Magnesium hydroxide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-03-02 to 2010-09-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-03-02 to 2010-09-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

March 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The EPA Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commision Regulation (EC) No 440/2008 Part B:Methods for the Determination of Toxicity and other Heallth Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

October 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050( repeated Dose 28-day oral toxicity study in rodents)

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

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

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories france, L'Arbresle Cedex, France.
- Age at study initiation: Approximately 11 weeks
-Weight at study initiation: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on days 1 and 4. Live pups were weighed on days 1 and 4 of lactation.
-Health check: A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.
-Housing: Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18cm).
Mating: Females were caged together with males on a one-to-one basis in Macrolon cages ( MIII type, height 18cm)
Post-mating: Males were housed in their home cage (macrolon cages, MIV type, height 18cm) with a maximum of 5 animals per cage. Fema les were individually housed in Macrolon cages (MIII type, height 18cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages( MIII type, height 18cm)
General: Sterilised sawdust as bedding material and paper as cage-enrichment were supplied. During activity monitoring animals were ho used individually in Macrolon cages (MIII type; height 15cm) with sterilised sawdust as bedding material. No cage-enrichment was provide d during activity monitoring.
-Number of animals: 40 females and 40 males.
- Diet : free access to pelleted rodent diet.
- Water : Free access to tap water.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
Animals were housed in a controlled environment.
- Temperature (°C): 21 ± 3°C ( actual range: 19.7-21.7 °C)
- Humidity (%): A relative humidity of 40-70% (actual range: 34-73%)
- Air changes (per hr): Approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day.

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.

Vehicle:

water

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at NOTOX and on information provided by the sponsor.
- Method of Formulation: Formulations were prepared daily within 6 hours prior to dosing and were homogenised to a visually accepted level.
-Rationale for dose levels: Based on the results of a 10-day dose range finding study the dose levels for this combined 28-day oral gavage with reproduction/developmental toxicity screening test were selected to be 110, 330 and 1000 mg/kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

-Formulation analysis showed that the formulations were prepared accurately and homogenously.
Chemical analysis of dose preparations:
-Analyses were conducted during the treatment phase, according to a validated method. Samples of formulations were analysed for homogenity and accuracy of preparation. The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonsrated if the coefficient of variation was <= 10%.
Analytical conditions: Instrument Agilent 7500CE (Agilent Technologies, Tokyo, Japan)
Cone: Nickel or Platinum
Plasma forward power: 1500 W
Peripump flow rate: 0.08 rps
Nebulizer
Type :MicroMist
Material:quartz
Spray chamber
Material: quartz
Temperature: 15 °C
Torch :2.5 mm i.d.
Detection of Magnesium
Reaction gas: none
Integration time: 0.1 seconds per replicate
Replicates per analysis: 5
Quantitation m/z: 24

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-45 days i.e. during two weks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Females 41, 46, 48, 49 ( Group 1, table 1), 53, 59 ( Group 2, table 1), 61,62, 68 ( Group 3, table 1) and 76 ( Group 4, table 1) were not dosed during littering.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals are dosed up to the day prior to scheduled necropsy.
-Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

110 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

330 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

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

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:Based on the results of a 10-day dose range finding study the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 110, 330 and 1000 mg/kg.
- Rationale for animal assignment: This species and strain of rat has been recognised as appropriate for general and reproduction toxicity studies. The animal model has been proven to be susceptible to the effects of reproductive toxicants.

Observations and examinations performed and frequency:

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

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4 of lactation.

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


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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from the 5 animals/sex/group (see table 2) immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes isoflurane anaesthesia was used for blood collection
- Animals fasted: Yes animals were fasted overnight before blood sampling but w ater was provided.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected overnight ( approximately 15-24 hrs) from the 5 selected males/group ( see table 2).
- Metabolism cages used for collection of urine: Yes urine was collected into a specimen vial, using a metabolism cage.
- Animals fasted: Yes animals were deprived of food but water was provided.

Sacrifice and pathology:

PATHOLOGY: Yes
-Necropsy parental animals: All animals were fasted overnight prior to planned necropsy, but water was provided. Animals survivng to the scheduled necropsy were deeply anaesthetised using iso-flurane vapor and subsequently exsanguinated.
Necropsy was coducted on the following days:
Condition Day of necropsy
Females which delivered Lactation Days 5-6
Males Following completion of the mating period ( 29 days of dose administration).

All animals were subjected to macroscopic examination (including examination of the body surface, orifices and cranial, thoracic and abdominal tissues and organs and their contents), with special attention being paid to the reproductive organs. Pups survivng to planned termination were decapitated on lactation Day 5 or 6.
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
-The preserved organs and tissues of the selected 5 animals/sex of groups 1 and 4( see table 1).
-The additional slides of the testes of the selected 5 males of Groups 1 and 4 to examine staging of spermatogenesis( see table 2).
-All gross lesions of all animals ( all dose groups).
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 3; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 4) was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Clinical signs:

no effects observed

Description (incidence and severity):

