Magnesium diethyl dicarbonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 March 2010 - 14 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to guidelines.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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)

Deviations:

no

Test guideline 2

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

Deviations:

no

Test guideline 3

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

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

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

Details on test material:

- Name of test material: Magnesium hydroxide
- Molecular formula: Mg(OH)2
- Molecular weight: 58.32
- Stability in water: Stable
- Description: White powder
-Test substance storage: At room temperature in the dark
-Stability under storage conditions: Stable
-Solubility in water: 0.00014 mol/L
-Hygroscopic: Yes, store in closed vessel
-pH: ±10 (10%/H20)

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
-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.

Administration / exposure

Route of administration:

oral: gavage

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 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 ( 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

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg
Basis:
other: control

Doses / concentrations 2

Remarks:

Doses / Concentrations:
110 mg/kg/day
Basis:
nominal in water

Doses / concentrations 3

Remarks:

Doses / Concentrations:
330 mg/kg/day
Basis:
nominal in water

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal in water

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.

Examinations

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.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:No mortality occurred during the study period. 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.

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

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

HAEMATOLOGY: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 treament-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 CHEMISTRY: 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.

URINALYSIS: 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 treatment-related trend.

ORGAN WEIGHTS: 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 1000mg/kg was considered to be due to a low weight for female no. 72. Individual wieghts of other female 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 weight ratios of males at 110 and 1000mg/kg occurred in the absence of a dose related trend and mean remained within the normal range for rats of this strain and age. These changes were therefore considered to be of no toxicological relevance.


GROSS PATHOLOGY: HISTOPATHOLOGY: The following slides were examined by a pathologist:
-The preserved organs and tissues of the selected 5 animals/sex of groups 1 and 4.
-The additonal slides of the testes of the selected 5 males of Groups 1 and 4 to examine staging of spermatogenesis.
-All gross lesions of all animals ( all dose groups).
All abnormalities were described and included in the report.

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

MACROSCOPIC EXAMINATION: Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a reult of any treatment. Incidental findings among control and treated animals included red foci on the stomach glandular mucosa or thymus, red discoloration of the mandibular lymh 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 clitoralglands, 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.

MICROSCOPIC EXAMINATION: There were no treatment related micrscopic findings.Recorded microscopic findings were within the range of background pathology encountered in Wistar (Hans) 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.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical 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: 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

 

 

Applicant's summary and conclusion

Conclusions:

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg/day was determined.

Executive summary:

Parental Results: 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.