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Diss Factsheets

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:
24 October 2006 - 04 April 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
12-aminododecanoic acid
EC Number:
211-754-7
EC Name:
12-aminododecanoic acid
Cas Number:
693-57-2
Molecular formula:
C12H25NO2
IUPAC Name:
12-aminododecanoic acid
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Description: White powder
Storage conditions: 4 °C in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Sprague-Dawley
- Age at study initiation: approximately 8 weeks.
- Weight at study initiation: males: 257 - 318 g; females: 198 - 255 g.
- Housing: Initially, all animals were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. During the mating phase for the non-recovery dose groups, animals were transferred to similar cages 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 polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes (Datesand Ltd. Cheshire, UK). Recovery animals were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper.
- Diet (e.g. ad libitum): ad libitum. A pelleted diet (Rodent PMI 5002 (Certified) diet, BCM IPS Limited, London, UK) was used throughout the study.
- Water (e.g. ad libitum): ad libitum. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 14 days.

The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
Environmental enrichment was provided in the form of wooden chew blocks (B & K Universal Ltd, Hull, UK) and cardboard fun tunnels (Datesand Ltd, Cheshire, UK) except for mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 15% relative
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
The stability and homogeneity of the test material formulations were determined analytically and show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately at 4°C in the dark.
The vehicle was chosen due to the complex nature of the test material and its limited solubility in organic and aqueous media.

Treatment Group Dose Level Treatment Volume Concentration
(mg/kg/day) (mL/kg) (mg/mL)
Control 0 4 0
Low 50 4 12.5
Intermediate 250 4 62.5
High 1000 4 250
Recovery Control 0 4 0
Recovery High 1000 4 250

The test material was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg/day of Arachis oil BP.

The volume of test and control material administered to each animal was based on the most recent scheduled bodyweight and was adjusted at regular intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical Analysis of Test Material Formulations, Methods and Results
METHOD OF ANALYSIS
Due to the complex nature of the test material and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed. The concentration of 12-Aminododecanoic acid in the test material formulations was determined using a gravimetric technique.

-Samples
The test material formulations were weighed into tared glass sintered crucibles and then rinsed with acetone to leave a test material residue. The samples were then dried in an oven at approximately 105 °C before being allowed to cool over silica gel in a desiccator and re-weighed.
-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 +4 °C in the dark for fourteen days.
The analytical procedure is considered to be non stability indicating, however the residue was subjected to an FTIR scan and the results suggest the test material is stable in the formulation.
-Verification of Test Material Formulation Concentrations
The test material formulations were sampled and analysed within two days of preparation

Homogeneity of Test Material Formulations:

Nominal Concentration Sampling Location Concentration Found (mg/mL)
(mg/mL) 1 2 3 Mean
Top 13.2 12.7 13.4 13.1
12.5 Middle 11.5 13.1 13.2 12.6
Bottom 13.5 13.1 13.4 13.3
Top 250 245 246 247
250 Middle 246 242 236 241
Bottom 248 235 224 236


Stability of Test Material Formulations:

Nominal Concentration Concentration Found Initially Concentration Found After Storage for Fourteen Days
(mg/mL) (mg/mL) (mg/mL) (expressed as % of initial)
12.5 13.0 11.5 88
250 241 248 103


Verification of Concentration of Weekly Test Material Formulation:

Week No. Nominal Concentration Concentration Found
(mg/mL) (mg/mL) (expressed as % of nominal)
0 ND -
12.5 12.1 97
1 62.5 58.8 94
250 243 97

0 ND -
12.5 12.7 101
2 62.5 61.1 98
250 246 98

0 ND -
12.5 12.7 99
3 62.5 62.0 99
250 245 98

0 ND -
12.5 12.9 103
4 62.5 65.3 104
250 248 99

0 ND -
12.5 12.5 100
5 62.5 61.0 98
250 246 98

0 ND -
12.5 13.3 107
6 62.5 64.0 102
250 246 98

0 ND -
12.5 10.8 87
7 62.5 61.2 98
250 241 96

ND = none detected
- = not applicable

METHOD VALIDATION
Specificity
A blank Arachis Oil BP (control) was analysed. The analysis of the blank Arachis Oil BP (control) produced no signal that interfered with the signal due to the test material.