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

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes occurred in haematological parameters of treated rats. Any statistically significant changes were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. These changes consisted of lower red blood cell counts in males at 1000 mg/kg, higher mean corpuscular volume (MCV) and/or higher mean corpuscular haemoglobin levels (MCH) in males at 110 and 1000 mg/kg, higher reticulocyte counts in females at 110 mg/kg (due to female no.52) and lower mean corpuscular haemoglobin concentration (MCHC) in females at 110 and 100 mg/kg.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes in clinical biochemistry parameters distinguished treated animals from control animals:
-Lower total protein levels in males at 330 and 1000 mg/kg,
-Lower albumin levels in males at 1000 mg/kg
-Lower calcium levels in males at 330 and 1000 mg/kg.
Means of these changes only just exceeded or remained within the range considered normal for rats of this age and strain.
Any other statistically significant changes were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related trend and/or remained within the range considered normal for rats of this age and strain. These included lower or higher urea levels in males at 110 and 330 mg/kg respectively, higher potassium values in males at 110 and 330 mg/kg, and higher cholesterol and potassium levels in females at 110 and 330 mg/kg respectively.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

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

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in organ weights and organ to body weight ratios.
The significantly lower absolute thymus weight observed for females at 1000 mg/kg was considered to be due a low weight for female no. 72. Individual weights of other females of this dose group remained within the range observed among other treated females. The higher testes to body weight ratio of males at 110 mg/kg and the lower liver to body weig ht ratios of males at 110 and 1000 mg/kg occurred in the absence of a dose-related trend and means remained within the normal range for rats of this age and strain. These changes were therefore considered to be of no toxicological relevance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Incidental findings among control and treated animals included red foci on the stomach glandular mucosa or thymus, red discoloration of the mandibular lymph node and thymus, reduced size of the thyroid, a gray-white focus on or dark red discoloration of the papillary process of the liver, papillary process grown together with the left lateral lobe, enlarged clitoral glands, greenish soft nodule or tan focus on the clitoral glands, a yellowish hard or soft nodule on the tail of the epididymis, pelvic dilation of the kidneys and alopecia. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain and did not show a dose-related trend. These necropsy findings were therefore considered to be of no toxicological relevance.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

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

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Table 4: Clinical signs Males parental animals

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

 

Table 5: Clinical signs Females parental animals

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

Key:

G: Median value of the highest individual daily grades

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

-        : observation performed, no sign detected

Table 6: Body weights (g) Summary: Parental Males/Females 

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

 

Table 7: Food consumption (G/Animal/Day) Males/Females

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

 

 

Conclusions:

Based on the results obtained from a combined 28-day repeated dose study with the reproduction/developmental toxicity screening test conducted according to OECD 422, a NOAEL of 1000 mg/kg/day was determined.

Executive summary:

In a combined 28-day repeated dose study with the reproduction/developmental toxicity screening test conducted according to OECD 422, magnesium hydroxide (99.9% purity) was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 0, 110, 330 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41- 45 days).

Formulation analysis showed that the formulations were prepared accurately and homogenously.

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

relevance.

Overall, no toxicologically relevant changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

Based on these results, the NOAEL is considered to be 1000 mg/kg/day, the highest dose administered in this study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

March 1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 1995

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Magnesium hydroxide

EC Number:

215-170-3

EC Name:

Magnesium hydroxide

Cas Number:

1309-42-8

Molecular formula:

H2MgO2

IUPAC Name:

magnesium dihydroxide

Specific details on test material used for the study:

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

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Route=oral

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

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

Details on mating procedure:

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Duration of treatment / exposure:

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

Frequency of treatment:

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

Details on study schedule:

Male number paired with, mating date, confirmation of pregnancy, and delivery date were all recorded. Pregnant females were all examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care ( such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

110 mg/kg bw/day

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

330 mg/kg bw/day

Remarks:

Mid dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose

No. of animals per sex per dose:

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

Control animals:

yes, concurrent no treatment

Details on study design:

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

Examinations

Parental animals: Observations and examinations:

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

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

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

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

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

Sperm parameters (parental animals):

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

Litter observations:

STANDARDISATION OF LITTERS
- Each litter was examined.

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

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

Postmortem examinations (parental animals):

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

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

Postmortem examinations (offspring):

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

Statistics:

For each group the following calculations were performed:

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

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

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

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

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

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

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

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

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

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

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

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

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

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

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

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

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

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

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

REPRODUCTIVE PERFORMANCE: No toxicologically relevant effects on gestation index and duration, parturation, maternal care and early postnatal pup development ( mortality, clinical signs, body weight and macroscopy) were observed.
The gestation index was 100% for all groups and the duration of gestation was similar between control and treated groups.
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

No observed adverse effect level

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Effect levels (P1)

Remarks on result:

not measured/tested

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

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

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

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

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Histopathological findings:

no effects observed

Other effects:

not examined

Description (incidence and severity):

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no developmental toxicity was observed at any dose level

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Remarks on result:

not measured/tested

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 4: Clinical signs Males parental animals:

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

 

Table 5: Clinical signs Females parental animals:

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

Key:

G: Median value of the highest individual daily grades

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

- observation performed, no sign detected

Table 6: Reproduction Data

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

 

Table 7: Body weights (gram) Summary

Parental Males/Females:

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

 

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

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

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

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

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

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

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