival of the offspring during lactation period. The incidence of litters with still-born pups, pups found dead in cage or cannibalized pups were very small and/or comparable across the treated and the control groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The values of mean litter body weights and mean litter body weight gain for the offspring of female rats treated with Reaction Mixture of MnEDTA, MnDTPA and MnHEEDTA did not differ significantly (p>0.05) from those of the concurrent control group females during the post-natal lactation period except in high dose group pups. However, at one time point on day 2 post-natal lactation period, there was a significant lowering (p<0.05) of body weight gain. Since, the same group pups had gained weight at all other time points up to 13 days which was comparable statistically to that of control animals and hence of no toxicological significance.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Pooled serum samples collected from one or two of the surplus pups per litter on day 4 after birth were assessed for total T4 while individual serum samples collected from one or two of the surplus pups per litter on day 13 of lactation were assessed for total T4.
The test item, at and up to the dose level of 1000 mg/kg of body weight, did not induce any changes in the total T4 on day 4 after birth and also on day 13 of lactation.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Average absolute weights of thyroid gland from day 13 pups of treatment group i.e., G2 (0.0084g), G3 (0.0076 g) and G4 (0.0088 g) were found to be comparable with the control group (0.0088g).

Gross pathological findings:

no effects observed

Description (incidence and severity):

The incidence of normal foetuses and litters observed in this study was 100% in control group and in all treatment groups. There were no any external abnormalities observed in external genitalia or any other organs of pups.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The AGD (Anogenital distance) of each pup was measured daily from the postnatal day (PND) 0 to
PND 4. The anogenital distance was recorded in millimeters.
Treatment of dams with Reaction Mixture of MnEDTA, MnDTPA and MnHEEDTA did not have any adverse effect on the anogenital distance for male and female pups as the average of the anogenital distance (mm) was found to be comparable to that of control male and female pups from the PND 0 to PND 4.

The number of nipples / areolae in male pups was counted on PND 12. Among all male pups from
control and treated groups, there was no retention of nipple in any male pup.
Thus, the test item did not influence the nipple retention in male pups at post natal day 12.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

clinical biochemistry

gross pathology

other: anogenital distance, nipple retention

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Groups of ten male and fifteen female Wistar rats were administered with the test item Reaction Mixture of MnEDTA, MnDTPA and MnHEEDTA daily by oral gavage at the doses of 250, 500 and 1000 mg/kg of body weight to evaluate its systemic toxicity and effects on reproduction and development. Males were dosed for a period of four weeks and females were dosed for 50 to 60 days (this included two weeks prior to mating, the variable time to conception, the duration of pregnancy and thirteen days after delivery). The findings of this study were as follows:
* no treatment related deaths; no neurotoxic potential;
* no clinical abnormalities;
* no effect on average body weight and body weight gain during premating, mating, gestation and lactation period in females and no effect on average body weight and body weight gain during premating and mating period in males.
* no effect on the average daily food intake in treated rats;
* no effects on the haematological parameters;
* no effects on the blood chemistry parameters; no effects on total T4 levels;
* no alterations in the absolute and relative organ weights;
* no remarkable gross pathological alterations in tissues/organs;
* no histopathological alterations, suggestive of systemic toxicity, at 1000 mg/kg dose level;
* no effects on organ weight data, gross and histopathological alterations in reproductive organs;
* no effects on reproductive performance, gestation, parturition, lactation and litter data.

Based on the findings of this study, it is concluded that, the No-Observed-Adverse-Effect-Level (NOAEL) of Reaction Mixture of MnEDTA, MnDTPA and MnHEEDTA in Wistar rats, following oral administration for a period of four weeks for males and 50 to 60 days for females for systemic toxicity and for reproductive and developmental toxicity was found to be equal to or greater than 1000 mg/kg body weight.

Executive summary:

‘Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening test of Reaction Mixture of MnEDTA, MnDTPA and MnHEEDTA in Wistar rat’ was performed in compliance with the Organization for Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals (No. 422, Section 4: Health Effects) “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test”, adopted by the council on 29 July 2016 and in accordance with the mutually agreed Study Plan (No. P/17190/CRRDT/17).

Groups of ten male and fifteen female Wistar rats were administered with the test item Reaction Mixture of MnEDTA, MnDTPA and MnHEEDTA daily by oral gavage at the doses of 250, 500 and 1000 mg/kg of body weight. A concurrent control group of ten males and fifteen females receiving the vehicle, i.e. analytical grade water at 5 ml/kg was also maintained. Males were dosed for a period of four weeks, up to and including the day before scheduled sacrifice. Females were dosed (50 to 60 days) throughout the study. This included two weeks prior to mating, the variable time to conception, the duration of pregnancy and thirteen days after delivery, up to and including the day before scheduled sacrifice.