Accuracy

Samples of Arachis Oil BP were accurately fortified with known amounts of test material, and analysed:
Fortification Concentration Found Recovered Mean Recovery
(mg/g) (mg/g) (%) (%)
12.6 12.9 103 103
12.9 13.6 104
244 234 96 97
244 239 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:
Up to 54 consecutive days (42 days for the recovery group).
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 250 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose in the treatment groups.
5 animals per sex per dose in the recovery groups.
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were chosen based on the results of a preliminary range-finder.

The test material was administered by gavage to three groups each of ten male and ten female rats for up to fifty-four consecutive days, at dose levels of 50, 250 and 1000 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for forty-two consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating and at termination on five selected non-recovery males and females from each dose group. In addition, haematology and blood chemistry were evaluated after the fourteen day treatment free period on all recovery animals. Urinalysis was evaluated on five selected non-recovery males at the end of the treatment period and for all recovery group males at the end of the treatment free period.

Recovery Groups
-Groups of five male and five female rats were dosed according to dose group continuously up to the point of sacrifice of non-recovery males at which time treatment was discontinued.
-The males and females were maintained without treatment for a further fourteen days.
-Urinalysis was performed for all males during the final week of recovery.
-After fourteen days of recovery males and females were killed and examined macroscopically.

Examinations

Observations and examinations performed and frequency:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before and after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). During the treatment-free period, recovery animals were observed twice daily (once daily at weekends). All observations were recorded.

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females per non-recovery dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose-built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin colour
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behaviour Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation

Functional Performance Tests
-Motor Activity: Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period).

-Forelimb/Hindlimb Grips Strength: An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

- Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli:
Grasp response Touch escape
Vocalisation Pupil reflex
Toe pinch Startle reflex
Tail pinch Blink reflex
Finger approach

-Bodyweight
Individual bodyweights were recorded on Day 0 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Recovery animals were weighed on Day 1 (prior to dosing) and then weekly until termination.

-Food Consumption
During the maturation period, weekly food consumption was recorded for each cage of non-recovery adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum. Weekly food consumptions were performed weekly for each cage of recovery adults throughout the study period.

Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively for males throughout the study period and for females during maturation and the first two weeks of gestation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the final week of gestation and during lactation.

-Water Consumption
Water intake was measured gravimetrically throughout the study period, via the daily weighing of water bottles.

-Laboratory Investigations
Haematological and blood chemical investigations were performed on five males and five females selected from each non-recovery test and control group on Day 14 (day prior to pairing) and at termination (Day 43). In addition haematological and blood chemical investigations were performed on all recovery group animals after the fourteen day treatment free period at termination (Day 57). Blood samples were obtained from the lateral tail vein at Day 14 and by cardiac puncture at termination. Animals were not fasted prior to sampling. Urinalytical investigations were performed on five non-recovery males from the control and each test group during the final week of treatment and on all recovery males during the final week of the recovery period. Urine samples were collected overnight by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.

- Haematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices:
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count:
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic): Cresyl blue stained slides were prepared but reticulocytes were not assessed
Methaemoglobin (Meth)

Prothrombin time (CT) was assessed by ‘Thrombomax HS with calcium’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

- Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Calcium (Ca++)
Glucose Inorganic phosphorus (P)
Total protein (Tot.Prot.) Aspartate aminotransferase (ASAT)
Albumin Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation) Alkaline phosphatase (AP)
Sodium (Na+) Creatinine (Creat)
Potassium (K+) Total cholesterol (Chol)
Chloride (Cl-) Total bilirubin (Bili)

-Urinalysis
The following parameters were measured on collected urine:
Volume Ketones
Specific Gravity Bilirubin
pH Urobilinogen
Protein Reducing Substances
Glucose Blood
Sacrifice and pathology:
Adult non-recovery males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult non-recovery females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.

