Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 January 2017 - 27 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Current requirements of the Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 114
- Purity: 98.4%
- Expiration date of the lot/batch: Restest 12 October 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Sealed container, refrigerated (2 to 8 °C), in the dark
- Storage condition of formulated test material: Sealed container, room temperature (15 to 25 °C), in the dark
- Stability under test conditions: Stable for 48 days

VEHICLE
- Vehicle: Polyethylene glycol 400 (PEG 400)
- Supplier: Merck

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Sex: female
- Age at mating: 8 to 10 weeks old at mating
- Weight at study initiation (gestation day 3): 171.5 and 252.0 g
- Fasting period before study: No
- Housing: Animals were housed individually. Bedding, Aspen wood chips, was changed weekly. Animals were provided with wooden Aspen chew blocks and nesting materials as forms of environmental enrichment.
- Diet: ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 9-27 January 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Preparation: Formulations were stirred continuously before dosing commenced (excluding those for the control article [vehicle]) and throughout dosing.
- Rate of preparation of dose (frequency): Formulations were prepared daily.
- Storage temperature of prepared doses: Formulations were stored at room temperature (15 to 25 °C) in a sealed container, protected from the light.

DOSE VOLUME: 10 mL/kg bw

VEHICLE
- Concentration in vehicle: 0, 4, 12.5 and 40 mg/mL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations of the test material, prepared at 4 and 40 mg/mL (in Polyethylene Glycol 400 (PEG400)), were analysed to determine homogeneity and stability. Formulations were determined to be homogeneous and stable for 50 days when stored at 2 to 8 °C.
Formulations prepared for use on the first and last day of dosing were analysed to determine the achieved concentration. The mean % target range for the preparation of formulations was 90 to 110 % of nominal. Results were within this range. Test material was not detected in Group 1 control samples. The analytical procedure was validated and all acceptance criteria for the method were met.

Triplicate samples of 3 mL were removed from the top, middle and bottom of the test material formulations per Group and control formulations and were analysed by gas chromatography (GC) to determine concentration. Samples were diluted in methanol and analysed against a standard.

GC SYSTEM PARAMETERS
Column (analytical): DB1, 30 m x 0.32 mm id, 0.25 µm df
Column (guard): Deactivated, 1 m x 0.32 mm id
Inlet liner: Single taper, deactivated split liner with glass wool, e.g. Restek Sky® 23309
Carrier gas: Helium @ 2.5 mL/minute constant flow
Oven temperature program: 80 °C hold for 10 minutes; 10 °C/minute t 150 °C, hold for 2 minutes; 25 °C/minute to 300 °C, hold for 10 minutes
Injection volume: 1 µL
Run time: 25 minutes
Inlet temperature: 300 °C
Split ratio: 10:1
FID temperature: 350 °C
Detector gas flow rates: hydrogen 40 mL/minute; Air: 350 mL/minute; Makeup (nitrogen) 35 mL/minute
Injector wash solvent: Methanol (3 washes pre and post injection). 3 sample washes and 6 sample pumps prior to injection
Details on mating procedure:
Mating was carried out at the supplier's laboratory. Animals were delivered to testing facility on gestation day 3.
- Impregnation procedure: cohoused
- Length of cohabitation: 1 night
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
Daily from gestation day 6 - 20
Frequency of treatment:
Daily
Duration of test:
Females were maintained to gestation day 21
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
40 mg/kg bw/day
Dose / conc.:
125 mg/kg bw/day
Dose / conc.:
400 mg/kg bw/day
No. of animals per sex per dose:
22 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high dose level of 400 mg/kg/day was selected. Maternal toxicity was expected on the basis of some increased organ weights and haematological and splenic effects (secondary to the haematological effects) and microscopic evaluation of the kidney (considered part of the process producing relative weight increases) noted at 200 and 1000 mg/kg bw/day in a 28 day rat gavage study (Hita Research Laboratories, Japan Study Number D-3213). These parameters were not evaluated in this study.
The intermediate dose of 125 mg/kg/day was selected as it approximates the top dose/root 10. It was expected to be a maternal no observed effect level (NOEL) on the basis of a 28 day gavage study (Hita Research Laboratories, Japan Study Number D 3213).
The low dose of 40 mg/kg/day was selected as it approximates the mid dose/root 10 and was included in the study as a potential NOEL if effects were observed at the intermediate-dose level.
- Rationale for animal assignment (if not random): Animals were assigned to dose groups based on their body weights at gestation day 0.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from the start of dosing until necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on GD 3, 6, 9, 12, 15, 17, 20, and 21.
Carcass Weight = Terminal body weight - uterus weight
Total Weight Change = Day 21 body weight - Day 3 body weight

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Determined daily from GD 3; calculated as g/animal/day.

