Magnesium hydroxide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

No reproductive/developmental toxicity was observed in a repeated dose reproduction screening test conducted with the target substance magnesium hydroxide up to the highest dose of 1000 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-03-02 to 2010-09-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Name of the test material used in the report: Magnesium hydroxide
- Appearance: white powder
- Batch No.: 20BR0026
- Purity: 99.90%
- Storage: at room temperature in the dark
- Expiry date: 2012-01-31

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
- Age at study initiation: Approximately 11 weeks
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18cm)
Mating: Females were caged together with males on a one-to-one basis in Macrolon cages(MIII type, height 18cm)
Post-mating: Males were housed in their home cage ( Macrolon cages, MIV type, height 18cm) with a maximum of 5 animals per cage. Females were individually housed in Macrolon cages ( MIII type, height 18cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages ( MIII type, height 18cm)
General: Sterilised sawdust as bedding material and paper as cage enrichment. During activity monitoring animals were housed individually in Macrolon cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cage-enrichment was provided during activity monitoring.
- Diet: Free access to pelleted rodent diet.
- Water: Free access to tap water.
- Acclimation period: At least 5 days prior to the start of treatment.

ENVIRONMENTAL CONDITIONS
Animals were housed in a controlled environment.
- Temperature (°C): 21 ±3°C (actual range: 19.7-21.7°C)
- Humidity (%): A relative humidity of 40-70% (actual range: 34-73%)
- Air changes (per hr): 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day. Temporary fluctuations from the light/dark cycle ( with a maximum of 1 hour) occured due to performance of pupillary tests and/or opthalmoscopic examinations in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Route=oral

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at Notox and on information provided my the sponsor.

PREPARATION OF DOSING SOLUTIONS:
- Formulations were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level.
-Storage conditions: At ambient temperature.
-Method: Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
CHEMICAL ANALYSIS OF DOSE PREPARATIONS:
- Analyses were conducted during the treatment phase, according to a validated method. Samples of formulations were analysed for homogeneity and accuracy of preparation. The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was < 10%..

Details on mating procedure:

- M/F ratio per cage: Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating.
- Length of cohabitation: Once mating had occurred, the males and females were separated.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged: Females were individually housed in Macrolon cages (MIII type, height 18cm). the females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes,placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Accuracy and homogeneity were determined for formulations prepared for use during treatment.
-Duplicate samples (approx 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasksof 50ml. For determination of accuracy, samples were taken at 50% height or at 90%, 50% and 10% height. The latter set of samples was also used for the determination of the homogeneity of the samples.
The volumetric flasks were filled up to the mark with 4% aqueous HNO3. The solutions were further diluted with 4% aqueous HNO3 to obtain concentrations within the calibration range.

Duration of treatment / exposure:

EXPOSURE PERIOD
-Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Females 41, 46, 48, 49 (group 1, table 1), 53,59 (group 2, table 1), 61, 62, 68 (group3, table 1) and 76 ( group 4, table 1) were not dosed during littering.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Details on study schedule:

Male number paired with, mating date, confirmation of pregnancy, and delivery date were all recorded. Pregnant females were all examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth. Any 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 examined.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Dose / conc.:

110 mg/kg bw/day

Remarks:

Low dose

Dose / conc.:

330 mg/kg bw/day

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose

No. of animals per sex per dose:

Four groups of ten male and ten female Wistar (Hans) rats were exposed by oral gavage to the test substance.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on the results of a 10-day dose range finding study.
- Rationale for animal selection: This species and strain of rat has been recognised as appropriate for general and reproduction toxicity studies.

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily detailed clinical observations were made in all animals immediately after dosing. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating , or housed individually.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 17 and 20 post-coitum and during lactation on days 1 and 4.