In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted.

Recovery animals were killed by intravenous overdose of sodium pentobarbitone followed by exanguination fourteen days after the termination of treatment (Day 57).

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.

- Organ Weights
The following organs, removed from the five selected males and parental females from each group that were killed at the end of the study, were dissected free from fat and weighed before fixation.:
Adrenals Liver
Brain Spleen
Heart Kidneys
Thymus

The reproductive organs shown below were weighed from all animals that were killed at the end of the study:
Ovaries
Epididymides
Testes

- Histopathology
Samples of the following tissues were preserved from five males and five females from each dose group, in buffered 10% formalin.
Adrenals Mammary gland
Aorta (thoracic) Muscle (skeletal)
Bone & bone marrow (femur including stifle joint) Pancreas
Bone & bone marrow (sternum) Oesophagus
Brain (including cerebrum, cerebellum and pons) Rectum
Caecum Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Skin (hind limb)
Eyes Spinal cord (cervical)
Gross lesions Spleen
Heart Stomach
Ileum Thyroid/parathyroid
Jejunum Trachea
Kidneys Thymus
Liver Urinary bladder
Lungs (with bronchi) # Vagina
Lymph nodes (cervical and mesenteric)

# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

The tissues shown below were also removed from the remaining animals:
Coagulating gland Prostate
Epididymides* Seminal vesicles
Ovaries Testes *
Pituitary Uterus/Cervix

* = preserved in Bouin’s fluid and then in 70% IMS after forty-eight hours

All tissues were despatched to Propath UK Ltd (Principal Investigator: N Candy). The tissues from five selected 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 5 µm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues from the remaining control and 1000 mg/kg/day were also processed.

Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of kidney from five animals per sex in the low and intermediate groups and all recovery group animals.

Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.
Statistics:
The following parameters were subjected to statistical analysis:
Bodyweight and bodyweight change
Food consumption during gestation and lactation
Litter data
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.
Non-parametric methods were 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)

Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:
-Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
-Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.

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:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Daily gravimetric measurements revealed an increase in water consumption for animals of either sex treated with 1000 or 250 mg/kg/day, but not for animals of either sex treated with 50 mg/kg/day.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Females treated with 1000 or 250 mg/kg/day showed elevations in plasma urea during blood chemical analysis carried out at Day 14. Females treated with 1000 mg/kg/day continued to show the elevation in plasma urea at termination.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see below
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below
Histopathological findings: neoplastic:
not examined
Details on results:
-Mortality
There were no unscheduled deaths during the study.
-Clinical Observations
There were no toxicologically significant clinical observations detected.
-Behavioural Assessment
Weekly open field arena observations did not reveal any treatment-related effects during the study.
All inter and intra groups variations in urination, defecation and transfer arousal scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.
-Functional Performance Tests
No treatment-related effects were detected in the functional performance parameters investigated. Statistical analysis of the data did not reveal any significant inter group differences.
- Sensory Reactivity Assessments
No treatment-related effects were detected in sensory reactivity.
All inter and intra group differences in sensory reactivity scores were considered to be result of normal variation for arts of the strain and age used and were of no toxicological importance.
-Bodyweight
No treatment related effects on bodyweight or bodyweight development was detected.
-Food Consumption
No treatment-related effect on dietary intake or food efficiency was detected for all treated animals throughout the study period.
-Water Consumption
Daily gravimetric measurements revealed an increase in water consumption for animals of either sex treated with 1000 or 250 mg/kg/day, but not for animals of either sex treated with 50 mg/kg/day.

