Magnesium glycerophosphate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 11-2018 to March 20-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted on July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Dose levels for this study were selected based on Dose Range Finding study (RCC Study No. 138) observation and results
Three dose groups are included in this study i.e. 100, 300 and 1000 mg/kg body weight.

Test material

Test material information

Constituent 1

Reference substance name:

Magnesium glycerophosphate

EC Number:

213-149-3

EC Name:

Magnesium glycerophosphate

Cas Number:

927-20-8

Molecular formula:

C3H9O6P.Mg

IUPAC Name:

magnesium glycerophosphate

Test material form:

solid: particulate/powder

Details on test material:

Batch: INVG003917

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:INVG003917
- Expiration date of the lot/batch:June 10, 2021




STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (20 to 30°C)
- Stability under test conditions: stable at 0 and 6 hours
- Solubility and stability of the test substance in the solvent/vehicle:Corn oil
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: NO

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Laboratories India Private Limited
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age on first Treatment: 13-14 Weeks
- Body weight when treated:
Males - 280.7 to 321.3 g
Females - 210.4 to 250.7 g
- Fasting period before study: Not Applicable
- Housing: In groups of three and two animals of same sex per cage in Polycarbonate cages (Approximate internal dimensions of 365 mm x 202 mm x 180 mm height) with paddy husk bedding.
- Diet (e.g. ad libitum): Teklad Certified Global 14% Protein Rodent Maintanence Diet. (Lot No. – 2014C-051418MA) was provided ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 21 days under laboratory conditions, after veterinary examination. only animals without any visible signs of illness were used for the study

DETAILS OF FOOD AND WATER QUALITY:
Food and water is checked for proximate and contaminant analysis. the reports were archived at RCC India

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8°C to 23.7°C
- Humidity (%): 57 to 66%
- Air changes (per hr): >10
- Photoperiod (hrs dark / hrs light):12 hours light :12hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): CORN OIL
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:

Details on mating procedure:

- M/F ratio per cage:
- Length of cohabitation: 9
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

• The linear relationship between actual magnesium content derived theoretically and determined by titrimetry was substantiated by regression coefficient (r2 = 0.9999)
• The test item was found to be homogeneously dispersed in corn oil, which was evinced from the mean recovery of test item from different layers (Top, middle and bottom layers of corn oil) was within 95% to 119% for the three different doses (20 mg/mL, 60 mg/mL and 200 mg/mL)
• The dose concentration verification was carried out for test item fortified in corn oil pertaining to low dose (20mg/mL) and high dose (200mg/mL). The mean recovery of 102-108% augments that the test item dose prepared in corn oil was within the guideline specification.

Duration of treatment / exposure:

57 DAYS

Frequency of treatment:

DAILY

Details on study schedule:

Study Initiation Date May 11, 2018
Experimental Starting Date June 08, 2018
Acclimatization Start Date June 08, 2018
Administration / Treatment
Males
Premating period
Mating & Post mating period
Females
Premating period
Mating period
Gestation period
Lactation period

June 29, 2018 to July 12, 2018
July 12, 2018 to July 30, 2018

June 29, 2018 to July 12, 2018
July 12, 2018 to July 21, 2018
July 13, 2018 to August 11, 2018
August 03, 2018 to August 25, 2018
Blood collection for T4 & TSH Male – July 30, 2018
Female – August 07, 2018 to August 25, 2018
Necropsy
Males July 30, 2018
Females August 17, 2018 to August 26, 2018
Pups August 16, 2018 to August 25, 2018
Experimental Completion Date October 30, 2018
Study Completion Date March 20, 2019

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Remarks:

LOW DOSE

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Remarks:

INTERMEDIATE DOSE

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

HIGH DOSE

No. of animals per sex per dose:

12 /SEX/DOSE

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were in terms of the test item. Dose levels for this study were selected based on Dose Range Finding study (RCC Study No. 138) observation and results. Hence as per the sponsor, three dose groups are included in this study i.e. 100, 300 and 1000 mg/kg body weight.

