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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19 March 2004 to 19 April 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
This study was a Dose Range Finding Study for a Main teratology study. Only 8 females per conditions were used. Fetuses examination was not complete, only external observation was performed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
This study was a Dose Range Finding Study for a Main teratology study. Only 8 females per conditions were used
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[(2-nitrophenyl)amino]ethanol
EC Number:
225-555-8
EC Name:
2-[(2-nitrophenyl)amino]ethanol
Cas Number:
4926-55-0
Molecular formula:
C8H10N2O3
IUPAC Name:
2-[(2-nitrophenyl)amino]ethanol
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Supplied by the Sponsor, Lot No. 17
- Expiration date of the lot/batch: 11 December 2005
- Purity test date: 11 December 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: Stable at 0.4 and 200 mg/mL during 24 hours at room temperature in vehicle. Stable for 15 days at 5±3 deg Celsius.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Formulations (solutions) were prepared at least every two weeks. . Prepared solutions were stored refrigerated; the actual temperature range in the storage refrigerator from the day of the first preparation to the last day of dosage administration was 4 deg Celsius to 8 deg Celsius. No more details

FORM AS APPLIED IN THE TEST (if different from that of starting material)
In solution in PEG-400 polyethylene glycol 400

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc. (USA)
- Age at study initiation: 16 weeks (females) / 73-79 days (at arrival the 14 and 22 September 2003)
- Weight at study initiation: 220-245 g (female) / 483-698g (males)
- Fasting period before study: not specified
- Housing: Rats were individually housed in stainless steel, wire-bottomed cages, except during the cohabitation period. During cohabitation, each pair of male and female rats were housed in the male rat’s cage.
- Diet (e.g. ad libitum): Rats were given ad libitum access to Certified Rodent Diet® #5002 (PMI® Nutrition International, St. Louis, Missouri, USA) in individual feeders.
- Water (e.g. ad libitum):Local water that had been processed by passage through a reverse osmosis membrane was available to the rats ad libitum from an automatic watering access system and/or individual water bottles attached to the cages. Chlorine was added to the processed water as a bacteriostat.
- Acclimation period: 5 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 deg Celsius
- Humidity (%): 30 to 70%
- Air changes (per hr): a minimum of 10 cycles per hour
- Photoperiod (hrs dark / hrs light): 12h light / 12 hours dark

IN-LIFE DATES: From: 22 January 2004 To: 19 April 2004

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (solutions) were prepared at least every two weeks. . Prepared solutions were stored refrigerated; the actual temperature range in the storage refrigerator from the day of the first preparation to the last day of dosage administration was 4 deg Celsius to 8 deg Celsius. No more details

VEHICLE
- Justification for use and choice of vehicle (if other than water): No justification
- Concentration in vehicle: 0, 5, 15, 40, 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): T42604 and Y11608
- Purity:100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity was determined for the low and high concentrations using samples collected from the top, middle and bottom of the dosage formulations on the first day of preparation. Mean concentration results from these samples were calculated, and homogeneity relative standard deviation (RSD) was calculated by determining the percent RSD of the three mean values. All of the results were within the acceptable range of ≤5% RSD. The values obtained were 0.7% and 1.8% RSD for the 5 mg/mL and 100 mg/mL formulations, respectively.

Samples collected from dosage formulations prepared on 1 and 14 April 2004 for concentration analysis were within the acceptable limits of ±15% error.

Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: 5 days (maximum)
- Further matings after two unsuccessful attempts: not specified
- Verification of same strain and source of both sexes: not specified
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Day 6 to day 20 of gestation
Frequency of treatment:
once daily, seven days a week
Duration of test:
21 Days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
8 females per sex per dose
Control animals:
yes
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The highest dosage level was selected to induce some overt developmental and/or maternal toxicity (clinical signs or a decrease in body weight) but not death or severe suffering. At least one intermediate dosage level was selected to produce minimal observable toxicity. The lowest dosage level should not have produced any evidence of either maternal or developmental toxicity. A descending sequence of dosage levels was selected with the purpose of demonstrating any dosage-related response and to establish a no-observed-adverse-effect level (NOAEL).