HAEMATOLOGY: Yes
- Time schedule for examinations: Terminal blood samples for haematology (1 x 0.5 mL [EDTA], nominal) and coagulation (1 x 0.5 mL [trisodium citrate], nominal) were withdrawn from the abdominal aorta immediately prior to exsanguination from 11 animals per group.
- Parameters examined: Haemoglobin concentration, red blood cell count, packed cell volume, reticulocyte count, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, red cell distribution width, haemoglobin distribution width, total and differential white cell count, platelet count, platelet crit, mean platelet volume, platelet distribution width, prothrombin time, activated partial thromboplastin time and fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for examinations: Terminal blood samples for clinical chemistry (1 x 0.6 mL [lithium heparin] were withdrawn from the abdominal aorta immediately prior to exsanguination from 11 animals per group.
- Parameters examined: Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, sodium, potassium, calcium, inorganic phosphate, chloride, total protein, albumin, globulin, albumin:globulin ratio, total cholesterol, glucose, urea, total bilirubin and creatinine.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Food was not removed prior to scheduled necropsies. Females were sacrificed on GD 21 by cervical dislocation following isoflurane anaesthesia; death was confirmed by exsanguination. For females subjected to clinical pathology (haematology and clinical chemistry) following collection of the terminal blood samples animals were terminally sacrificed (under anaesthesia) by exsanguination.
- Examinations: Animals were weighed before necropsy. Animals were examined macroscopically. All lesions were recorded and retained in the relevant fixative. The ovaries and uteri were removed and examined. The uterus of any apparently non-pregnant female was immersed in a 10 % ammonium sulphide solution to reveal any evidence of implantation. For females subjected to clinical pathology. The spleen was dissected, freed from fat and other contiguous tissue, and weighed before fixation in 10 % neutral-buffered formalin.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Terminal body weight (recorded for adjusted gravid uterus weight calculations only and have not been reported). Early intrauterine deaths were classified as those which showed decidual or placental tissue only. Late intrauterine deaths showed embryonic or foetal tissue in addition to placental tissue. Dead foetuses were classified as those which appeared to have died shortly before necropsy. Implantations and foetuses were allocated numbers relating to their position in utero.
Fetal examinations:
- External examinations: Yes: all per litter
Approximately one half of the foetuses in each litter were dissected, and the viscera were examined. They were then eviscerated, and the carcasses were cleared in potassium hydroxide. The skeletons were stained with Alizarin Red S, retained in glycerol/propylene glycol, and examined for skeletal abnormalities.
The remaining foetuses were fixed in Bouin's fluid, and the viscera examined. The foetuses fixed for visceral examination were retained in the relevant fixative.
Fetal abnormalities were classified as malformations (rare and/or potentially lethal defects) and variations (commonly occurring non-lethal abnormalities).
Statistics:
Body weight, body weight gains, food consumption, clinical pathology parameters, absolute organ weights and organ:terminal body weight ratios were analysed using analysis of variance (ANOVA).
The number of corpora lutea, implantation sites, early and late resorptions, live foetuses, and % pre- and post-implantation loss were analysed using the Kruskal Wallis and Wilcoxon rank sum test.
Mean foetal weight (male, female, and combined) was analysed using analysis of covariance (ANCOVA), with litter size (live and dead foetuses) as the covariate. The number of corpora lutea, implantation sites, early and late resorptions, dead foetuses, live foetuses, percent pre- and post-implantation loss, and percent male foetuses were analysed using the Kruskal-Wallis and Wilcoxon rank sum test.
Gravid uterine weights and corrected body weights (carcass weights) were analysed using analysis of variance (ANOVA).
The percentage of foetuses affected (mean litter percent) was analysed using the Kruskal-Wallis and Wilcoxon rank sum test.
Proportion of litters affected was analysed using a one-sided upper tail Fisher’s exact test.
Indices:
% Pre-Implantation Loss = ((Number of corpora lutea - number of implantations) / Number of corpora lutea) x 100
% Post-Implantation Loss = ((Number of implantations - number of live embryos) / Number of implantations) x 100
% Male Foetuses = (Number of male foetuses / Number of foetuses of determined sex) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Immediately following administration, incidences of mouth rubbing, salivation, and paddling were observed for animals of all groups. These observations are noted in animals with this route of administration and are considered to be due to unpalatable vehicle and/or test articles and is not adverse.
Throughout the observation period, incidences of red/pink/orange stained fur, thinning fur, and minimal sores/lesions were noted in animals in all dose groups including control. One vehicle control female was also noted to have pale teeth. These are common findings often observed in animals of this age and strain and were considered not related to treatment.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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:
effects observed, treatment-related
Description (incidence and severity):
Decreases in haemoglogin, red blood cell count and packed cell volume of 4-5 % were observed in animals administered 400 mg/kg/day, and were accompanied by increases in absolute numbers and the proportion of reticulocytes (39 and 45 % increases, respectively). Of these, the decreased haemoglobin concentration and increased reticulocyte count attained statistical significance.
All other changes were considered within normal background levels.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant increases in aspartate and alanine aminotransferase activities (60 and 33 % increases, respectively) were noted in animals in the 400 mg/kg bw/day group.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increase in mean relative spleen weight of 23 % was noted for top-dose females. Increases in relative spleen weight at the mid and low doses were ca. 10 % or less and not dose related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The gastrointestinal tract finding of abnormal dark/red or red contents was noted in some females that littered early. This was likely placental material. This is an anticipated finding as the consumption of placental material following parturition is an anticipated behaviour in female rats.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
1, 3, 1 or 2 females administered 0, 40, 125 or 400 mg/kg/day were found to be non-pregnant and one female administered 400 mg/kg/day was found to have no viable foetuses. Pregnancy rates were reasonably consistent between the groups and within background data ranges. These rates of non-pregnancy were, therefore, considered incidental.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 125 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Kidney dilation was noted in litters from all groups including controls, but only the incidence for litters of females administered 125 mg/kg/day attained statistical significance. The incidences noted were outside the background data ranges for all treated groups; however, no dose relationship occurred, and given the high background incidence likely represents an enhancement of an existing underlying physiological process rather than a direct toxicological effect of treatment. These incidences are, therefore considered of no toxicological significance, if not incidental.
Incidences of thymic remnant attained statistical significance in animals administered 400 mg/kg/day. However, these incidences are within the background range for this laboratory and, therefore, were not considered adverse.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Summary of Reproductive Performance