-Haematology
Statistically significant reductions in haemoglobin, haematocrit and erythrocyte count were detected for males treated with 1000 mg/kg/day at Day 14 of the study; this resulted in reductions being detected in mean cell haemoglobin and mean cell volume. The reductions in haematocrit, mean cell haemoglobin and mean cell volume were also detected for 250 mg/kg/day males at Day 14. No such observations were detected for treated females during the Day 14 haematological analysis.
Haematological analysis carried out for non-recovery dose groups at termination (Day 43 for males and Day 5 post partum for females) revealed statistically significant reductions in haemoglobin, mean cell haemoglobin and mean cell volume for animals of either sex treated with 1000 mg/kg/day. Females from this treatment group showed reductions in haematocrit. The reduction in mean cell haemoglobin and mean cell volume extended in the male 250 mg/kg/day.
Recovery group females treated with 1000 mg/kg/day showed statistically significant reductions in haemaglobin and haematocrit.

-Blood Chemistry
Females treated with 1000 or 250 mg/kg/day showed elevations in plasma urea during blood chemical analysis carried out at Day 14. Females treated with 1000 mg/kg/day continued to show the elevation in plasma urea at termination.

- Urinalysis
No toxicologically significant effects were detected in treated animals when compared to controls. Statistical analysis of the quantitative data did not reveal any significant intergroup differences.

Pathology
-Organ Weights
Females treated with 1000 mg/kg/day had elevated absolute kidney weights, whilst these females, 1000 mg/kg/day males and females treated with 250 mg/kg/day had elevated kidney weight relative to bodyweight. Elevations in absolute kidney weight were also detected for animals of either sex treated with 1000 mg/kg/day after the fourteen day treatment free period. Females treated with 1000 mg/kg/day also displayed elevated kidney weights relative to terminal bodyweight.

-Histopathology
The following treatment-related changes were observed:
KIDNEY: A greater incidence and generally higher grades of severity of groups of basophilic tubules, the presence of tubular dilatation, hypertrophy of the epithelium of collecting ducts, and hyperplasia of the renal pelvic/papillary epithelium, were all observed in relation to treatment for animals of either sex treated with 1000 mg/kg/day. In addition, and for females only at this dose level, cortico-medullary mineralisation was observed and acute inflammatory cell foci were seen in the renal papilla. Similar changes but with lesser prevalence and generally higher grades of severity were seen for animals of either sex treated with 250 mg/kg/day but not at 50 mg/kg/day.
Hypertrophy of the collecting duct epithelium remained among Recovery 1000 mg/kg/day animals of either sex following an additional fourteen days without treatment. Other treatment-related changes had largely regressed although globular accumulations of eosinophilic material, similar in appearance to α-2 microglobulin deposits, were observed for four male rats in the Recovery 1000 mg/kg/day group but not among Recovery Control male rats. α-2 Microglobulin is exclusive to the kidney of male rats.

Effect levels

Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: kidney, haematological and blood chemical changes

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Results Tables

Key:

* = statistically different from control group p<0.05

** = statistically different from control group p<0.01

*** = statistically different from control group p<0.001

 

Haematology for Males - Group Mean Values

Non-Recovery Day 14

Dose Level (mg/kg/day)

Number of Animals

Hb

(g/dL)

RBC (1012/L)

Hct

(%)

MCH

(pg)

MCV

(fL)

MCHC (g/dL)

WBC (109/L)

0 (Control)

5  mean

         sd 

15.8

0.5

7.49

0.21

44.7

1.2

21.1

0.3

59

1

35.3

0.4

11.8

3.2

50

5  mean

         sd 

15.1

0.6

7.44

0.35

42.9

1.6

20.4

0.6

57

1

35.5

0.4

11.6

3.1

250

5  mean

         sd 

14.9

0.7

7.54

0.27

42.1*

2.0

19.9**

0.2

56**

1

35.5

0.6

12.3

2.0

1000

5  mean

         sd 

14.0***

0.4

6.97*

0.19

39.7***

0.8

20.1**

0.5

57*

1

35.2

0.6

14.5

4.4

 

Non-Recovery Day 43

Dose Level (mg/kg/day)

Number of Animals

Hb

(g/dL)

RBC (1012/L)

Hct

(%)

MCH

(pg)

MCV

(fL)

MCHC (g/dL)

WBC (109/L)

0 (Control)

