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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOEL reproductive = 100 mg/kg/day, NOEL systemic = 150 mg/kg/day male/female, NOAEL = 1000 mg/kg/day male/female (rat), OECD 421

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 22 January 2009 and 13 May 2009.

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 421 (Reproduction / 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:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

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 applicable.

- Housing:
Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Harlan Laboratories UK Ltd). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

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

- Diet (e.g. ad libitum):
Ad libitum, Rodent 2081C Teklad Global Certified Diet. Harlan Laboratories UK Ltd.

- Water (e.g. ad libitum):
Ad libitum, Mains drinking water.

- 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 dark/12 hours light


IN-LIFE DATES:
Males: From: Day 1 To: Day 42
Females: From: Day 1 To: Day 54 (Day 4 post partum)

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).

- Storage temperature of food:
Not applicable (gavage study).

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Not stated.

- Concentration in vehicle:
1%

- Amount of vehicle (if gavage):
approximately 10 ml/kg

- Lot/batch no. (if required):
Not stated.

- Purity:
Not stated.

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.

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

no= none detected
- = not applicable

- 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.

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.

Details on study schedule:

- Chronological Sequence of Study:

i) Groups of ten male and ten female animals (approximately 12 weeks old) were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.

ii) On Day 15, all animals were paired on a 1 male:1 female basis within each dose group for a maximum of fourteen days.

iii) Following evidence of mating (designated as Day 0 post coitum) , the males were returned to their original cages and females were transferred to individual cages.

iv) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.

v) The male dose groups were killed and examined macroscopically on Day 43.

vi) At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically.

Dose / conc.:

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

Dose / conc.:

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

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:
The dose levels were based on a previous 28 day study (study number 1456/0070). Cross reference to section 7.5.1 Repeated dose toxicity: oral for the robust study summary.

- Rationale for animal assignment: Random.

Positive control:

Not conducted.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Yes
- Time schedule:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing and one hour after dosing at weekends (except for females during parturition where applicable).

- 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.

- Clinical observations checked in table 2 and 3 were included.

BODY WEIGHT:
Yes

- Time schedule for examinations:
Day 1 and then weekly until termination of male.
Day 0,7,14,20 post coitum
Day 1 & 4 post partum for female.

- Bodyweight observations checked in table 5 were included.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Not applicable, gavage study.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
Water consumption

A possible treatment-related effect was detected on Day 2 therefore gravimetric measurements were initiated from Day 3 through to study termination.

OTHER:

Oestrous cyclicity (parental animals):

A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
During Histopathology following terminal kill the male epididymides were examined for the following parameters:
Coagulating glands:
-Reduced scretory content in vesicle 1 and 2.

Epididymides:
-Reduced spermatozoal content in Epididymis 1 and 2.
-Spermatocoel granuloma
-Chronic inflammatory cells

Mammary gland:
-number of sections

Prostate:
-Chronic inflammatory cell foci
-Prostatitis

Seminal vesicles:
-Reduced secretory content in vesicle 1 and 2

Testes:
-Atrophy testis 1
-Atrophy testis 2

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 5 postpartum:
No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded and offspring were individually identified within each litter by a tattoo on Day 1 post partum.
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Day 1 and 4 post partum
iii) Sex of offspring on Day 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring and litter weights on Day 1 and 4 post partum


GROSS EXAMINATION OF DEAD PUPS:
Full external and internal examination and macroscopic abnormalities were recorded.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals:
The male dose groups were killed and examined macroscopically on Day 43.
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 of post partum.

- Maternal animals:
At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically. Any females that failed to acheive pregnancy or produce a litter were killed on or after Day 26 postcoitum.


GROSS NECROPSY
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
For all females the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 1% ammonium polysulphide solution.


HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively:
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

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [.?] days of age.
Not applicable

Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.