 

Dose Group (mg/kg)

0

40

125

400

Total No. of females

22

22

22

22

Pregnant females

21

19

21

20

Non-pregnant females

1

3

1

2

Pregnant with total litter loss (NVF)

0

0

0

1

Females with live foetuses

21

19

21

19

NVF = No viable foetuses

 

Table 2: Summary of Caesarean Section Data

 

 

Dose Group (mg/kg)

0

40

125

400

Excluding female with no viable foetuses

No. pregnant at caesarean section

N

21

19

21

19

Corpora lutea

N

Mean

SD

21

12

1.9

19

12

2.0

21

12

1.8

19

12

1.5

Implantation sites

N

Mean

SD

21

11

1.7

19

11

1.4

21

11

2.4

19

11

2.4

Pre-implantation loss

N

Mean

SD

21

1

1.2

19

1

2.5

20*

1

2.0

19

1

1.6

Pre-implantation loss (%)

N

Mean

SD

21

9.9

9.56

19

8.2

13.49

20*

9.9

17.59

19

8.6

15.03

Early resorptions

N

Mean

SD

21

1

0.9

19

1

0.8

21

1

1.1

19

1

1.0

Late resorptions

N

Mean

SD

21

0

0.0

19

0

0.2

21

0

0.2

19

0

0.0

Total resorptions

N

Mean

SD

21

1

0.9

19

1

0.9

21

1

1.2

19

1

1.0

Dead foetuses

N

Mean

SD

21

0

0.0

19

0

0.0

21

0

0.7

19

0

0.0

Post-implantation loss

N

Mean

SD

21

1

0.9

19

1

0.9

21

1

1.3

19

1

1.0

Post-implantation loss (%)