5  mean

         sd

14.4

0.5

7.82

0.36

43.0

1.6

18.5

0.5

54

1

35.5

0.2

7.3

2.1

50

5  mean

         sd 

14.1

0.5

7.84

0.15

42.3

1.5

18.0

0.4

53

1

33.4

0.5

6.2

3.4

250

5  mean

         sd 

14.1

0.6

8.17

0.27

42.4

2.1

17.3**

0.4

51**

1

33.4

0.3

9.8

2.9

1000

5  mean

         sd

13.3*

0.6

7.71

0.22

40.3

1.7

17.3**

0.4

52*

1

33.1

0.1

8.5

3.5

 

Haematology for Females - Group Mean Values

Non-Recovery Day 5post partum

Dose Level (mg/kg/day)

Number of Animals

Hb

(g/dL)

RBC (1012/L)

Hct

(%)

MCH

(pg)

MCV

(fL)

MCHC (g/dL)

WBC (109/L)

0 (Control)

5  mean

         sd 

12.2

0.6

6.22

0.40

37.2

2.1

19.7

0.4

60

2

32.9

0.6

7.4

0.9

50

5  mean

         sd 

12.0

0.7

6.29

0.38

36.3

2.6

19.1

0.6

57

2

33.0

0.5

6.8

2.7

250

5  mean

         sd

11.8

0.9

6.12

0.75

36.0

3.0

19.4

1.3

59

3

32.8

0.4

8.0

2.3

1000

5  mean

         sd

10.7**

0.3

5.82

0.25

32.5*

1.1

18.3*

0.5

55*

1

32.8

0.2

8.5

2.9

 

Blood Chemistry for Females – Group Mean Values

Non-Recovery Day 14

Dose Level (mg/kg/day)

Number of Animals

Urea

(mg/dL)

Glucose

(mg/dL)

Tot. Prot.

(g/dL)

Albumin

(g/dL)

A/G Ratio

Na+ (mmol/L)

K+ (mmol/L)

Cl- (mmol/L)

0 (Control)

5 mean

        sd  

39

2

165

13

6.40

0.48

3.86

0.25

1.52

0.08

146

2

5.25

0.50

105

1

50

5 mean

        sd  

41

3

165

13

6.37

0.25

3.75

0.20

1.43

0.11

146

1

4.79

0.59

104

2

250

5 mean

        sd 

48*

6

157

8

6.38

0.22

3.75

0.11

1.43

0.04

150

3

4.93

0.50

104

4

1000

5 mean

        sd 

65

23

152

11

6.03

0.21

3.54

0.16

1.42

0.13

148

1

5.08

0.54

105

1

 

Dose Level (mg/kg/day)

Number of Animals

Ca++ (mmol/L)

P (mmol/L)

ASAT (IU/L)

ALAT (IU/L)

AP (IU/L)

Creat (mg/dL)

Chol (mg/dL)

Bili (mg/dL)

0 (Control)

5 mean

        sd 

2.84

0.08

1.9

0.2

74

5

45

6

308

108

0.91

0.05

76

17

0.08

0.05

50

5 mean

        sd 

2.81

0.15

2.0

0.2

81

4

44

7

294

112

0.88

0.06

70

12

0.11

0.08

250

5 mean

        sd 

2.79

0.10

2.0

0.2

79

4

37

7

336

91

0.94

0.01

60

5

0.11

0.05

1000

5 mean

        sd 

2.86

0.16

1.8

0.2

75

3

36*

3

461

161

1.09**

0.12

65

13

0.12

0.03

 

Non-Recovery Day 5post partum

Dose Level (mg/kg/day)

Number of Animals

Urea

(mg/dL)

Glucose

(mg/dL)

Tot. Prot.