FOOD CONSUMPTION: Yes
- Food consumption: Weekly, except for males and females which were housed together for mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

FUNCTIONAL OBSERVATIONS:
The following tests were performed on the selected 5 animals/sex/group:
-hearing ability, pupillary reflex, static righting reflex and grip strength.
-motor activity test.
During the motor activity test, males were caged individually and females were caged with their pups. The selected males were tested during week 4 of treatment and the selected females were tested during lactation. In order to avoid hypothermia of pups, dams were removed from the pups for not more than 30-40 minutes.

Sperm parameters (parental animals):

Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.

Litter observations:

STANDARDISATION OF LITTERS
- Each litter was examined.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
Mortality/ Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined.
Clinical signs: At least once daily, detailed clinical observations were made in all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: sex was determined for all pups on Days 1 and 4 of lactation.

GROSS EXAMINATION OF DEAD PUPS:
Yes, if possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: The day of necropsy for all survivng males was following completion of the mating period ( 29 days of dose administration)
- Maternal animals: The day of necropsy for females which delivered was lactation days 5-6.
GROSS NECROPSY
- All animals were subjected to macroscopic examination (including examination of the body surface, orifices and cranial, thoracic and abdominal tissues and organs and their contents), with special attentionbeing paid to reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [3] were prepared for microscopic examination and weighed, respectively.
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4( table 2)
-The additional slides of the testes of the selcted 5 males of Groups 1 and 4 to examine staging of spermatogenesis ( table 2)
-All gross lesions of all animals

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed by decapitation on lactation Day 5 or 6.
All pups were sexed and descriptions of all external abnormalities were recorded. The sromach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

Statistics:

For each group the following calculations were performed:

Mating (%) - (Number of females mated/Number of females paired) x 100

Fertility Index (%) – (Number of pregnant females/Number of females paired) x 100

Conception index (%) – (Number of pregnant females/Number of females mated) x 100

Gestation index (%) – (Number of females bearing live pups/ Number of pregnant females) x 100

Duration of gestation – Number of days between confirmation of mating and the beginning of parturition.

Percentage live males at first litter check – (Number of live male pups at first litter check/ Number of live pups at First Litter Check) x 100

Percentage live females at First Litter Check – (Number of live female pups at First Litter Check/ Number of live pups at First Litter Check) x 100

Percentage of postnatal loss Days 0-4 of lactation – (Number of dead pups on Day 4 of lactation/ Number of live pups at First Litter Check) x 100

Viability index (%) – Number of live pups on Day 4 of lactation/ Number of pups born alive) x 100

The following statistical methods were used to analyse the data:
-If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
-The steel-test was applied if the data could not be assumed to follow a normal distribution.
-The Fisher Exact-test was applied to frequency data.
All test were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were noted during the observation period. Incidental findings that were noted in single females included alopecia or piloerection. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after allowance for body weight was similiar between treated and control animals.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes occurred in haematological parameters of treated rats. Any statistically significant changes were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes in clinical biochemistry parameters distinguished treated animals from control animals:
-lower total protein levels in males at 330 and 1000 mg/kg,
-lower albumin levels in males at 1000 mg/kg,
-lower calcium levels in males at 330 and 1000 mg/kg.

Means of these changes only just exceeded or remained within the range considered normal for rats of this age and strain.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes in urinary parameters distinguished treated males from control males:
- Lower sodium excretion (mmol/TPV) at 330 and 1000 mg/kg,
- Lower potassium excretion (mmol/TPV) at 1000 mg/kg,
- Higher calcium concentration (mmol/L) at 1000 mg/kg.
Means of these changes only just exceeded or remained within the range considered normal for rats of this age and strain. The significant higher specific gravity seen at 330 mg/kg was not considered to be toxicologically relevant as it occurred in the absence of a dose-related trend.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment related microscopic findings.
Recorded microscopic findings were within the range of background pathology encountered in Wistar (Han) rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats. The spermatogenic staging profiles were normal for all group 1 and group 4 males evaluated.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

REPRODUCTIVE PERFORMANCE: No toxicologically relevant effects on gestation index and duration, parturation, maternal care and early postnatal pup development ( mortality, clinical signs, body weight and macroscopy) were observed.
The gestation index was 100% for all groups and the duration of gestation was similar between control and treated groups.
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.