N

Mean

SD

21

8.1

9.27

19

7.9

8.95

21

8.0

12.64

19

4.9

9.39

Live foetuses

N

Mean

SD

21

10

2.1

19

10

1.6

21

10

2.5

19

10

2.5

Gravid uterine weight (g)

N

Mean

SD

21

68.9

12.60

19

70.5

10.70

21

67.9

16.20

19

69.0

16.13

Corrected body weight (carcass weight; g)

N

Mean

SD

21

243.8

22.73

19

252.1

16.00

21

247.3

17.42

19

246.0

19.47

Corrected weight change days 3 to 21 (g)

N

Mean

SD

21

49.0

8.85

19

51.1

9.43

21

51.3

8.72

19

50.7

11.38

Total weight change days to 21 (g)

N

Mean

SD

21

117.9

15.59

19

121.6

12.54

21

119.2

16.97

19

119.7

19.70

No. of females with live foetuses

N

21

19

21

19

Mean number of male foetuses per litter

N

Mean

SD

21

5

1.6

19

5

1.8

21

5

1.6

19

5

1.9

Mean number of female foetuses per litter

N

Mean

SD

21

5

2.1

19

5

1.5

21

5

1.9

19

5

1.7

% Male foetuses

N

Mean

SD

21

48

16.4

19

49

14.1

21

47

11.4

19

51

13.0

Mean foetal weight (g)

N

Mean

SD

21

5.31

0.465

19

5.37

0.264

21

5.29

0.381

19

5.18

0.399

Mean weight male foetuses (g)

N

Mean

Adj. mean

SD

21

5.46

5.46

0.504

19

5.54

5.53

0.279

21

5.40

5.39

0.344

19

5.32

5.34

0.425

Mean weight female foetuses (g)

N

Mean

Adj. mean

SD

21

5.13

5.13

0.424

19

5.22

5.22

0.274

21

5.19

5.18

0.465

19

5.01

5.03

0.412

Including female with no viable foetuses

No. pregnant at caesarean section

N

21

19

21

20

Corpora lutea

N

Mean

SD

21

12

1.9

19

12

2.0

21

12

1.8

20

11

2.6

Implantation sites

N

Mean

SD

21

11

1.7

19

11

1.4

21

11

2.4

20

10

3.0

Pre-implantation loss

N

Mean

SD

21

1

1.2

19

1

2.5

20*

1

2.0

20

1

1.6

Pre-implantation loss (%)

N

Mean

SD

21

9.9

9.56

19

8.2

13.49

20*

9.9

17.59

20

8.2

14.76

Early resorptions

N

Mean

SD

21

1

0.9

19

1

0.8

21

1

1.1

20

1

1.0

Late resorptions

N

Mean

SD

21

0

0.0

19

0

0.2

21

0

0.2

20

0

0.0

Total resorptions

N

Mean

SD

21

1

0.9

19

1

0.9

21

1

1.2

20

1

1.0

Dead foetuses

N

Mean

SD

21

0

0.0

19

0

0.0

21

0

0.7

20

0

0.0

Post-implantation loss

N

Mean

SD

21

1

0.9

19

1

0.9

21

1

1.3

20

1

1.0

Post-implantation loss (%)

N

Mean

SD

21

8.1

9.27

19

7.9

8.95

21

8

12.64

20

9.6

23.15

Live foetuses

N

Mean

SD

21

10

2.1

19

10

1.6

21

10

2.5

20

10

3.4

Gravid uterine weight (g)

N

Mean

SD

21

68.9

12.60

19

70.5

10.70

21

67.9

16.20

19

69.0

16.13

Corrected body weight (carcass weight; g)

N

Mean

SD

21

243.8

22.73

19

252.1

16.00

21

247.3

17.42

19

246.0

19.47

Corrected weight change days 3 to 21 (g)

N

Mean

SD

21

49.0

8.8

19

51.1

9.43

21

51.3

8.72

19

50.7

11.38

Total weight change days to 21 (g)