- Rationale for animal assignment (if not random):
Upon arrival, rats were assigned to individual housing on the basis of computer-generated random units. Healthy, mated female rats were assigned to five dosage groups (Groups I through V), eight rats per dosage group, by using a computer-generated (weight-ordered) randomization procedure based on body weights recorded on DG 0.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked : viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily and weekly during acclimation period

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly during the acclimation period, on DG 0 and daily during the dosage and postdosage periods.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
Calculated as g food/animal/day
Feed consumption values were recorded on DGs 0, 6, 9, 12, 15,18, 20 and 21.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
- Organs examined: the thoracic, abdominal and pelvic viscera

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: The number and distribution of corpora lutea were recorded. The uterus of each rat was excised and weighed (if gravid). The uterus of each rat was examined for pregnancy, number and distribution of implantation sites, live and dead fetuses and early and late resorptions.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
All data were tabulated, summarized and/or statistically analyzed using the Argus Automated Data Collection and Management System, the Vivarium Temperature and Relative Humidity Monitoring System, Microsoft® Excel (part of Microsoft® Office 97/2000/XP), Quattro Pro 8 and/or The SAS System (version 6.12). Averages and percentages were calculated. Litter values were used where appropriate.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical observations considered to be related to the test substance included discolored (orange) skin, fur and/or urine in all groups administered the test substance and excess salivation which occurred only in the 500 mg/kg/day dosage group.

All other clinical observations were considered to be unrelated to the test substance because:
1) the clinical observation was observed in only one rat in any dosage group; 2) the incidences were not dosage dependent; and/or 3) the observations occurred in the rat that was sacrificed due to moribund condition. These observations included gasping, dyspnea, soft or liquid feces, localized alopecia (underside) and dehydration.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Rat 8393 in the 500 mg/kg/day dosage group was sacrificed due to moribund condition approximately two hours after the 13th dosage of test substance on DG 18. Necropsy revealed yellow discoloration of the intestines, pancreas, stomach, mesentery and abdominal adipose tissue. All other tissues appeared normal. The litter consisted of 12 fetuses and one early resorption. Based on the examination that was conducted one hour post-dosage that indicated open-mouthed breathing, increased breathing and the rat maintaining an extended neck, the moribund condition probably resulted from an intubation accident. All other rats survived to scheduled sacrifice.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights were comparable among the five dosage groups through DG 21. Gravid uterine weights were increased in the groups administered the test substance, in comparison with the control group value; however, the increases were not dosage dependent. Corrected maternal body weights (DG 21 body weight minus the gravid uterine weight) were comparable among the dosage groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Absolute and relative feed consumption values were generally comparable among the five dosage groups during the dosage period (calculated as DGs 6 to 21) and the gestation period (DGs 0 to 21).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
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:
no effects observed
Other effects:
not examined
Details on maternal toxic effects:
No Caesarean-sectioning or litter parameters were affected by dosages of the test substance as high as 500 mg/kg/day. The litter averages for corpora lutea, implantations, litter sizes, live fetuses, early and late resorptions, fetal body weights, percent resorbed conceptuses, and percent live male fetuses were comparable among the five dosage groups. No dam had a litter consisting of only resorbed conceptuses, and there were no dead fetuses.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
maternal abnormalities
mortality

Results (fetuses)

Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
All fetuses appeared normal at gross external examination.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall observations

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

TABLE 1 : CLINICAL OBSERVATIONS - SUMMARY

 

------------------------------------------------------------------------------------------------------------------------------------

 

 MAXIMUMPOSSIBLEINCIDENCE         128/8          128/8           128/8          128/8                                   125/8

 

MORIBUNDSACRIFICED                  0                0                 0                0                                     1b

 

ORANGE URINE

0/

0

85/

8

109/

8

114/

8

119/

8b

ORANGE FUR

0/

0

17/

6

110/

8

117/

8

121/

8b

ORANGE SKIN

0/

0

6/

1

10/

4

18/

7

42/

8b

EXCESS SALIVATION

0/

0

0/

0

0/

0

0/

0

3/

3b

GASPING

0/

0

0/

0

0/

0

0/

0

1/

1b

DYSPNEA

0/

0

0/

0

0/

0

0/

0

1/

1b

SOFT OR LIQUID FECES

3/

1

5/

3

1/

1

0/

0

0/

0

LOCALIZED ALOPECIA:UNDERSIDE

0/

0

0/

0

9/

1

0/

0

0/

0

DEHYDRATION

1/

1

0/

0

0/

0

0/

0

0/

0

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MAXIMUM POSSIBLE INCIDENCE = (DAYS x RATS)/NUMBER OF RATS EXAMINED PER GROUP ON DAYS 6 THROUGH 21 OF PRESUMED GESTATION.