GROSS NECROPSY
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not applicable

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)

Reproductive indices:

- Mating Performance and Fertility:
The following parameters were calculated from the individual data during the mating period of the parental generation:

i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
For each group the following were calculated:

Mating Index (%) = (Number of animals mated / Number of animals paired) x 100

Pregnancy Index (%) = (Number of pregnant females / Number of animals paired) x 100

- Gestation and Parturition Data:
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation:

i)Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii)Parturition Index
The following was calculated for each group:

Parturition Index (%) = (Number of females delivering live offspring / Number of pregnant females) x 100

Offspring viability 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

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
A summary incidence of daily clinical observations is given in Table 2 and Table 3. Individual clinical observations are presented in Appendices 1 and 2.
Instances of noisy respiration were evident for males treated with 1000 mg/kg/day throughout the study. Two males showed gasping respiration on Day 5 and Day 40, with one of these males also showing a decreased respiratory rate on Day 5. Episodes of staining around the mouth/snout/ano-genital region and increased salivation were also detected in 1000 mg/kg/day males. A further male from this treatment group had hunched posture between Days 14 and 28. Animals of either sex treated with 1000 mg/kg/day also had diarrhoea 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.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Bodyweight:
Group mean bodyweights, bodyweight changes and standard deviations for males are given in Table 4 (statistically significant differences are indicated). Group mean bodyweights and bodyweight changes and standard deviations during the maturation, gestation and lactation phases for females are given in Table 5. Cumulative bodyweight changes are presented graphically in Figures 1 and 2. Individual data are given in Appendix 3 and Appendix 4.

Males
Males treated with 1000 mg/kg/day showed a statistically significant reduction in bodyweight gain during the first two weeks of treatment (p<0.001 week 1 only) and during the final week of treatment (p<0.05). Actual bodyweight losses were evident in the majority of these males during the first and final weeks of treatment with two males continuing to show a bodyweight loss in Week 3. Subsequently the cumulative bodyweight gains for 1000 mg/kg/day males were significantly reduced throughout the treatment period. No such effects were detected in males treated with 150 or 15 mg/kg/day

Females
No adverse effect on bodyweight gain was detected for females throughout the two week maturation period or during the gestation or lactation phase of the study.
Statistical analysis did not reveal any significant intergroup differences.

Food Consumption and Food Efficiency:
Group mean food consumptions for males are given in Table 6 and group mean food consumptions during the maturation, gestation and lactation phases for females are given in Table 7. These are presented graphically in Figures 3 and 4. Individual cage values for males prior to and after mating and females prior to mating are presented in Appendices 5 and 6. Individual values for females during gestation and lactation are presented in Appendix 7.
Food efficiency for males is presented in Table 8. Food efficiency for females during maturation and early gestation is given in Table 9.

Males
No adverse effect on food consumption was detected for males throughout the treatment period.

Females
No adverse effect on food consumption was detected for females throughout the two week maturation period or during the gestation or lactation phase of the study.

Water Consumption:
Group mean weekly water consumptions for males and females are given in Tables 10 and 11. Individual data are given in Appendices 8 and 9.
An overt intergroup difference in water intake was detected on Day 2, therefore measurements were initiated from Day 3 onwards.
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.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Not applicable, gavage study.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The distribution of pre-coital intervals for treated animals was comparable to controls with majority of animals showing positive evidence of mating within four days of pairing.
Please see table 12

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Not stated.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Reproductive performance:
A summary incidence of adult performance in presented in Table 1.

Mating:
Group values for mating performance are presented in Table 12. Individual data are given in Appendix 10.
There were no treatment-related effects on mating performance. The distribution of pre-coital intervals for treated animals was comparable to controls with the majority of animals showing positive evidence of mating within four days of pairing (i.e. the first oestrus opportunity).

Fertility:
Group values for fertility are given in Table 12. Individual data are given in Appendix 10.
There were no treatment-related effects on fertility.

Gestation Length:
Group values for gestation length are given in Table 12. Individual gestation lengths are given in Appendix 10.
There were no significant intergroup differences in gestation lengths or parturition for treated females, with the majority of females giving birth following 22 to 23½ days of gestation. The distribution for treated females was comparable to controls.

Litter Responses:
In total all ten females from control, nine females from 50 mg/kg/day, eight females from 250 mg/kg/day and nine females from 1000 mg/kg/day dose groups gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.
The following assessment of litter response is generally based on those litters reared to termination on Day 5 of lactation/age, although data available for females showing total litter loss has also been taken into consideration, where considered appropriate.

Offspring Litter Size and Viability:
Group mean corpora lutea and implantation counts, litter size, implantation, survival indices and sex ratio are given in Tables 13 to 15. Individual data are given in Appendices 11 to 13.
The mean numbers of corpora lutea observed for treated females did not indicate any adverse effect of treatment at 15, 150 or 1000 mg/kg/day. Subsequent resultant litter size at Day 1 for treated animals was similar to controls. Post-natal survival was unaffected in all treated groups with litter size at Day 4 again being similar to controls.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Group mean absolute and bodyweight-relative male reproductive organ weights and standard deviations for test and control group adult males are presented in Table 21. Individual data are given in Appendix 19.
No adverse effects were detected in the organ weights measured.
Statistical analysis did not reveal any significant intergroup differences.

