Magnesium hydroxide sulfate (Mg6(OH)10(SO4)), hydrate (1:3)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 421 (Reproductive / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19/08/2008; Date of signature: 04/03/2009

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

483-390-9

EC Name:

-

Cas Number:

12508-61-1

Molecular formula:

H16Mg6O17S

IUPAC Name:

hexamagnesium(2+) trihydrate decahydroxide sulfate

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Harlan Laboratories UK Ltd.

- Age at study initiation:
(P) approximately 12 weeks old; (F1) Not applicable.

- Weight at study initiation:
(P) Males: 330-385g; Females: 200-236g; (F1) Not applicable.

- Fasting period before study:
Not stated.

- Housing:
- Use of restrainers for preventing ingestion (if dermal):
Not applicable.

- Diet (e.g. ad libitum):
Ad libitum

- Water (e.g. ad libitum):
Ad libitum

- Acclimation period:
13 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
21±2 deg C

- Humidity (%):
55±15%

- Air changes (per hr):
At least 15 air changes per hour.

- Photoperiod (hrs dark / hrs light):
12 hours dar/12 hours light


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% Carboxy methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Preparation of Test Material:
For the purpose of the study, the test material was prepared at the appropriate concentrations as a suspension in 1% Carboxy methylcellulose. The stability and homogeneity of the test material formulations were determined by Harlan Laboratories Ltd. Shardlow, UK. Results are given in Appendix 20 and show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4ºC in the dark.
Samples were taken of each test material formulation and were analysed for concentration of MOS-HIGE at Harlan Laboratories Ltd. Shardlow, UK. The method used for analysis of formulations and the results obtained are given in Appendix 20. The results indicate that the prepared formulations were within acceptable limits for the purpose of this study.

DIET PREPARATION
- Rate of preparation of diet (frequency):
Not applicable, gavage study.

- Mixing appropriate amounts with (Type of food):
Not applicable, gavage study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- METHOD OF ANALYSIS:
- Summary.
The concentration of MOS-HIGE in the test material formulations was determined by Atomic Absorption Spectroscopy (AAS) using an external standard technique.

- Samples.
The test material formulations were diluted with 10 % nitric acid to give a final, theoretical test material concentration of approximately 0.001 mg/ml.

- Standards.
Due to the non linear response of the detector, standard solutions of test material were prepared in 10% Nitric acid at a number of concentrations ranging from 0.0005 to 0.002 mg/ml. All results were calculated from the relevant standard curve.

- Procedure.
The standard and sample solutions were analysed by AAS using the following conditions:
Element: hollow cathode magnesium detection lamp
Lamp Current: 5 mA
Wavelength: 285 nm
Flame description: lean blue flame

- Homogeneity Determinations.
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.

- Stability Determinations.
The test material formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark for fourteen days.

- Verification of Test Material Formulation Concentrations.
The test material formulations were sampled and analysed within four days of preparation.

- RESULTS:
- Homogeneity of Test Material Formulations.

Nominal Sampling Concentration Found (mg/ml)
Concentration Location
(mg/ml) 1 2 3 Mean
1.5 Top 1.33 1.35 1.36 1.35
Middle 1.36 1.39 1.39 1.38
Bottom 1.37 1.33 1.41 1.37
100 Top 97.4 98.3 99.3 98.3
Middle 97.6 99.1 107 101
Bottom 95.6 103 100 99.7

- Stability of Test Material Formulations.
Nominal Concentration Concentration Found After Storage
Concentration Foundfor Fourteen Days
(mg/ml) Initially (mg/ml) (mg/ml) (expressed as % of initial)
1.5 1.37 1.43 104
100 99.7 102 102

- Verification of Concentration of Weekly Test Material Formulation:
Week Number Nominal Concentration Found
Concentration (mg/ml) (expressed as % of nominal)
(mg/ml)
1 0 ND -
1.5 1.37 91
15 14.6 97
100 94.7 95
2 0 ND -
1.5 1.23 82
15 15.1 101
100 95.0 95
3 0 ND -
1.5 1.35 90
15 14.6 97
100 103 103
4 0 ND -
1.5 1.43 95
15 15.2 101
100 102 102
5 0 ND -
1.5 1.46 97
15 15.6 104
100 103 103
6 0 ND -
1.5 1.37 91
15 15.0 100
100 111 111
7 0 ND -
1.5 1.38 92
15 14.1 94
100 97.5 98

- METHOD VALIDATION:
- Specificity.
The diluent solvent 10 % nitric acid and a blank 1 % Carboxy methylcellulose (control) were analysed. The results are shown in the following table :
Sample Concentration Found
10 % nitric acid None detected
1 % CMC(control) None detected

Analysis of the solvent and a blank 1% Carboxy methylcellulose (control) produced no signal that interfered with the signal due to the test material.