N

Mean

SD

21

117.9

15.59

19

121.6

12.54

21

119.2

16.97

19

119.7

19.70

No. of females with live foetuses

N

21

19

21

20

Mean number of male foetuses per litter

N

Mean

SD

21

5

1.6

19

5

1.8

21

5

1.6

20

5

2.2

Mean number of female foetuses per litter

N

Mean

SD

21

5

2.1

19

5

1.5

21

5

1.9

20

5

2.0

% Male foetuses

N

Mean

SD

21

48

16.4

19

49

14.1

21

47

11.4

20

49

17.1

Mean foetal weight (g)

N

Mean

SD

21

5.31

0.465

19

5.37

0.264

21

5.29

0.381

19

5.18

0.399

Mean weight male foetuses (g)

N

Mean

Adj. mean

SD

21

5.46

5.46

0.504

19

5.54

5.53

0.279

21

5.40

5.39

0.344

19

5.32

5.34

0.425

Mean weight female foetuses (g)

N

Mean

Adj. mean

SD

21

5.13

5.13

0.424

19

5.22

5.22

0.274

21

5.19

5.18

0.465

19

5.01

5.03

0.412

*Number examined reduced due to excluded data

 

Table 3: Summary of Foetal Observations

 

 

Dose Group (mg/kg)

0

40

125

400

Malformation in foetal external - live

Number of dams/foetus

22/209

22/188

22/206

22/194

Number examined litter/foetus

21/209

19/188

21/206

19/194

Eye; eye bulge; small (malformation)

 

% Litter

% Foetal

1/1

5

0.43

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

Variation in foetal external - live

Tail; fleshy tab

 

% Litter

% Foetal

1/1

5

0.43

2/3

11

1.61

1/1

5

0.43

0/0

0

0.00

Malformation in foetal fresh visceral - live

Number of dams/foetus

22/209

22/188

22/206

22/194

Number examined litter/foetus

21/105

19/96

21/103

19/97

Eye; anophthalmia

 

% Litter

% Foetal

1/1

5

0.95

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

General; situs inversus

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

1/1

5

1.05

Thyroid; absent

 

% Litter

% Foetal

1/1

5

0.95

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

Gonad; small

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

1/1

5

2.63

Variation in foetal fresh visceral - live

Number of dams/foetus

22/209

22/188

22/206

22/194

Number examined litter/foetus

21/105

19/96

21/103

19/97

Blood vessel; umbilical artery; left-sided

 

% Litter

% Foetal

7/8

33

7.10

7/9

37

9.04

13/20

62

18.89

11/14

58

15.05

Kidney; dilated

 

% Litter

% Foetal

0/0

0

0.00

1/1

5

0.88

0/0

0

0.00

0/0

0

0.00

Liver; liver lobe; discoloured

 

% Litter

% Foetal

0/0

0

0.00

1/1

5

1.05

1/1

5

0.95

0/0

0

0.00

Liver; liver lobe; supernumerary

 

% Litter

% Foetal

1/2

5

1.90

2/2

11

1.75

1/1

5

1.19

0/0

0

0.00

Thoracic cavity; fluid filled

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

1/1

5

0.95

0/0

0

0.00

Thymus; thymic remnant

 

% Litter

% Foetal

0/0

0

0.00

1/1

5

1.05

2/2

10

1.75

0/0

0

0.00

Thyroid; small

 

% Litter

% Foetal

1/1

5

0.95

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

Ureter; dilated

 

% Litter

% Foetal

0/0

0

0.00

2/2

11

2.19

1/1

5

1.19

0/0

0

0.00

Abdomen; abdominal cavity; fluid filled

 

% Litter

% Foetal

4/6

19

5.00

1/1

5

0.88

3/3

14

2.82

0/0

0

0.00

Malformation in foetal fixed visceral - live

Number of dams/foetus

22/209

22/188

22/206

22/194

Number examined litter/foetus

21/104

19/92

21/103

19/97

Diaphragm; hole

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

1/1

5

0.75

Eye; macrophthalmia

 

% Litter

% Foetal

2/3

10

3.97

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

Eye; microphthalmia

 

% Litter

% Foetal

1/1

5

1.19

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

Variation in foetal fixed visceral - live

Number of dams/foetus

22/209

22/188

22/206

22/194

Number examined litter/foetus

21/104

19/92

21/103

19/97

Blood vessel; umbilical artery; left-sided

 