Key result

Dose descriptor:

NOAEL

Remarks:

No observed adverse effect level

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproduction/developmental toxicity was observed at any dose level

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

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:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Remarks on result:

not measured/tested

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental clinical symptoms of pups consisted of blue spot on the back and scabbing of the snout or back. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Two pups of the control group, three pups at 110 mg/kg, and one pup at 330 mg/kg were found dead or missing during lactation. The missing pups were most likely cannibalised. No pups were found dead or missing at 1000 mg/kg. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were considered to have been unaffected by treatment.

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:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental macroscopic findings for pups that were found dead included autolysis and absence of milk in the stomach. Scabbing on the snout was noted for one surviving pup. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Histopathological findings:

no effects observed

Other effects:

not examined

Description (incidence and severity):

The number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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:

other: no developmental toxicity was observed at any dose level

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

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:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

Table 4: Clinical signs Males parental animals:

		Pre-Mating		Reproduction period
Sign (Max Grade)	Week	1	2	1	2	3	4
Location	day	1,2,3,4,5,6,7	1,2,3,4,5,6,7	1,2,3,4,5,6,7	1,2,3,4,5,6,7	1,2,3,4,5,6,7	1,2,3,4,5,6,7,1,2,3
Group 1 (control) No clinical signs noted		-	-	-	-	-	-
Group 2 (110 mg/kg) No clinical signs noted		-	-	-	-	-	-
Group 3 (330 mg/kg) No clinical signs noted		-	-	-	-	-	-
Group 4 (1000 mg/kg) No clinical signs noted		-	-	-	-	-	-

 

Table 5: Clinical signs Females parental animals:

		Pre-Mating		Reproduction Period
Sign (Max Grade)	Week	1	2	1	2	3	4
Location	Day	1,2,3,4,5,6,7	1,2,3,4,5,6,7	1,2,3,4,5,6,7	1,2,3,4,5,6,7	1,2,3,4,5,6,7	1,2,3,4,5,6,7,1,2,3
Group 1 (control) No clinical signs noted		-	-	-	-	-	-
Group 2 (110 mg/kg) Skin/ fur  Alopecia	G:   %:	-   -	-   -	-   -	1,1,1,1,1,1,1   1,1,1,1,1,1,1	1,1,1,1,1,1,1   1,1,1,1,1,1,1	1,1,1,1,1,1,1   1,1,1,1,1,1,1
Group 3 (330 mg/kg) Skin/fur  Piloerection	G:   %:	-   -	-   -	1   1	-   -	-   -	-   -
Group 4 (1000 mg/kg) No clinical signs noted		-	-	-	-	-	-

Key:

G: Median value of the highest individual daily grades

%: Percent of affected animals (0= less than 5%, 1= between 5% and 15%, A= more than 95%)

- observation performed, no sign detected

Table 6: Reproduction Data

	Group 1 (Control)	Group 2 (110 mg/kg)	Group 3 (330 mg/kg)	Group 4 (1000 mg/kg)
Females paired	10	10	10	10
Females Mated	10	10	10	10
Non-pregnant	0	0	0	0
Pregnant females	10	10	10	10
Females with living pups on Day 1	10	10	10	10
Mating Index (%) (females mated/females paired) * 100	100.0	100.0	100.0	100.0
Fertility Index (%) (Pregnant females/females paired) * 100	100.0	100.0	100.0	100.0
Conception index (%) (Pregnant females/females mated) * 100	100.0	100.0	100.0	100.0
Gestation index (%) (Females with living pups on Day 1/pregnant females) * 100	100.0	100.0	100.0	100.0

 

Table 7: Body weights (gram) Summary

Parental Males/Females:

		Group 1 Control		Group 2 110 mg/kg		Group 3 330 mg/kg		Group 4 1000 mg/kg
		Male	Female	Male	Female	Male	Female	Male	Female
Pre-mating
Day 1	Mean	314	180	312	175	319	177	317	180
Week 1	St.Dev	19.8	7.9	17.3	4.9	22.6	4.5	29.2	11.2
	N	10	10	10	10	10	10	10	10
Day 8	Mean	337	190	334	186	342	186	337	188
Week 2	St.Dev	23.1	12.0	17.7	6.6	22.7	7.2	28.1	8.1
	N	10	10	10	10	10	10	10	10
Mating period
Day 1	Mean	353	195	346	193	356	192	351	195
Week 1	St.Dev	29.0	12.2	19.1	7.5	24.9	6.5	30.1	7.6
	N	10	10	10	10	10	10	10	10
Day 8	Mean	365		354		365		356
Week 2	St.Dev	33.1		21.7		25.1		32.5
	N	10		10		10		10
Day 15	Mean	385		368		381		376
Week 3	St.Dev	34.2		22.1		26.9		34.7
	N	10		10		10		10

 

Table 8: Food consumption (G/Animal/Day) Males/Females Parental:

		Group 1 Control		Group 2 110 mg/kg		Group 3 330 mg/kg		Group 4 1000 mg/kg
		Males	Females	Males	Females	Males	Females	Males	Females
Pre mating
Days 1-8	Mean	23	14	23	14	23	14	22	14
Weeks 1-2	St.Dev	0.0	0.4	1.9	0.2	0.2	0.3	0.5	0.3
	N (cage)	2	2	2	2	2	2	2	2
Days 8-15	Mean	24	15	23	16	24	15	24	15
Weeks 2-3	St.Dev	0.0	0.4	1.2	0.6	0.6	0.1	0.0	1.3
	N (cage)	2	2	2	2	2	2	2	2
Mean f Means over pre mating: mean		24	15	23	15	24	14	23	15
Mating Period
Days 1-8	Mean	26	-	26		26		27
Weeks 1-2	St.Dev	0.8	-	3.2		0.9		0.3
	N (cage)	2	0	2		2		2
Days 8-15	Mean	24		23		24		25
Weeks 2-3	St.Dev	0.3		1.3		1.2		0.4
	N (cage)	2		2		2		2
Mean of means over mating period: mean		25		25		25		26

Conclusions:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test no adverse effects were found after oral administration of magnesium hydroxide in male and female Wistar rats and in the male and female pups. Based on the results, a NOAEL of 1000 mg/kg/day was determined.

Executive summary:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422), the test item magnesium hydroxide (99.9% purity) was administered orally to 10 male and 10 female Wistar rats/dose in water by gavage at dose levels of 0, 110, 330 and 1000 mg/kg bw/day. The animals were treated daily with the test item formulation on 7 days per week.

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Some females were not dosed during littering.

A number of clinical biochemistry and urinary changes were noted at 330 and 1000 mg/kg in males which included lower total protein, albumin and calcium levels in blood, and lower sodium and potassium excretion and higher calcium concentration in urine. Means of these changes only just exceeded or remained within the range considered normal for rats of this age and strain. Moreover, there were no histopathological correlates in eg. liver or kidneys that would support these changes. Therefore, these changes were considered not to be of toxicological relevance.

Overall, no toxicologically relevant changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).Based on these results, a NOAEL of 1000 mg/kg/day, the highest dose administered in this study, was determined.

 

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP guideline study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a combined repeated dose reproductive toxicity screening tests conducted according to OECD No. 422 and GLP no effects on male and female fertility were observed up to the highest dose tested of 1000 mg/kg bw/day. In addition, the absence of effects of magnesium ions on the reproductive organs of males and females was also reported in 90-day dietary studies in rats and mice with magnesium chloride hexahydrate up to the highest dose levels tested.

Magnesium hydroxide is a solid and in general, solids need to be dissolved before they can be absorbed and become bioavailable. However, the water solubility of magnesium hydroxide is relatively low (~ 2 mg/L). The water solubility approximates the bioavailability of a substance. In addition, magnesium salts are generally absorbed so slowly that their oral administration only results in a bioavailability of 30-50 % [Stendig-Lindberg G., 1991 (see IUCLID section 7.1)]. When administered orally, Mg(OH)2 dissociates to magnesium cations (Mg2+). About 5–15% of the dissociated Mg2+cations are absorbed from the gastrointestinal tract (through the epithelial lining of the small intestine).