- Accuracy.
Samples of 1 % Carboxy methylcellulose were accurately fortified with known amounts of test material, and analysed:
Fortification Concentration Found % Recovered Mean Recovery
(mg/g) (mg/g) (%)
1.40 1.51 108 106
1.44 1.50 104

92.7 94.5 102 100
103 101 98

The analytical method has been considered to be sufficiently accurate for the purpose of this study. The test sample results have not been corrected for recovery.

- Conclusion.
The analytical method has been satisfactorily validated in terms of specificity and accuracy for the purposes of the study.

Details on mating procedure:

- M/F ratio per cage:
1:1 during mating phase of test.
mated females were then housed individually

- Length of cohabitation:
up to 14 days, until mated.

- Proof of pregnancy:
A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).

- Further matings after two unsuccessful attempts:
Not applicable

- After successful mating each pregnant female was caged (how):
Females were housed individually during the period of gestation and lactation.

- Any other deviations from standard protocol:
None stated.

Duration of treatment / exposure:

The test material was administered by gavage to three groups each of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, for up to fifty-four consecutive days, (including a two week maturation phase, pairing, gestation and early lactation for females).

Frequency of treatment:

The test material was administered daily.

Duration of test:

The test material was administered by gavage to three groups each of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, for up to fifty-four consecutive days, (including a two week maturation phase, pairing, gestation and early lactation for females).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10 males and 10 females per dose group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Not stated.

- Rationale for animal assignment (if not random):
Random.

- Other:

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS:
Yes

- Time schedule:
Daily

- Cage side observations checked in tables 2 and 3 were included. Individual clinical observations are presented in Appendices 1 and 2.


DETAILED CLINICAL OBSERVATIONS:
Yes

- Time schedule:
Daily

- Cage side observations checked in table 2 were included.
BODY WEIGHT:
Yes

- Time schedule for examinations:
Weekly

- Cage side observations checked in table 5 were included.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data
Not applicable. gavage study.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
Not applicable. gavage study.


POST-MORTEM EXAMINATIONS:
Yes
- Sacrifice on gestation day #
maternal animals were sacrificed on day 5 post partum.

- Organs examined:
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin.
Coagulating gland
Epididymides *
Gross lesions
Ovaries
Mammary tissue
Pituitary
Prostate
Seminal vesicles
Testes *
Uterus/Cervix
Vagina
All tissues were despatched to Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, UK (Principal Investigator: E Richards). The tissues from control and 1000 mg/kg/day dose group animals and those animals dying during the study, were prepared as paraffin blocks, sectioned at nominal thickness of 5µm and stained with haematoxylin and eosin for subsequent microscopic examination.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.
* = preserved in Bouin’s fluid and then transferred to 70% Industrial Methylated Spirits (IMS) approximately
forty-eight hours later


OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination:
Yes

Examinations included:
- Gravid uterus weight:
Yes

- Number of corpora lutea:
Yes

- Number of implantations:
Yes

- Number of early resorptions:
No data

- Number of late resorptions:
No data

- Other:

Fetal examinations:

- External examinations:
Yes: all per litter

- Soft tissue examinations:
Yes: all per litter

- Skeletal examinations:
Yes: all per litter

- Head examinations:
Yes: all per litter

Statistics:

The following parameters were subjected to statistical analysis:
Bodyweight and bodyweight change
Food consumption for females during gestation and lactation
Litter data
Sex ratio
Implantation losses and viability indices
Offspring bodyweight and bodyweight change
Offspring surface righting
Adult absolute and bodyweight relative organ weights
The following statistical procedures were used:
Data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.
The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
Probability values (p) are presented as follows:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p ≥ 0.05 (not significant)

Indices:

- Litter Responses:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
i) Implantation Losses (%)
Group mean percentile pre-implantation and post implantation loss were calculated for each female/litter as follows:

%pre-implantation loss = ((Number of Corporalutea - Number of implantation sites) / Number of implantation sites) x 100

% Post-implantation loss + ((Number of implantation sites - Total number of live offspring) / Number of implantation sites) x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:

Live Birth Index (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) x 100

Viability Index (%) = Number of offspring alive on Day 4 / Number of offspring alive on Day 1) x 100

iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:

(Number of male offspring / Total number of offspring) x 100

Indicies information is continued in the remarks section.

Historical control data:

None stated. Concurrent control groups were used during the test.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Water Consumption.
Animals of either sex treated with 1000 mg/kg/day showed an increase in water consumption throughout the treatment period. No such effects were detected for animals of either sex treated with 150 or 15 mg/kg/day.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOEL

Effect level:

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

Basis for effect level:

other: The effects detected in 1000 mg/kg/day females was confined to an increase in water consumption and in the absence of any changes this effect was considered not to represent an adverse health effect.