% Litter

% Foetal

9/14

43

13.55

7/11

37

11.40

10/15

48

13.46

10/21

53

21.23

Brain; brain ventricle; dilated

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

1/1

5

1.05

Eye; lens; altered texture

 

% Litter

% Foetal

1/1

5

1.59

0/0

0

0.00

0/0

0

0.00

2/2

11

2.37

Eye; retina; fold

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

1/1

5

0.68

0/0

0

0.00

Kidney; dilated

 

% Litter

% Foetal

3/3

14

4.29

5/9

26

9.47

11/17

52*

18.90

6/7

32

6.29

Liver; liver lobe; supernumerary

 

% Litter

% Foetal

5/5

24

4.41

8/9

42

10.18

4/4

19

4.81

7/9

37

9.09

Thymus; thymic remnant

 

% Litter

% Foetal

2/2

10

1.90

4/6

21

5.79

3/3

14

2.43

7/7

37*

7.29

Ureter; dilated

 

% Litter

% Foetal

1/1

5

2.38

1/1

5

1.05

0/0

0

0.00

1/1

5

0.75

Malformation in foetal skeletal - live

Number of dams/foetus

22/209

22/188

22/206

22/194

Number examined litter/foetus

21/105

19/96

21/103

19/97

Skull; mandible; fused

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

1/1

5

0.95

0/0

0

0.00

Skull; mandible; single incisor socket

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

1/1

5

0.95

0/0

0

0.00

Skull; orbital socket; small

 

% Litter

% Foetal

1/1

5

0.95

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

Skull; palatine; cleft

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

1/1

5

0.95

0/0

0

0.00

Vertebra; cervical arch; absent

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

1/1

5

1.05

Vertebra; cervical arch; small

 

% Litter

% Foetal

1/1

5

0.95

0/0

0

0.00

0/0

0

0.00

1/1

5

1.05

Vertebra; cervical centrum; absent

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

1/1

5

1.05

Vertebra; thoracic centrum; hemicentric

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

1/1

5

0.79

1/1

5

1.05

Rib; abnormal attachment

 

% Litter

% Foetal

1/1

5

0.95

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

Rib; branched

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

1/1

5

1.05

Rib; fused

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

1/1

5

1.05

Variation in foetal skeletal - live

Number of dams/foetus

22/209

22/188

22/206

22/194

Number examined litter/foetus

21/105

19/96

21/103

19/97

Skull; frontal; incomplete ossification

 

% Litter

% Foetal

1/1

5

1.19

0/0

0

0.00

1/1

5

0.79

0/0

0

0.00

Skull; frontal; unossified line

 

% Litter

% Foetal

1/1

5

0.79

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

Skull; hyoid; incomplete ossification

 

% Litter

% Foetal

1/1

5

0.95

1/1

5

0.88

1/2

5

1.59

0/0

0

0.00

Skull; hyoid; unossified

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

1/1

5

0.95

0/0

0

0.00

Skull; interparietal; incomplete ossification

 

% Litter

% Foetal

5/8

24

8.33

2/2

11

1.93

4/6

19

5.08

6/10

32

9.24

Skull; parietal; incomplete ossification

 

% Litter

% Foetal

3/7

14

6.98

2/3

11

3.63

3/7

14

5.87

6/10

32

9.27

Skull; parietal; unossified line

 

% Litter

% Foetal

1/1

5

0.79

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

Skull; presphenoid; incomplete ossification

 

% Litter

% Foetal

1/1

5

0.95

0/0

0

0.00

1/1

5

0.95

0/0

0

0.00

Skull; squamosal; incomplete ossification

 

% Litter

% Foetal

2/3

10

2.86

0/0

0

0.00

2/2

10

1.75

1/1

5

0.88

Skull; supraoccipital; incomplete ossification

 

% Litter

% Foetal

3/3

14

3.10

0/0

0

0.00

2/3

10

2.54

3/3

16

3.12

Skull; suture; sutural bone

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

2/2

10

1.90

½

5

2.11

Skull; zygomatic arch; incomplete ossification

 

% Litter

% Foetal

2/3

10

3.10

2/2

11

1.93

2/3

10

2.54

0/0

0

0.00

Skull; zygomatic arch; ossification bridge

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

1/1

5

0.79

0/0

0

0.00

Pelvic girdle; iliac alignment; abnormal

 