GROSS PATHOLOGY (PARENTAL ANIMALS)
A summary incidence of necropsy findings for offspring and adults is given in Tables 18 to 20. Individual data are given in Appendices 16 to 18.
Offspring
Neither the incidence, type or distribution of macroscopic findings observed at necropsy of decedent offspring nor offspring killed at scheduled termination (Day 5 of age) indicated any adverse effect of maternal treatment.
Adults
No toxicologically significant macroscopic abnormalities were detected.
One male treated with 1000 mg/kg/day had small testes and seminal vesicles at necropsy. A further two males from this treatment group also showed small seminal vesicles. In the absence of any histological correlates the intergroup differences were considered to be of no toxicological importance. One female treated with 150 mg/kg/day had an enlarged uterus containing two malformed offspring. One female treated with 15 mg/kg/day had a right ovary encased in a fluid filled sac. In the absence of a true dose related response or any histological correlates the intergroup differences were considered to be of no toxicological importance. One control male had small and flaccid testes and a mass on the cauda of the left epididymis. In the absence of treatment, this was considered to be incidental.

HISTOPATHOLOGY (PARENTAL ANIMALS)
A summary incidence of histopathological findings is given in Table 22 and Table 23. Individual data are given in Appendices 20 and 21.
No treatment related microscopic abnormalities were detected.
All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

OTHER FINDINGS (PARENTAL ANIMALS):
Mortality:
There were no unscheduled deaths.

Key result

Dose descriptor:

NOEL

Remarks:

systematic toxicity

Effect level:

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

Sex:

male/female

Basis for effect level:

other: Overall effects: the oral administration of MOS-HIGE to rats by gavage at dose levels of 15, 150 and 1000mg/kg/day resulted in treatment related effects in animals of either sex treated with 1000 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

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 associated changes this effect was considered not to represent an adverse health effect.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
Group mean corpora lutea and implantation counts, litter size, implantation, survival indices and sex ratio are given in Tables 13 to 15. Individual data are given in Appendices 11 to 13.
The mean numbers of corpora lutea observed for treated females did not indicate any adverse effect of treatment at 15, 150 or 1000 mg/kg/day. Subsequent resultant litter size at Day 1 for treated animals was similar to controls. Post-natal survival was unaffected in all treated groups with litter size at Day 4 again being similar to controls.

CLINICAL SIGNS (OFFSPRING)
In total all ten females from control, nine females from 50 mg/kg/day, eight females from 250 mg/kg/day and nine females from 1000 mg/kg/day dose groups gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.
The following assessment of litter response is generally based on those litters reared to termination on Day 5 of lactation/age, although data available for females showing total litter loss has also been taken into consideration, where considered appropriate.

BODY WEIGHT (OFFSPRING)
Offspring Growth and Development:
Group mean values for total litter weights, offspring bodyweight change, surface righting reflex and a summary incidence of clinical signs are given in Tables 13, 16 and 17. Individual values and observations are given in Appendix 11, 14 and 15.
Mean litter weights and bodyweight gains were considered to have been unaffected by maternal exposure.
The percentage of offspring who successfully showed surface righting reflex on Day 1 and the type, incidence and distribution of clinical signs in the offspring to termination on Day 5 of age was unaffected by maternal exposure.

SEXUAL MATURATION (OFFSPRING)
Not applicable, offspring sacrificed on Day 5 post partum.

ORGAN WEIGHTS (OFFSPRING)
Not applicable

GROSS PATHOLOGY (OFFSPRING)
Neither the incidence, type or distribution of macroscopic findings observed at necropsy of decedent offspring nor offspring killed at scheduled termination (Day 5 of age) indicated any adverse effect of maternal treatment.

HISTOPATHOLOGY (OFFSPRING)
Not applicable


OTHER FINDINGS (OFFSPRING)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Reproductive effects observed:

not specified

Discussion

The oral administration of MOS-HIGE to rats for a period of up to fifty-four Days at dose levels of 15, 150 and 1000 mg/kg/day resulted in treatnt-related effects.