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: The effects detected in 1000 mg/kg/day females was confined to an increase in water consumption and in the absence of any changes this effect was considered not to represent an adverse health effect.

3

Dose descriptor:

NOEL

Effect level:

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

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No treatment-related effects were detected in the reproductive parameters measured and as such the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg/day.

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

not specified

Basis for effect level:

other: no effects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The oral administration of MOS-HIGE to rats by gavage at dose levels of 15, 150 and 1000 mg/kg/day resulted in treatment-related effects in animals of either sex treated with 1000 mg/kg/day. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 150 mg/kg/day. The effects detected in 1000 mg/kg/day females was confined to an increase in water consumption and in the absence of any associated changes this effect was considered not to represent an adverse health effect. The ‘No Observed Adverse Effect Level’ (NOAEL) for females was therefore considered to be 1000 mg/kg/day.
No treatment-related effects were detected in the reproductive parameters measured and as such the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg/day.

Executive summary:

Introduction.

The study was perford to screen for potential adverse effects of the test material on reproduction including offspring developnt and provides an initial hazard assessnt for effect on reproduction. The study complies with the recomndations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developntal Toxicity Screening Test” (adopted).

This study is designed to comply with Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods.

The test material was administered by gavage to three groups each of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, for up to fifty-four consecutive days, (including a two week maturation phase, pairing, gestation and early lactation for females) at dose levels of 15, 150 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (1% Carboxy methylcellulose).

Clinical signs, bodyweight developnt, dietary intake and water consumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatnt group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were perford on all surviving offspring, together with litter size and offspring weights and assessnt of surface righting reflex.

Adult males were terminated on Day 43, and all females and offspring on Day 5post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was perford.

Results.

Adult Responses:

Mortality.

There were no unscheduled deaths.

Clinical Observations.

Instances of noisy and gasping respiration were evident for males treated with 1000 mg/kg/day throughout the treatment period, with one male also showing a decreased respiratory rate on one occasion. Episodes of staining around the mouth/snout/ano-genital region and increased salivation were also detected in 1000 mg/kg/day males and one male from this treatment group had hunched posture between Days 14 and 28. Diarrhoea was evident at 1000 mg/kg/day from Day 14 (males) and Day 15 (females) onwards. No toxicologically significant clinical signs were detected in animals of either sex treated with 150 or 15 mg/kg/day.

Bodyweight.

Males treated with 1000 mg/kg/day showed a reduction in bodyweight gain during the first two weeks of treatment and during the final week of treatment. Actual bodyweight losses were evident in the majority of males during the first and final week of treatment. No adverse effects on bodyweight change were detected for females treated with 1000 mg/kg/day or animals of either sex treated with 150 or 15 mg/kg/day.

Food Consumption and Food Efficiency.

No adverse effect on food consumption or food efficiency was detected for animals of either sex treated with 1000, 150 or 15 mg/kg/day when compared to controls.

Water Consumption.

Animals of either sex treated with 1000 mg/kg/day showed an increase in water consumption throughout the treatment period. No such effects were detected for animals of either sex treated with 150 or 15 mg/kg/day.

Reproductive Performance:

Mating.

There were no treatment-related effects on mating for animals of either sex treated with 1000, 150 or 15 mg/kg/day.

Fertility.

There were no treatment-related effects on conception rates for animals of either sex treated with 1000, 150 or 15 mg/kg/day.

Gestation Length.

There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

Litter Responses:

Offspring Litter Size and Viability.

Of the litters born, litter size at birth and subsequently on Day 1 and 4post partumwere comparable to controls.

Offspring Growth and Development.

Offspring bodyweight gain and litter weights at birth and subsequently on Day 1 and 4post partumwere comparable to controls.

Litter Observations.

No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Pathology:

Necropsy.

No toxicologically significant effects were detected.

Organ Weights.

No toxicologically significant effects were detected in the organ weights measured.

Histopathology.

No treatment-related effects were detected.

Conclusion.

The oral administration of MOS-HIGE to rats by gavage, at dose levels of 15, 150 and 1000 mg/kg/day, resulted in treatment-related effects in animals of either sex treated with 1000 mg/kg/day. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 150 mg/kg/day. The effects detected in 1000 mg/kg/day females was confined to an increase in water consumption and in the absence of any associated changes this effect was considered not to represent an adverse health effect. The ‘No Observed Adverse Effect Level’ (NOAEL) for females was therefore considered to be 1000 mg/kg/day.