N/N = TOTAL NUMBER OF OBSERVATIONS/NUMBER OF RATS WITH OBSERVATION.

a.   Dosage occurred on days 6 through 20 ofgestation.

b.   Dam 8393 was moribund sacrificed on day 18 ofgestation.


 

 

ORAL (GAVAGE) DOSAGE-RANGE DEVELOPMENTAL TOXICITY STUDY OF GTS03975 IN RATS (SPONSOR'S STUDY NUMBER:2747-53983)

 

TABLE 2 : NECROPSY OBSERVATIONS - SUMMARY

 

------------------------------------------------------------------------------------------------------------------------------------

DOSAGE GROUP

I

II

III

IV

V

DOSAGE (MG/KG/DAY)a

0

25

75

200

500

------------------------------------------------------------------------------------------------------------------------------------  

RATS EXAMINED

MORIBUND SACRIFICED                  N        8                    8               8                      8                  8

N        0                   0               0                         0                 1c

 APPEARED NORMAL                       N  0                   0               0                         0                 7

                   

 

INTESTINE, PANCREAS, STOMACH, MESENTERY AND ABDOMINAL ADIPOSE:

YELLOW

                                  N     8                  8               8                      8                  1c

                                     

    

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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a.   Dosage occurred on days 6 through 20 ofgestation.

b.   Refer to the individual clinical observations table (Table 9) for external observations confirmed atnecropsy.

c.   Dam 8393 was moribund sacrificed on day 18 ofgestation.

 

 

 

 

TABLE 3: MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS - SUMMARY

 

------------------------------------------------------------------------------------------------------------------------------------ DOSAGEGROUP             

                                               I        II             III              IV                                        V

DOSAGE(MG/KG/DAY)a                     0              25              75             200                                        500

------------------------------------------------------------------------------------------------------------------------------------  RATSTESTED             

                              N         8               8               8               8                          8

PREGNANT                  N             8               8               8               8                          8

MATERNAL BODY WEIGHT (G)

DAY 0            MEAN±S.D.   232.9±7.9    233.6±7.1     233.8±6.7    234.1±8.1    233.8 ±6.5

DAY 6            MEAN±S.D.   266.4±14.2    267.2±12.3    262.8±9.1    269.2±10.0    265.8 ±8.5

DAY 7            MEAN±S.D.   266.4±10.4    267.0±9.2     265.1±8.8    265.8±9.3    268.5 ±9.5

DAY 8            MEAN±S.D.   266.1±12.9    272.4±10.6    268.5±7.3    272.9±11.7    273.0 ±11.0

DAY 9            MEAN±S.D.   274.0±12.3    272.5±15.9    277.9±6.0    275.4±10.5    274.2 ±8.9

DAY10           MEAN±S.D.   281.9±13.6    278.2±14.8    278.8±5.9    282.6±15.2                  280.1 ±7.8

DAY11           MEAN±S.D.   289.1±13.0    290.1±15.6    285.4±7.7    293.8±11.3                  285.1 ±9.1

DAY12           MEAN±S.D.   294.5±11.1    296.0±14.8    290.9±10.2    297.8±14.3                  291.5 ±7.7

DAY13           MEAN±S.D.   299.4±13.3    303.2±14.8    295.2±10.0    302.8±15.4                  295.1 ±9.5

DAY14           MEAN±S.D.   305.5±14.2    311.1±16.8    300.8±11.0    307.1±17.8                  300.2 ±9.6

 

DAY15           MEAN±S.D.   311.9±16.8    320.0±15.8    309.9±11.0    313.8±20.5                  309.6 ±10.6

 