Magnesium (Mg2+) is the fourth most abundant cation in the human body. The average adult's body contains about 24 g of magnesium and the normal range of magnesium in human serum is 1.5–2.5 mEq/L (18.2–30.3 mg/L) and about two-third of magnesium is present as free cation and one-third is bound to plasma proteins (EMA, 1995). The half-life of magnesium ions in humans after i.v. administration has been reported to be 4 h. Magnesium is mainly excreted by the kidney and under normal conditions, 3-5% of the filtered ion is very quickly excreted in the urine. Magnesium clearance increases roughly linearly with the concentration of magnesium in the serum. In addition, small amounts of magnesium are also excreted in milk and saliva [Behmer et al., 1990; Lu et al., 2000; Massry et al., 1969 (see IUCLID section 7.1); EMA, 1995].

Furthermore, external human exposure to magnesium can occur via its natural presence in food, for example in fruits, vegetables, meats or seafoods. Based on the available information on concentrations of magnesium in some of these products, the European Medical Agency (EMA) reported in its review (EMA 1995), that 20-40 mEq (0.25 -0.5 g) magnesium is ingested daily in typical diet. Also, in 2015, following a request from the European Commission, an European Food Safety Agency (EFSA) expert group on Dietetic Products, Nutrition and Allergies (NDA) defined Adequate Intakes (AIs) for magnesium based on observed magnesium intakes in healthy populations in the European Union (EU). For adults, an AI for magnesium was set at 350 mg/day for men and 300 mg/day for women. Magnesium hydroxide is also used as food additive, as such it is authorized in Europe (E528) and categorized by the U.S. Food and Drug Administration (FDA) as a GRAS (generally recognized as safe) food ingredient. It is also approved for use as a nutritional supplement and a pH-control agent in foods.

Based on the above-mentioned observations and due to the role of magnesium as a common and natural food ingredient, and ubiquitous metabolic product/substrate in mammals with proven low toxicity, it can safely be expected that magnesium hydroxide would not contribute to any systemic effects. Furthermore, these observations are common textbook knowledge and hence can be considered as adequately and reliably documented. Thus, and in accordance with REACH Annex XI, section 1.1 it is scientifically not justified to conduct an extended-one generation reproductive toxicity study in rats with the target substance magnesium hydroxide.

Effects on developmental toxicity

Description of key information

A developmental toxicity study in rats with magnesium chloride hexahydrate at dose levels that just did not lead to maternal toxicity (as concluded from a dose range finding study) did not show any test substance developmental toxicity. Thus, in this study, a NOAEL of >800 mg/kg bw/day was determined, which is equal to 230 mg/kg bw/day magnesium hydroxide.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

For details and justification of read-across please refer to the attached report in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Species:

rat

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 specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not specified

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

There were no differences between the groups with regard to general condition and death. Furthermore, no significant differences between the control group and magnesium chloride hexahydrate groups were observed with respect to body weight and food consumption. Regarding the number of corpora lutea, number of implants, number of living fetuses, sex ratio, fetal weight and mortality of implants/fetuses, no significant differences between control group and the magnesium chloride hexahydrate groups were observed.

Key result

Dose descriptor:

NOAEL

Effect level:

> 800 mg/kg bw/day

Based on:

test mat.