% Litter

% Foetal

9/19

43

20.16

10/21

53

21.67

7/13

33

11.92

5/9

26

8.33

Sternebra; dumbbell ossification

 

% Litter

% Foetal

0/0

0

0.00

1/1

5

1.05

0/0

0

0.00

1/1

5

1.05

Sternebra; incomplete ossification

 

% Litter

% Foetal

10/18

48

15.87

5/6

26

6.05

8/16

38

14.57

7/13

37

12.82

Sternebra; increased ossification

 

% Litter

% Foetal

0/0

0

0.00

1/1

5

0.88

2/2

10

1.75

0/0

0

0.00

Sternebra; misaligned ossification centres

 

% Litter

% Foetal

1/1

5

1.59

1/1

5

1.05

2/2

10

1.75

1/1

5

1.05

Sternebra; unossified

 

% Litter

% Foetal

1/1

5

0.95

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

Supernumerary rib; present

 

% Litter

% Foetal

21/85

100

80.04

19/65

100

69.47

20/63

95

61.94

17/54

89

53.78

Vertebra; cervical arch; incomplete ossification

 

% Litter

% Foetal

2/2

10

2.14

0/0

0

0.00

1/1

5

1.19

3/3

16

2.81

Vertebra; cervical centrum; incomplete ossification

 

% Litter

% Foetal

9/19

43

17.94

8/10

42

11.32

10/18

48

17.14

13/23

68

21.67

Vertebra; cervical centrum; misaligned

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

1/1

5

1.05

Vertebra; cervical centrum; unilateral ossification

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

1/1

5

1.05

Vertebra; cervical centrum; unossified

 

% Litter

% Foetal

12/22

57

21.75

7/10

37

11.58

11/24

52

22.98

7/14

37

13.07

Vertebra; lumbar centrum; asymmetric ossification

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

1/1

5

1.19

1/1

5

0.75

Vertebra; lumbar centrum; bipartite ossification

 

% Litter

% Foetal

1/1

5

1.19

0/0

0

0.00

2/2

10

1.98

0/0

0

0.00

Vertebra; lumbar centrum; dumbbell ossification

 

% Litter

% Foetal

1/3

5

3.57

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

Vertebra; lumbar centrum; incomplete ossification

 

% Litter

% Foetal

1/1

5

0.95

1/1

5

1.05

1/1

5

1.19

1/1

5

0.75

Vertebra; sacral arch; ossification bridge

 

% Litter

% Foetal

5.7

24

6.98

4/8

21

7.89

10/16

48

15.48

7/10

37

11.84

Vertebra; sacral centrum; incomplete ossification

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

1/1

5

1.05

Vertebra; thoracic centrum; bipartite ossification

 

% Litter

% Foetal

1/2

5

2.38

0/0

0

0.00

1/1

5

1.19

1/1

5

1.05

Vertebra; thoracic centrum; dumbbell ossification

 

% Litter

% Foetal

2/3

10

3.17

3/3

16

2.81

2/2

10

1.75

1/1

5

1.05

Vertebra; thoracic centrum; incomplete ossification

 

% Litter

% Foetal

2/3

10

3.97

1/1

5

1.05

4/6

19

5.40

2/2

11

1.63

Vertebra; thoracic centrum; misaligned

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

1/1

5

0.79

1/1

5

1.05

Forelimb; phalanx; incomplete ossification

 

% Litter

% Foetal

2/3

10

2.86

0/0

0

0.00

2/4

10

3.33

3/4

16

4.26

Forelimb; phalanx; unossified

 

% Litter

% Foetal

1/2

5

1.90

1/1

5

1.05

2/2

10

2.14

0/0

0

0.00

Hindlimb; metatarsal; incomplete ossification

 

% Litter

% Foetal

5/7

24

6.39

6/11

32

10.00

4/4

19

3.54

7/13

37

11.87

Hindlimb; metatarsal; unossified

 

% Litter

% Foetal

5/9

24

8.89

2/2

11

2.19

5/8

24

7.86

4/6

21

6.63

Hindlimb; phalanx; incomplete ossification

 

% Litter

% Foetal

14/30

67

27.14

14/36

74

37.98

15/30

71

28.73

14/34

74

33.55

Hindlimb; phalanx; unossified

 