Clinically observable signs developed in 1000 mg/kg/day males throughout the treatment period. Instances of noisy respiration were evident throughout the study followed by further respiratory pattern changes identified as gasping respiration and a decreased respiratory rate. Increased salivation around the time of dosing was evident in 1000 mg/kg/day males from Day 2 onwards together with staining around the mouth/snout/ano-genital region and hunched posture. Animals of either sex treated with 1000 mg/kg/day also had diarrhoea from Day 14 (males) and Day 15 (females) onwards. Bodyweight development was also adversely effected in 1000 mg/kg/day males. Significant actual bodyweight losses were evident in the majority of these males during the first and final week of treatment and subsequently the cumulative bodyweight gains for 1000 mg/kg/day males were significantly reduced throughout the treatment period. Two males continued to show a bodyweight loss in Week 3. No adverse effect on food consumption was detected however an increase in water consumption was evident in animals of either sex treated with 1000 mg/kg/day throughout the treatment period. 

No such effects were detected in animals of either sex treated with 150 or 15 mg/kg/day.

No macroscopic or microscopic abnormalities were detected in treated animals. 

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 Developnt.

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. 

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 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:

The key study, conducted on the registered material in accordance with standardised guidelines and under GLP conditions, is a high quality study. In addition, a literature study is provided to support the key data. The quality is therefore considered to be high.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The key study was performed to screen for potential adverse effects of the test material on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study was conducted in accordance with the standardised guideline OECD 421 under GLP conditions. It was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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 development, 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 treatment 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 performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

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

In the adults, there were no unscheduled deaths. 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.

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.

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.

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.

There were no treatment-related effects on mating or conception rates for animals of either sex treated with 1000, 150 or 15 mg/kg/day. There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

No toxicologically significant effects were detected at necropsy. No toxicologically significant effects were detected in the organ weights measured or during histopathological investigations.

Of the litters born, litter size at birth and subsequently on Day 1 and 4post partum were comparable to controls. Offspring bodyweight gain and litter weights at birth and subsequently on Day 1 and 4post partum were comparable to controls. No clinically observable signs of toxicity were detected for offspring from all treatment groups.

The oral administration of the test material 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.

A supporting study is provided in order to further address the potential toxicity of the test material. It is a literature report conducted on a read across material and was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

 

The study was conducted to determine whether sulfate in the drinking water affects the reproductive performance of female mice during gestation and lactation over two parities.

Sixty random-bred ICR mice were randomly assigned to six sulfate treatments. Sodium sulfate was added to deionised, distilled water to give sulfate levels in ppm as follows: 0 (control), 0 (Na control), 625, 1250, 2500 and 5000 ppm. All treatments (except the control) contained the same Na content (2392 ppm) by varying sodium bicarbonate content.

The treated water was available ad libitum beginning 1 week prior to breeding and was continued throughout the experiment. At 21 days postpartum, the pups were weaned and the litters and dams were weighed individually. The dams then were rebred at first oestrus immediately following weaning. This procedure was carried out over two parities. Only animals that whelped during each parity were used in the analysis. Water consumption was measured daily during the 2nd and 3rd weeks of gestation and the 1st and 2nd weeks of lactation.

Mice receiving only the deionised, distilled water drank less (P < 0.05) than mice receiving the other treatments at all times measured. Animals offered the 0 (Na control) water drank more (P < 0.05) water than mice of the other sulfate treatments. There was no difference (P > 0.10) in litter size, litter weaning weight, or gestational and lactational weight gain of the dam among water treatments.

Although levels of sulphate up to 5000 ppm and levels of sodium up to 2392 ppm in the drinking water cubicly altered water consumption, they did not affect litter size, litter weaning weight, or gestational and lactational weight gain of the dam when sulfate ingestion was continued over two parities.

Therefore, under the conditions of this study, the results indicate that if litter size and weaning weight are used as indicators of maximum reproductive performance, water containing up to 5000 ppm of sulfate is not detrimental to the gestating mouse. With ad libitum water consumption, sulfates in the water do not affect reproductive performance.

Effects on developmental toxicity

Description of key information

NOEL reproductive = 100 mg/kg/day, NOEL systemic = 150 mg/kg/day male/female, NOAEL = 1000 mg/kg/day male/female (rat), OECD 421

Link to relevant study records

Reference

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

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

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:

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).

Dose / conc.:

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

Dose / conc.:

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

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:

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.

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.

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.

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.

Dose descriptor:

NOEL

Effect level:

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

Basis for effect level:

other: developmental toxicity

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.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

not specified

Basis for effect level:

other: no effects

Abnormalities:

not specified

Developmental effects observed:

not specified

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. 

