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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restricitions (Lack of data on test substance).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
- lack of data on test substance
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Crl:CD BR VAF/Plus
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Stone Ridge, NY, USA
- Age at study initiation: approximately 9 - 10 weeks
- Weight at study initiation: 200 - 274 g (female)
- Housing: The animals were housed in suspended stainless steel and wire mesh cages with absorbent paper below the cages. The females were housed separately during the study period except during mating.
- Diet: Purina Certified Rodent Chow No. 5002, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 13 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0 - 24.4
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 03 Oct 1994
To: 08 Nov 1994

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: polyethylene glycol (PEG 400)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The undiluted test substance was thoroughly mixed in vehicle prior to dispensing. The dosing solutions were prepared weekly.

VEHICLE
- Lot/batch no. (if required): 122H1109
- Physical state: colorless liquid
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis for homogenity of the test substance dilutions were performed on the lowest and the highest concentrations expected during the course of the study. The relative standard deviation ranged from 0.72 to 3.19%. Concentrations were analysed in the first and the third dosing mixture preparation. The analytical results for all test solutions were within 7% of the nominal concentrations for weeks 1 and 3.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
After confirmation of pregnancy, each mated female was returned to its cage and new females were placed into the males' cages until a required number of pregnant females was obtained.
Duration of treatment / exposure:
gestation days (GD) 6 - 15
Frequency of treatment:
daily, 7 d/week
Duration of test:
21 d (GD 0 - 21)
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 500 and 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
25 females per dose, 50 males in total
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: Mated females were assigned to dose groups in the order of mating. Accordingly, the first confirmed mated female was assigned to Group 1, the next to Group 2 and so on until all mated animals for a given day were assigned to dose groups. On subsequent days, the next group in sequence was filled by the first confirmed mated female on that day and so on. Assignments were made until all groups were filled with confirmed mated females.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the treatment period and once daily at all other times during the study period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to selection and daily during gestation

BODY WEIGHT: Yes
- Time schedule for examinations: prior to selection and on GD 0, 6, 9, 12, 15, 18 and 21

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: concurrently with body weight examinations

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on GD 21
- all females were examined by gross necropsy
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
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
Bartlett's Test was performed to determine if the dose groups had equal variance (1% level of significance). If equal, the testing was done using parametric methods, otherwise nonparametric techniques were used. Parametric procedures: a standard one way ANOVA (F distribution) was used. If significant differences among the means were indicated, Dunnett's Test was used to determine which treatment groups differed significantly from control. In addition to the ANOVA, a standard regression analysis for linear response in the dose groups was performed that also tested for linear lack of fit in the model. Nonparametric procedures: the test of equality of means was performed using the Kruskal-Wallis Test. If significant differences among the means were indicated, Dunn's Summed Rank Test was used to determine which treatment groups differed significantly from the control. In addition, Jonckheere's Test for monotonic trend in the dose response was performed. All tests were conducted at the 5% and 1% level of significance.
Fetal weight was analyzed by a standard nested analysis of covariance (fetuses nested within dams and dams nested within doses and litter size (both sexes combined)). If differences in groups were identified, the Least Significant Difference technique was used to determine which groups differed from the control group. Male and female fetuses were tested separately (the covariate was combined sexes in each analysis). Fetal malformation and variation incidence data were analyzed for statistical significance as follows: a standard chi-square analysis was performed to determine if the proportions of incidences differ between the groups tested. If any one cell had an expected value less than 5, this step was not reported. Next, each treatment group was compared to the control group using a 2x2 Fisher Exact Test. Thirdly, Armitage's test for linear trend in the dosage groups was performed. All tests were reported at the 5% or 1% level of significance.
Indices:
Preimplantation loss = (no. of Corpora Lutea - no. of Implantation Sites) / no. of Corpora Lutea * 100

Postimplantation loss = (no. of Implantation Sites - no. of live foetuses) / no. of Implantation Sites * 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality:
No treatment-related mortality occurred. On GD 7, one female of the 500 mg/kg bw/d group was found dead. At necropsy, discolored and consolidated lungs were the only findings. The death of this female was probably a result of an accidental gavage error.

Clinical signs:
No treatment-related clinical signs were observed during the study period. The most frequently observation was soft stool, that occurred in treated and control animals and was therefore considered as a response to the vehicle rather than to the test substance. Other findings were scabs, little sign of stool, rales and alopecia in one or more groups. One female of the high dose group had a subcutis mass in the cervical area on GD 21. In summary, these clinical findings were considered to be incidental and unrelated to treatment with the test substance.

Body weight:
Treatment with the test substance had no statistically significant effect on mean body weight and mean body weight change at any interval.

Food consumption:
There were no statistically significant differences in mean food consumption between treated and control animals at any interval.

Post-mortem examinations:
There were no necropsy findings that were considered to be treatment-related. One high dose female had a subcutaneous mass in the cervical area and one control and one high dose female had dilated renal pelvis. In summary, these necropsy findings were considered to be incidental and unrelated to treatment.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
maternal
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse or toxicologically relevant effects were observed up to and including the highest tested dose level.