Examinations

Parental animals: Observations and examinations:

Reproductive Indices
Mating Performance and Fertility
No treatment-related effects were detected on mating performance. All the female animals of with vehicle control group (G1- 0 mg/kg body weight), low dose group (G2- 100 mg/kg body weight intermediate (G3- 300 mg/kg body weight) and high dose group (G4-1000 mg/kg body weight) showed positive evidence of mating. No significant difference was observed in the pre-coital interval of female animals between vehicle control (G1- 0 mg/kg body weight), low (G2- 100 mg/kg body weight), Intermediate (G3- 300 mg/kg body weight) and high dose group (G1- 1000 mg/kg body weight). Oestrous cyclicity was checked for all the female animals. Regular oestrous cycle was observed in all the animals of different groups in the study.
Mating index for all the groups was 100 % (as confirmed by presence of sperm in the vaginal smear).
The pregnancy index for control, low dose, intermediate dose and high dose groups were 83.33 %, 91.7 %, 83.3% and 75.0%, respectively. No test item related changes in litter born, litter size and subsequently on days 1 and 4 post partum were observed.
There were no treatment-related effects observed in fertility of treated animals in any of the treated groups.
Gestation and Parturition
There were no differences in gestation lengths. The gestation length for treated females was comparable to controls. All pregnant animals showed gestation lengths between 21 to 24 days.
The parturition index for vehicle control (G1- 0 mg/kg body weight), low dose (G2- 100 mg/kg body weight), intermediate dose (G3- 300 mg/kg body weight) and high dose (G4- 1000 mg/kg body weight) groups were 83.3 %, 91.7 %, 83.3 % and 66.7%, respectively. No significant change in parturition was observed and no test item related effects could be detected.
Litter Responses:
Pre Natal & Post Natal Loss
The pre and post natal loss in all treated groups were comparable control group.
Litter Size and Live Birth
Of the litters delivered, in all the treatment groups, litter size at birth and subsequently on Day 1 and 4 postpartum were comparable to controls.
No nipple retention was found in any of the male litter from vehicle control, low, intermediate and high dose groups.
Sex Ratio
Sex ratio was calculated as percent (%) male and female. No significant difference in sex ratio was observed in any of the treated groups when compared with control.
Offspring Growth and Development
No toxicological relevance effect on the growth of offspring was observed.
No significant differences and no test item related effects in the Anogenital distances (AGD) between control and treated animals were observed.

Oestrous cyclicity (parental animals):

Regular oestrous cycle was observed in all the animals of different groups in the study.

Sperm parameters (parental animals):

The spermatogenesis stages in the testes were found to be normal in all animals (except animal number 50 with gross and microscopic findings) which was considered incidental

Litter observations:

Litter Responses:
Pre Natal & Post Natal Loss
The pre and post natal loss in all treated groups were comparable control group.
Litter Size and Live Birth
Of the litters delivered, in all the treatment groups, litter size at birth and subsequently on Day 1 and 4 postpartum were comparable to controls.
No nipple retention was found in any of the male litter from vehicle control, low, intermediate and high dose groups.
Sex Ratio
Sex ratio was calculated as percent (%) male and female. No significant difference in sex ratio was observed in any of the treated groups when compared with control.
Offspring Growth and Development
No toxicological relevance effect on the growth of offspring was observed.
No significant differences and no test item related effects in the Anogenital distances (AGD) between control and treated animals were observed.

Reproductive indices:

TABULAR SUMMARY REPORT OF EFFECTS ON REPRODUCTION/DEVELOPMENT
Parameters Group 1 Group 2 Group 3 Group 4
Pairs started (N) 12 12 12 12
Females showing evidence of copulation (N) 12 12 12 12
Females achieving pregnancy (N) 10 11 10 9
Conceiving Days 1 - 5 (N) 12 11 11 10
Conceiving Days ≥ 6 (N) 0 1 1 2
Pregnancy = 20 Days (N) 0 0 0 0
Pregnancy = 21 Days (N) 3 2 1 1
Pregnancy = 22 Days (N) 3 5 5 2
Pregnancy = 23 Days (N) 2 4 3 4
Pregnancy = ≥ 23 Days (N) 2 0 1 1
Dams with live young born (N) 10 11 10 8
Dams with live young at Day 4 pp (N) 10 11 10 8
Implants/dam (mean) 9.4 10.1 10.5 8.6
Live pups/dam at birth (mean) 8.9 9.7 10.1 8.2
Live pups/dam at Day 4 (mean) 8.9 9.6 10.1 8.2
Sex ratio % males at Day 1 post partum 54.8 56.5 58.3 49.4
Sex ratio % males at Day 4 post partum 54.8 56.5 58.3 49.4
Pup weight at Day 1 -Combined (g, mean) 5.46 5.37 5.71 5.60
Pup weight at Day 4 - Combined (g, mean) 7.58 7.99 8.49 8.26
Pup weight at Day 1 Male (g, mean) 5.43 5.34 5.66 5.61
Pup weight at Day 4 Male ((g, mean) 7.59 7.84 8.55 8.25
Pup weight at Day 1 Female (g, mean) 5.51 5.41 5.77 5.59
Pup weight at Day 4 Female (g, mean) 7.56 8.21 8.41 8.26
Male with nipple retention on day 13 0 0 0 0
ABNORMAL PUPS (Based on gross finding)
Dams with 0 (N) 0 0 0 0
Dams with 1 (N) 0 0 0 0
Dams with 2 (N) 0 0 0 0
Dams with more than 2 abnormal pups (N) 0 0 0 0