Remarks on result:

other: no effects at highest tested dose

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not specified

Anogenital distance of all rodent fetuses:

not specified

Changes in postnatal survival:

not specified

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One to 4 fetuses with gross malformations were found in each group. However, regarding the incidence rate, there was no significant difference between control group and magnesium chloride hexahydrate groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In the 800 mg/kg/day group, one fetus had skeleton malformations, however, with regard to the incidence rate; there was no significant difference between control group and the magnesium chloride hexahydrate groups. Furthermore, no significant differences between control group and magnesium chloride hexahydrate groups were observed as far as the incidence rate of skeletal variations is concerned. Regarding the incidence rate of fetuses with lumbar rib and additional rib bones, there were no significant differences. No significant differences were found in the number of ossification centers, metacarpal and metatarsal bones as well as sacral and tail vertebrae. The aforementioned numbers were examined as indicators for the progress of ossification.Four to 6 fetuses in each group showed malformations, however, no significant difference were observed between control group and magnesium chloride hexahydrate groups.

Visceral malformations:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Key result

Dose descriptor:

NOAEL

Effect level:

> 800 other: mg/kg/day

Basis for effect level:

other: no observed adverse effects at highest tested dose

Abnormalities:

not specified

Developmental effects observed:

not specified

No increased incidences of foetal malformation were noted, and no toxic signs were found in the pregnant rats and the foetuses.

Table 1. Fetal growth in pregnant rats treated with magnesium chloride hexahydrate

Dose (mg/kg/day)		0 (Control)	200	400	800
No. of litters		22	22	22	22
No. of corpora lutea		373	370	361	362
No. of implants		364	340	345	345
No. of live foetuses		346	326	324	332
Foetal weight (g)	Male	3.95	3.98	3.87	3.98
	Female	3.73	3.75	3.72	3.81
No. of dead implants		18	14	21	13
Mortality (%)		4.8	4.8	5.9	3.6

 

Table 2. Gross malformations in the foetuses from pregnant rats treated with magnesium chloride hexahydrate

Dose (mg/kg/day)	0 (Control)	200	400	800
No. of litters	22	22	22	22
No. foetuses examined	346	326	324	332
No. of litters with malformed foetuses	3	1	3	1
No. of foetuses with malformation	3	1	4	1

 

Conclusions:

In conclusion, magnesium chloride hexahydrate has no teratogenicity in rats when given by gavage at the doses examined in this study. The no observed adverse effect level for magnesium chloride hexahydrate was determined to be >800 mg/kg/day for both pregnant rats and rat fetuses.

Executive summary:

In a publication from Usami et al., teratogenicity of magnesium chloride hexahydrate was examined in rats. 22 male and female Wistar rats were orally treated with magnesium chloride hexahydrate (>95% purity) in water at doses of 0, 200, 400 and 800 mg/kg bw/day from Day 6 to Day 15 of pregnancy. The pregnant rats were sacrificed on day 20 of pregnancy and their fetuses were examined for malformation. It was found that magnesium chloride hexahydrate caused no increased incidences of fetal malformation and no toxic signs in the pregnant rats and the fetuses. Even a dose of 800 mg/kg bw/day, considered the maximum non-fatal dose of magnesium chloride hexahydrate in pregnant rats, did not increase the incidence of teratogenicity. The no observed adverse effect level for magnesium chloride hexahydrate was determined to be >800 mg/kg/day for both pregnant rats and rat fetuses, which is equal to 230 mg/kg bw/day magnesium hydroxide.

This information is used in a read-across approach in the assessment of the target substance. For details and justification of read-across please refer to the attached report in section 13 of IUCLID.

Reference 2

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Species:

rabbit

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

230 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Comparable to guideline study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a publication from Usami et al., teratogenicity of magnesium chloride hexahydrate was examined in rats. 22 male and female Wistar rats were orally treated with magnesium chloride hexahydrate in water at doses of 0, 200, 400 and 800 mg/kg bw/day from Day 6 to Day 15 of pregnancy. It was found that magnesium chloride hexahydrate caused no increased incidences of fetal malformation and no toxic signs in the pregnant rats and the fetuses. Even a dose of 800 mg/kg bw/day, considered the maximum dose of magnesium chloride hexahydrate in pregnant rats without a sign of maternal toxicity, did not increase the incidence of teratogenicity. The NOAEL for magnesium chloride hexahydrate was determined to be >800 mg/kg/day for both pregnant rats and rat fetuses, which is equal to 230 mg/kg bw/day magnesium hydroxide.