Effect on developmental toxicity: 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:

The key study, conducted on the registered material in accordance with standardised guidelines and under GLP conditions, is a high quality study. In addition, two literature studies are provided to support the key data. The quality is therefore considered to be high.

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

The key study was performed to screen for potential adverse effects of the test material on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study was conducted in accordance with the standardised guideline OECD 421 under GLP conditions. It was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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 development, 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 treatment 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 performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

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

In the adults, there were no unscheduled deaths. 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.

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.

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.

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.

There were no treatment-related effects on mating or conception rates for animals of either sex treated with 1000, 150 or 15 mg/kg/day. There were no differences in gestation lengths. The distribution for treated females was comparable to controls.

No toxicologically significant effects were detected at necropsy. No toxicologically significant effects were detected in the organ weights measured or during histopathological investigations.

Of the litters born, litter size at birth and subsequently on Day 1 and 4post partum were comparable to controls. Offspring bodyweight gain and litter weights at birth and subsequently on Day 1 and 4post partum were comparable to controls. No clinically observable signs of toxicity were detected for offspring from all treatment groups.

The oral administration of the test material 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.

Two supporting studies are provided in order to further address the potential toxicity of the test material. Both are literature reports conducted on read across materials; the first was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997) and the second was awarded a reliability score of 2 in accordance with the same criteria.

The first supporting study was conducted to examine the association between prenatal exposure to magnesium sulfate and foetal brain injury. This study was initiated to determine whether maternal subcutaneous administration of the test material during pregnancy might affect foetal rat body and brain weight and size.

Two groups of pregnant Long-Evans rats were included (control and dose group, 22 animals per group). At 17 days of gestation, animals were injected subcutaneously with a loading dose of 270 mg/kg of the test material or saline. This was followed by further injections of 27 mg/kg every 20 minutes for 4 hours (second loading dose was repeated).

Tail venous blood was collected for magnesium levels at the beginning and conclusion of the injection period. At gestation day 20, rats were perfused and Caesarean sections were performed. Foetuses were delivered, perfused transcardially and the brains obtained intact. Foetal body and brain weight and size were measured.

Maternal exposure to the test material injection protocol resulted in increased blood magnesium levels from 1.85 ± 0.2 to 7.45 ± 1.2 mg/dL (p < 0.01).

Maternal subcutaneous administration of the test material had no significant effect on foetal rat brain and body weight and size. These data support the presumed safety of prenatal exposure to magnesium.

The second supporting study was conducted to assess the potential teratogenic effects of magnesium chloride in a study conducted using methodology equivalent to that outlined in the standardised guideline OECD 414.

Groups of 22 pregnant female Wistar rats were administered the test material via oral gavage for 10 days, from day 6 to day 15 of pregnancy. The dose levels were 200, 400 and 800 mg/kg/day; a control group was administered the vehicle, distilled water, in the same fashion.

Clinical conditions were observed daily. The body weight and food consumption of pregnant animals were measured on days 0, 1, 3, 6, 9, 12, 15, 17 and 20. Animals were sacrificed on day 20 of gestation and the uterine contents examined.

Gross malformation and the sex of live foetuses were examined and body weights were measured. For approximately half of the live foetuses from each pregnant animal, Alizarin red S skeletal specimens were prepared. The remaining live foetuses were supplied for internal organ observation. Gross dissection was used for the observation of head and abdomen, microdissection was used for thorax.

No mortality and no changes in clinical condition were noted in any group. No significant difference was noted in body weight and food consumption between the control group and the groups treated with the test material.

No significant difference was noted in the number of corpora lutea, implantation sites, implantation rate, number of live foetuses, sex ratio, foetal weight and mortality of implants between the control group and the groups treated with the test material.

No significant differences were noted for gross malformations, skeletal malformations or internal organ malformations between foetuses of control group animals and those from treated dams. 

Under the conditions of this study, it is considered that the test material does not cause teratogenicity in the rat by oral administration. The test material did not cause an increase of the incidence of foetal malformation even at 800 mg/kg/day, which is considered as the maximum dose level to cause no death in the pregnant rat. Also, it is estimated that an increased incidence of foetal malformation will not occur at higher dose levels, because no change was noted in the incidence of extra rib which is considered to be a potential index of teratogenicity at low dose levels.

The no observed effect level in the pregnant rat under the conditions of this study is considered to be 800 mg/kg/day. The no observed effect level in the foetal rat is considered to be 800 mg/kg/day as no significant change relating to the test material administration was noted.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to reproductive toxicity.