DAY16           MEAN±S.D.   321.2±19.0    329.9±17.9    320.4±10.2    321.4±24.4                  317.6 ±12.6

 

DAY17           MEAN±S.D.   335.6±20.3    343.6±18.1    332.2±11.3    332.9±27.2                  330.9 ±12.9

 

DAY18           MEAN±S.D.   350.5±21.4    362.8±18.8    350.0±13.1    348.6±30.2                  339.4 ±23.3

 

DAY19           MEAN±S.D.   365.4±24.6    381.5±17.8    364.6±11.8    366.8±30.6                  365.8 ±15.4

[ 7]b

DAY20           MEAN±S.D.   378.9±25.9    397.6±22.3    383.0±9.6    383.0±33.0                  382.6 ±20.7

[ 7]b

DAY21           MEAN±S.D.   401.8±25.7    425.8±20.4    414.9±16.6    412.4±37.8                  406.8 ±23.9

[7]b

GRAVID UTERINE

WEIGHTS(G)       MEAN±S.D.    95.0±20.4    112.8±9.5     108.1±14.5    102.6±17.2                  104.3 ±16.2

[6]c                            [7]c           [7]c                      [6]b,c

      DAY21Cd       MEAN±S.D.   302.2±23.1    312.9±20.3    305.8±19.1    309.1±27.9                        306.7 ±7.2

[6]c                            [7]c           [7]c                                  [6]b,c

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DAY = DAY OF GESTATION

[ ] = NUMBER OF VALUES AVERAGED

a.   Dosage occurred on days 6 through 20 ofgestation.

b.   Excludes values for dam 8393, which was moribund sacrificed on day 18 ofgestation.

c.   Excludes values that were notrecorded.

d.   21C = Corrected maternal body weight (day 21 of gestation body weight minus the gravid uterineweight).

 

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of the study, there were no test substance related effects on body weights, body weight gains, gravid uterine weights, corrected maternal body weights or corrected maternal body weight gains or absolute or relative feed consumption values. No Caesarean-sectioning or litter parameters were affected by dosages of the test substance as high as 500 mg/kg/day. All fetuses appeared normal at gross external examination. Based on these data, dosages of 0 (Vehicle), 50, 200 and 500 mg/kg/day of the test item were selected for the developmental toxicity study in rats.
Executive summary:

The purpose of this GLP study was to provide information for the selection of dosages to be used in the developmental toxicity (embryo-fetal toxicity and teratogenic potential) study of the Test Item HC Yellow No. 2 administered orally via gavage to Crl:CD(SD) presumed pregnant female rats.

Forty presumed pregnant Crl:CD(SD) rats were randomly assigned to five dosage groups, eight rats per group. Solutions of the test substance or the vehicle, PEG 400, were administered orally (gavage) once daily to these naturally bred female rats on days 6 through 20 of presumed gestation (GD 6 - 20) at dosages of 0, 25, 75, 200 and 500 mg/kg bw/d. Viabilities, clinical observations, body weights and feed consumption values were recorded. All surviving rats were sacrificed on GD 21. The gravid uterus was excised, weighed and subsequently examined for the number and distribution of corpora lutea,

implantation sites and uterine contents. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Foetuses were weighed and examined for gross external alterations and sex.

One rat in the 500 mg/kg bw/d dosage group was sacrificed due to moribund condition GD 18. This mortality was non related to treatment and considered the result of an intubation accident. All other rats survived to scheduled sacrifice. Clinical observations considered to be related to the test substance included discoloured (orange) skin, fur and/or urine in all groups administered the test substance and excess salivation in the 500 mg/kg bw/d dosage group. No additional gross lesions were revealed by necropsy examination.

Under the experimental conditions of the study, there were no test substance related effects on body weights, body weight gains, gravid uterine weights, corrected maternal body weights or corrected maternal body weight gains or absolute or relative feed consumption values. No Caesarean-sectioning or litter parameters were affected by dosages of the test substance as high as 500 mg/kg/day. All fetuses appeared normal at gross external examination. Based on these data, dosages of 0 (Vehicle), 50, 200 and 500 mg/kg/day of HC Yellow No. 2 were selected for the developmental toxicity study in rats.