No further studies are warranted for this endpoint.

Magnesium hydroxide is a solid and in general, solids need to be dissolved before they can be absorbed and become bioavailable. However, the water solubility of magnesium hydroxide is relatively low (~ 2 mg/L). The water solubility approximates the bioavailability of a substance. In addition, magnesium salts are generally absorbed so slowly that their oral administration only results in a bioavailability of 30-50% [Stendig-Lindberg G., 1991 (see IUCLID section 7.1)]. When administered orally, Mg(OH)2 dissociates to magnesium cations (Mg2+). About 5–15% of the dissociated Mg2+ cations are absorbed from the gastrointestinal tract (through the epithelial lining of the small intestine). Magnesium (Mg2+) is the fourth most abundant cation in the human body. The average adult's body contains about 24 g of magnesium and the normal range of magnesium in human serum is 1.5–2.5 mEq/L (18.2–30.3 mg/L). About two-third of magnesium is present as free cation and one-third is bound to plasma proteins (EMA, 1995). The half-life of magnesium ions in humans after i.v. administration has been reported to be 4 h. Magnesium is mainly excreted by the kidney and under normal conditions, 3-5% of the filtered ion is very quickly excreted in the urine. Magnesium clearance increases roughly linearly with the concentration of magnesium in the serum. In addition, small amounts of magnesium are also excreted in milk and saliva [Behmer et al., 1990; Lu et al., 2000; Massry et al., 1969 (see IUCLID section 7.1); EMA, 1995]. Furthermore, external human exposure to magnesium can occur via its natural presence in food, for example in fruits, vegetables, meats or seafoods. Based on the available information on concentrations of magnesium in some of these products, the European Medical Agency (EMA) reported in its review (EMA, 1995), that 20-40 mEq (0.25 -0.5 g) magnesium is ingested daily in typical diet. Also, in 2015, following a request from the European Commission, an European Food Safety Agency (EFSA) expert group on Dietetic Products, Nutrition and Allergies (NDA) defined Adequate Intakes (AIs) for magnesium based on observed magnesium intakes in healthy populations in the European Union (EU). For adults, an AI for magnesium was set at 350 mg/day for men and 300 mg/day for women. Magnesium hydroxide is also used as food additive, as such it is authorized in Europe (E528) and categorized by the U.S. Food and Drug Administration (FDA) as a GRAS (generally recognized as safe) food ingredient. It is also approved for use as a nutritional supplement and a pH-control agent in foods. Based on the above-mentioned observations and due to the role of magnesium as a common and natural food ingredient, and ubiquitous metabolic product/substrate in mammals with proven low toxicity, it can safely be expected that magnesium hydroxide would not contribute to any systemic adverse effects. Furthermore, these observations are common textbook knowledge and hence can be considered as adequately and reliably documented. Also, supporting evidence that magnesium hydroxide has no potential to induce any reproductive/developmental toxicity is derived from a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD TG 422). No adverse effects were observed and the NOAEL for parental and reproductive toxicity is considered to be 1000 mg/kg bw/day. In addition, several repeated dose studies of 90-day and longer duration with the source substance magnesium chloride showed no effects on any reproductive organ up to the highest dose tested [Tanaka, 1994 and Takizawa, 2000 (see IUCLID section 7.5.1)]. By assessing the information provided in IUCLID section 7.10 in a weight of evidence approach it can be concluded that based on human experience from occupational exposure to magnesium hydroxide and medical uses of magnesium salts did not lead to adverse signs of reproductive/developmental toxicity. Thus, in accordance with REACH annex XI, section 1.1 it is scientifically not justified to conduct a developmental toxicity study in rabbits with the target substance magnesium hydroxide.

Justification for classification or non-classification

Based on the available data, the target substance magnesium hydroxide does not require classification for reproductive/developmental toxicity in accordance with the CLP Regulation 1272/2008.