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no mortalities observed in vehicle control group (0 mg/kg BW), low dose group (100 mg/kg BW), intermediate dose group (300 mg/kg BW) and high dose group (1000 mg/kg BW) animals in pre-mating, gestation and lactationperiodin female with exception of one animal (Dam No.:94) of high dose group which was found dead due to dystocia. Single incidance of this finding was not considred to be treatment and substance related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No significant changes in body weights and body weight gains (%) of males were observed when compared with vehicle control group (G1- 0 mg/kg body weight).
In female, no significant changes were observed in body weights during pre-mating, gestation and lactation period with exception of significant increase in body weight on day 0 of gestation in intermediate dose group (G3-300 mg/kg body weight) when compared with vehicle control group (G1- 0 mg/kg body weight). Significant increase in body weight gain (%) was observed in pre-mating period in low (G2-100 mg/kg body weight), intermediate (G3-300 mg/kg body weight) and high dose group (G4-1000 mg/kg body weight) on day 7; and day 14 in intermediate dose (G3-300 mg/kg body weight) group when compared with vehicle control group (G1-0 mg/kg body weight).
The significance observed in body weights and body weight gains (%) are could not be attributed to treatment as there was no dose response was observed.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant changes in feed consumption of the male and female were observed during pre-mating, gestation and lactation period

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

no effects observed

Ophthalmological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No significant changes in the T4 and TSH levels in any of the treated group parental male and female ainimal when compared with control group.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Single incidence of degenerative changes in the seminiferous tubules in testes, aspermia observed in epididymis in animal number 50 (intermediate dose) and dilated uterus with edema in animal number 94 (high dose) were considered incidental findings

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

food efficiency

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No mortality in the offspring in any of the treated animals was observed with exception of one offspring (Dam No: 41) which was cannibalized on Day 0 of Lactation period. This was not considered substance related (single incidance) and no other clinical sign of toxicity was observed any of the treated animals.

Body weight and weight changes:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No significance changes were observed in T4 and TSH levels of male and female offspring at lactation period in any of the treated groups when compared to vehicle control group.

Gross pathological findings:

no effects observed

Effect levels (F1)

Key result

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:

clinical signs

mortality

clinical biochemistry

gross pathology

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

The administration of Magnesium glycerophosphate to Wistar rats by oral gavage, at dose levels of 0, 100, 300 and 1000 mg/kg body weight/day, resulted in no treatment-related effects on clinical signs, body weights, feed consumption in high dose group.
No treatment-related effects on reproduction/ development such as mating index, gestation length, pre-natal loss, post-natal loss, sex ratio and offspring growth and development.
Single incidence of the mortality was observed in single dam of high dose group (G4 - Dam No. 94). There was a female pup of dam no. 41 from low dose group (G2) cannibalized on day 0.
There were no treatment-related effects observed in fertility of treated animals. No significant difference in the live birth was noted between control and treated groups. T4 and TSH analysis of both parent and offspring revealed no significant and toxicological relevant changes between treated and control group observed
Necropsy performed at the end of the treatment, did not reveal any macroscopic findings in males, females and pups.
No test item related changes were observed in organ weights and there were no macroscopic findings observed in any of the male and female animals and pups of low (G2), mid (G3) and high dose (G4) groups.
The test item Magnesium glycerophosphate did not produce any test item related microscopic changes in testes, epididymides and ovaries at the highest dose level tested (1000 mg/kg body weight).
No Observed Adverse Effect Level (NOAEL) for Reproduction / Developmental Toxicity Screening Test : 1000 mg/kg body weight

Executive summary:

This study was designed to investigate the potential adverse effects of the test item on male and female reproduction performance and is compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test", Adopted by the Council on 29^thJuly 2016”. The test item (formulated in Corn Oil) was administered by oral gavage to three groups, each of twelve male and twelve female wistar rats. Males were treated for up to 30 days, which includes pre-mating, mating and post-mating period. Females were treated for two weeks of pre-mating, mating period (1-9 days), three weeks gestation and 13 days lactation of dams, at dose levels of 100, 300 and 1000 mg/kg body weight/day. A control group of twelve males and twelve females were treated with vehicle (Corn Oil).The dose volume was 5 mL/kg body weight. Detailed histological examination was performed on testes using PAS-H stain with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure and epididymides of male animals and ovaries of female animals in order to identify treatment related effects.

RESULTS

Mortality and Clinical Signs:

There were no mortalities observed in vehicle control group (G1-0 mg/kg body weight), low dose group (G2-100 mg/kg body weight), Intermediate dose group (G3-300 mg/kg body weight) and high dose group (G4-1000 mg/kg body weight) animals in pre-mating, mating, gestation and lactation period in female animals with exception of one female animal (Dam No: 94) of high dose group (G4-1000 mg/kg body weight) which was found dead due to dystocia.Single incidence of this finding was not considered related to treatment. No mortality in the offspring in any of the treated animals was observed with exception of one offspring (Dam No: 41) which was cannibalized on Day 0 of Lactation period. Thiswas not considered treatment related and no other clinical sign of toxicity was observed any of the treated animals.

 

 

Feed Consumption:

No significant changes in feed consumption of the male and female were observed during pre-mating, gestation and lactation period.

Body Weight:

No significant and/or toxicologiccal relevant changes in body weights and body weight gains (%) of males and females were observed when compared with vehicle control group (G1- 0 mg/kg body weight).

Organ weight (%)

No significant difference in absolute and relative organ weight of males was observed in any of the dose groups tested when compared to vehicle control group animals.

No significant difference in absolute and relative organ weight of females was observed except increase in relative uterine weight in high dose when compared to vehicle control, low dose and intermediate group. In pups, no significant difference was observed in relative and absolute organ weight in both the sexes.

Reproductive Indices

Mating Performance and Fertility

No treatment-related effects were detected on mating performance. All the female animals of with vehicle control group (G1- 0 mg/kg body weight), low dose group (G2- 100 mg/kg body weight intermediate (G3- 300 mg/kg body weight) and high dose group (G4-1000 mg/kg body weight) showed positive evidence of mating. No significant difference was observed in the pre-coital interval of female animals between treated nad control animals. Oestrous cyclicity was checked for all the female animals. Regular oestrous cycle was observed in all the animals of different groups in the study. Mating index for all the groups was 100 % (as confirmed by presence of sperm in the vaginal smear). There was no siginifacant and relevant changes in the pregnancy index of treated and control animals. No test item related changes in litter born, litter size and subsequently on days 1 and 4 post partum were observed.

There were no treatment-related effects observed in fertility of treated animals in any of the treated groups.

Gestation and Parturition

There were no differences in gestation lengths. The gestation length for treated females was comparable to controls. All pregnant animals showed gestation lengths between 21 to 24 days. No significant change in parturition was observed and no test item related effects could be detected.

Litter Responses:

Pre Natal & Post Natal Loss

The pre and post natal loss in all treated groups were comparable control group. Of the litters delivered, in all the treatment groups, litter size at birth and subsequently on Day 1 and 4 postpartum were comparable to controls. No nipple retention was found in any of the male litter from vehicle control, low, intermediate and high dose groups. Sex ratio was calculated as percent (%) male and female. No significant difference in sex ratio was observed in any of the treated groups when compared with control. No toxicological relevance effect on the growth of offspring was observed. No significant differences and no test item related effects in theAnogenital distances (AGD) between control and treated animals were observed. No significance changes in the T4 and TSH levels in any of the treated group parental male and female animals were observed when compared with vehicle control group. No significance changes were observed in T4 and TSH levels of male and female offspring at lactation period in any of the treated groups when compared to vehicle control group. There were not reatment related microscopic findings in testes and epididymidesin males and in ovaries in females at high dose.

No Observed Adverse Effect Level (NOAEL) for Reproduction / Developmental Toxicity Screening Test       :       1000 mg/kg  body weight