The rats were examined daily for signs of toxicity, morbidity and mortality. They were subjected to detailed clinical examination before initiation of the study and weekly thereafter during the treatment period and at termination.Animals were additionally examined (five males and five females per group) for assessment of sensory reactivity, assessment of grip strength and motor activity. In males, these functional tests were made towards the end of their dosing period and in females during the last week of lactation (i.e. Lactation day 6-13). Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During pregnancy, females were weighed on days 0, 7, 14, 20 and then within 24 hours of parturition (day 1 post-partum), at day 4 and day 13 post-partum. During pre- mating, food consumption was measured weekly (on days 0, 7 and 14). During pregnancy it was measured on gestation day 0, 7, 14 and 20 and during lactation on days 4 and 13. Vaginal smears of all females were monitored daily during pretreatment, premating treatment period and during mating period until evidence of mating. After parturition, each litter was examined for number and sex of pups, still births, live births, runts and the presence of gross abnormalities. Litters were weighed within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 and day 13 post- partum. The anogenital distance of each pup was measured daily on the postnatal day (PND) 0 to PND 4. Pup body weight was recorded daily on PND 0 to PND 4 and at termination on day 13. The number of nipples / areolae in male pups were counted on PND 12. Total T4 assessments were carried out on few pups on day 4 and day 13 after birth.

Laboratory investigations were performed on male and female rats at baseline, at completion of premating period and at termination. All animals sacrificed terminally (males on day 29 and females at lactation day 14) were subjected to a detailed necropsy and weights of testes, epididymides, prostate, seminal vesicles with coagulating glands as a whole, levator ani plus bulbocavernous muscle complex, Cowper’s glands, glans penis of all adult males, the ovaries and uterus with cervix of all adult females were recorded.

In addition weights of liver, kidneys, adrenals, thymus, spleen, brain and heart from five adult males and females, randomly selected from each group were taken.

Thyroid gland from all adult male and female rats, from one male and one female pups of day 13 from each litter were also weighed. Histopathological evaluation was performed on all tissues in five male and five female rats from the control and high dose groups.

There was no incidence of any treatment related mortality amongst the rats treated with the Reaction Mixture of MnEDTA, MnDTPA and MnHEEDTA at any of the dose levels in both males and females. Treatment with Reaction Mixture of MnEDTA, MnDTPA and MnHEEDTA for males (28 days) and for females (50 to 60 days involving pregnancy and lactation period) did not induce any adverse clinical signs at any dose levels. No abortion/premature deliveries were observed during the study period.

The functional observations (neurological examinations) did not reveal any remarkable and treatment related incidence of neurological abnormalities. Also no findings, indicative of a neurotoxic potential of the test item, were encountered during these examinations.

Reaction Mixture of MnEDTA, MnDTPA and MnHEEDTA at and up to the dose of 1000 mg/kg dose group did not have any remarkable and adverse effects on the weight gain by the treated male and female rats in this study. The values of mean body weights and mean body weight gain for rats treated with test item at and up to 1000 mg/kg did not differ significantly (p>0.05) from those of the concurrent control group rats during premating, gestation period and the post-natal lactation period.

The test item did not have any adverse effect on the average daily food consumption by the male and female rats treated at any of the dose levels. The haematological and clinical chemistry parameters of male and female rats treated with the test item were found to be comparable to those of baseline values and also comparable to the control animals. The values of absolute and relative organ weights of male and female rats treated with test item were found to be comparable with those of the control group rats at the end of treatment period.

No treatment related gross pathological changes were noted. Histopathological examination was performed on tissues of the control and high dose group animals, where the changes in the high dose group were incidental or comparable to the control group or unrelated to treatment.

No evidence of immunological effect was observed.

The reproductive organ weight data, gross and histopathological examination of reproductive organs did not reveal any treatment related changes. Data regarding development and reproduction indicated no difference between the animals treated with the test item and the vehicle control.

Based on the findings of this study, it is concluded that, the No-Observed-Adverse-Effect-Level (NOAEL) of Reaction Mixture of MnEDTA, MnDTPA and MnHEEDTA in Wistar rats, following oral administration for a period of four weeks for males and 50 to 60 days for females for systemic toxicity and for reproductive and developmental toxicity was found to be equal to or greater than 1000 mg/kg body weight.