(g/dL)

Albumin

(g/dL)

A/G Ratio

Na+ (mmol/L)

K+ (mmol/L)

Cl- (mmol/L)

0 (Control)

5 mean

        sd  

47

3

136

13

5.93

0.20

3.39

0.05

1.34

0.10

152

1

5.25

0.50

105

1

50

5 mean

        sd 

44

10

139

5

6.17

0.37

3.45

0.15

1.28

0.13

150

2

4.79

0.59

104

2

250

5 mean

        sd 

51

15

143

7

5.75

0.31

3.20

0.17

1.25

0.05

152

4

4.93

0.50

104

4

1000

5 mean

        sd 

71**

9

131

16

5.80

0.18

3.15*

0.11

1.19

0.09

153

3

5.08

0.54

105

1

 

Dose Level (mg/kg/day)

Number of Animals

Ca++ (mmol/L)

P (mmol/L)

ASAT (IU/L)

ALAT (IU/L)

AP (IU/L)

Creat (mg/dL)

Chol (mg/dL)

Bili (mg/dL)

0 (Control)

5 mean

        sd 

2.51

0.13

1.8

0.2

71

5

62

6

229

60

0.91

0.04

69

9

0.15

0.01

50

5 mean

        sd 

2.48

0.26

1.8

0.2

72

9

64

18

260

168

0.90

0.06

72

16

0.15

0.01

250

5 mean

        sd 

2.44

0.16

1.4

0.5

73

12

53

20

209

76

0.92

0.08

56

4

0.14

0.02

1000

5/4mean

        sd 

2.45

0.14

1.6

0.3

74

7

60

11

288

144

0.93

0.04

61

6

0.09

0.05

†= one value for both individual calcium ions and potassium ions was omitted as they were considered erroneous

Group Mean Absolute Organ Weights and Standard Deviations (SD) – Males

Non-Recovery

Dose Level (mg/kg/day)

Number of Animals

Organ Weight (g)

Kidneys

Liver

Spleen

Testes

Thymus

0 (Control)

5 (10) mean 

sd

3.2643

0.4656

15.6226

2.1559

0.7634

0.1037

3.2414

0.4246

0.4376

0.1169

50

5 (10) mean 

sd

3.0927

0.4757

15.2546

3.2234

0.7029

0.1213

3.2038

0.3831

0.4689

0.1250

250

5 (10) mean 

sd

3.2472

0.1348

14.5800

0.6494

0.7005

0.0869

3.2319

0.3608

0.4845

0.0706

1000

5 (10) mean 

sd

3.6122

0.4802

14.3515

1.5678

0.7599

0.1705

3.3582

0.1933

0.4134

0.1441

( ) = number of animals used to calculate mean/sd bodyweights and reproductive organ weights

Group Mean Absolute Organ Weights and Standard Deviations (SD) – Females

Non-Recovery

Dose Level (mg/kg/day)

Number of Animals

Organ Weight (g)

Kidneys

Liver

Spleen

Testes

Thymus

0 (Control)

5 (10) mean 

sd

2.1723

0.2652

13.8595

1.8870

0.5708

0.0644

-

-

0.3138

0.0616

50

5 (10) mean 

sd

2.1211

0.2521

13.5250

1.3099

0.6444

0.0648

-

-

0.2798

0.0540

250

5 (10) mean 

sd

2.4516

0.2278

13.0556

0.9520

0.6697

0.0527

-

-

0.2377

0.0853

1000

5 (10) mean 

sd

3.4025***

0.4220

15.2667

1.8641

0.6529

0.0736

-

-

0.2086

0.0385

( ) = number of animals used to calculate mean/sd bodyweights and reproductive organ weights

Applicant's summary and conclusion

Conclusions:
The oral administration of the test material to rats by gavage, at dose levels of 1000, 250 and 50 mg/kg/day, resulted in treatment related effects at 1000 and 250 mg/kg/day. The kidney, haematological and blood chemical changes were considered to represent an adverse health effect. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 50 mg/kg/day.
Executive summary:

The oral administration of the test material to rats by gavage, at dose levels of 1000, 250 and 50 mg/kg/day, resulted in treatment related effects at 1000 and 250 mg/kg/day. The kidney, haematological and blood chemical changes were considered to represent an adverse health effect. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 50 mg/kg/day. The study was conducted in accordance with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).