% Litter

% Foetal

10/29

48

26.27

12/27

63

26.32

10/28

48

24.37

11/33

58

31.23

Rib; clubbed

 

% Litter

% Foetal

0/0

0

0.00

0/0

0

0.00

0/0

0

0.00

1/1

5

1.32

Rib; nodulated

 

% Litter

% Foetal

2/3

10

2.70

2/3

11

2.63

3/3

14

2.78

1/1

5

1.05

*Fisher 1-tail Ascending Test significant at the 0.05 level

Table 4: Selected Haematology Parameters

Dose Group (mg/kg) 0  40 125  400

Haemaglobin (g/dL)

 11.2 ± 0.50  11.1 ± 0.47  11.0 ± 0.63  10.6 ± 0.59*

Red blood cell count (10^12/L)

 6.00 ± 0.204  5.91 ± 0.274 5.92 ± 0.311 5.72 ± 0.310 

 Packed cell volume (%)

 34.4 ± 1.46  33.6 ± 1.34  33.5 ±1.85 32.9 ± 1.89 

Reticulocytes (%)

 3.1 ± 0.72  3.3 ± 0.87  2.7 ± 0.93 4.5 ± 1.53*

* p<0.05

Table 5: Selected Clinical Biochemistry Parameters

Dose Group (mg/kg)

0

40

125

400

Aspartate aminotransferase (IU/L)

55 ± 6.3

61 ± 11.5

67 ± 19.0 

 88 ± 42.0*

Alanine aminotransferase (IU/L) 

52 ± 9.4

58 ± 9.3

58 ± 5.0

69 ± 7.5**

* p<0.01

** p<0.001

Table 6: Summary of Organ weights

Dose Group (mg/kg)

 0

 40

 125

400

Terminal body weight (g)

 313.1 ± 31.98

 322.8 ± 18.39

 315.1 ± 29.87

 314.0 ± 27.22

Spleen (absolute) (g)

 0.493 ± 0.0918

 0.560 ± 0.0943

 0.522 ± 0.0635

 0.599 ± 0.0486*

Spleen (% body weight)

 0.157 ± 0.0217

 0.174 ± 0.0276

 0.166 ± 0.0163

 0.192 ± 0.0269*

* p<0.01

Applicant's summary and conclusion

Conclusions:
Based on the findings in this study, the female No Observed Adverse Effect Level (NOAEL) is considered to be 125 mg/kg/day, and foetal NOAEL is considered to be >=400 mg/kg/day.
Executive summary:

A study was conducted to determine the effects of the test material on the embryonic and foetal development of the rat in accordance with the standardised guidelines OECD 414, US EPA OPPTS 870.3700, the study was carried out under GLP conditions.

Four groups of 22 female time-mated Crl:WI(Han) rats were administered 0 (control article [vehicle]), 40, 125, or 400 mg/kg/day 4-OH TEMPO orally by gavage daily from Gestation Day (GD) 6 to 20, inclusive, at a volume of 10 mL/kg. The control article (vehicle) was Polyethylene glycol 400 (PEG 400).

Assessment of toxicity was based on evaluation of clinical observations, body weights, food consumption, and clinical pathology. On GD 21, females were sacrificed, and the progress and outcome of pregnancy were evaluated. Complete necropsies were performed on all animals, with a recording of macroscopic abnormalities for all tissues. Organ weights were conducted as indicated. Fetuses were evaluated for external, visceral, and skeletal malformations and variations.

Statistically significantly, but marginally decreased haemoglobin and increased reticulocytes were noted in animals administered the top dose of 400 mg/kg/day. These were correlated with a statistically significant increase in spleen weights. These changes, while attaining statistical significance are not considered to be toxicologically significant.

These were correlated with a statistically and toxicologically significant increase in spleen weights, and were indicative of toxicologically significant anaemic processes, showing the top dose chosen had been adequate. There were also marginal increases in aspartate- and alanine aminotranseferase activities, although in the absence of any liver weight findings, they are not considered intrinsically adverse in nature. 

No adverse effects on bodyweight, food consumption, clinical or postdose observations, macroscopic findings, or reproductive parameters were noted.

Based on the findings in this study the female No Observed Adverse Effect Level (NOAEL) is considered to be 125 mg/kg/day, and foetal NOAEL is considered to be 400 mg/kg/day.