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Fetal body weight:
There were no statistically significant differences between treated and control mean fetal body weights of either sex (see Table 1).

Implantation data:
There were no statistically significant differences in the mean live fetuses, mean live male or female fetuses, mean resorptions, mean implantation sites, mean corpora lutea, mean total dead fetuses, mean fetuses per implantation sites, mean dead fetuses per implantation sites, mean resorptions per implantation sites, % pre-implantation loss, % post-implantation loss, mean malformed fetuses, mean fetuses with variations, or mean affected (resorptions + dead + malformed fetuses per litter) fetuses between treated and control groups. (see Table 1)

Fetal observations:
No biologically or statistically significant differences in total or individual variations or malformations were observed between the litters of test substance groups and control group (see Table 2). In the control, 100, 500 and 1000 mg/kg bw/d group, 5, 2, 6 and 1 fetuses, respectively, were stunted.
External observations regarded as malformations were found in one fetus of the 100 mg/kg bw/d group (agnathia, astomia, low set ears, anophthalmia) and single findings of cleft palate, syndactyly and kinked tail in three foetuses of separate litters of the 500 mg/kg bw/d group. External variations were not observed in any group.
Visceral observations regarded as malformations were limited to single occurrences of anophthalmia in one foetus each of the low and mid dose group, cleft palate and dilated brain ventricles in the 100 mg/kg bw/d group and two occurrences of folded retina in the control group. Visceral variations included a single or low incidence of dilated renal pelves, distended ureters and/or convoluted ureter.
Skeletal observations regarded as malformations were limited to multiple skull bones malformed in one foetus of the 100 mg/kg bw/d group, short pubis in one foetus of the 1000 mg/kg bw/d group and one less presacral vertebrae in one foetus of the control, two foetuses in the low dose and one foetus in the high dose groups. Skeletal variations were observed throughout the groups and consisted primarily of hypoplastic, misshapen or unossified stemebrae, hypoplastic skull bones or pubis and rudimentary or misshapen ribs.
In summary and without a clear pattern of response, all malformations were considered incidental and not to be treatment-related. Statistically significant differences in incidences of variations were limited to a decrease in hypoplastic skull bones of the mid dose group and a decrease in unossified stemebrae of the high dose group compared with controls. All variations were of no biological importance.

Skeletal ossification:
There were no statistically significant differences in mean ossification sites between treated and control groups.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse or toxicologically relevant effects were observed up to and including the highest tested dose level.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Litter data and fetal body weight

 

 

Control

dose groups [mg/kg bw/day]

 

100

500

1000

No. of dams/litters examined

25

21

23

25

Mean no. of Corpora Lutea

16.8

16.8

17.7

16.8

± 2.28

± 2.50

± 3.50

± 1.82

n=25

n=20

n=23

n=25

Mean no. of Implantation Sites

15.76

15.9

16.09

15.52

± 2.24

± 2.62

± 2.25

± 2.49

n=25

n=21

n=23

n=25

Mean no. of resorptions

0.88

0.81

0.91

0.56

± 1.05

± 1.29

± 1.12

± 0.82

n=25

n=21

n=23

n=25

Pre-implantation loss [%]

5.7

6.7

8

7.7

± 8.1

± 8.2

± 8.2

± 12.9

n=25

n=20

n=23

n=25

Post-implantation loss per litter [%]

5.4

4.9

6

4.4

± 6.4

± 7.7

± 7.3

± 6.4

n=25

n=21

n=23

n=25

Mean no. of live fetuses

14.88

15.1

15.13

14.88

± 2.19

± 2.64

± 2.46

± 2.64

n=25

n=21

n=23

n=25

Mean no. of dead fetuses

0

0

0.04

0.08

 

 

± 0.21

± 0.28

n=25

n=21

n=23

n=25

Mean no. of female fetuses

7.72

7.48

8.04

7.8

± 2.28

± 3.11

± 2.29

± 1.78

n=25

n=21

n=23

n=25

Mean no. of male fetuses

7.16

7.62

7.09

7.08

± 1.93

± 2.25

± 2.09

± 2.58

n=25

n=21

n=23

n=25

Mean body weight (female fetuses) (g)

5.04

5.05

5.06

5.2

± 0.44

± 0.32

± 0.46

± 0.37

n=188

n=157

n=185

n=195

Mean body weight (male fetuses) (g)

5.33

5.27

5.39

5.44

± 0.52

± 0.39

± 0.38

± 0.38

n=175

n=160

n=163

n=177

Table 2: Fetal variations and malformations

 

 

Control

dose groups [mg/kg bw/day]

 

100

500

1000

No. of fetuses with external variations

0

0

0

0

n=372

n=317

n=347

n=358

No. of fetuses with external malformations

0

1

3

0

n=372

n=317

n=347

n=358

No. of fetuses with visceral or head variations

0

0

1

2

n=187

n=161

n=173

n=186/187*

No. of fetuses with visceral or head malformations

2

2

1

0

n=187

n=161

n=173

n=186/187*

No. of fetuses with skeletal variations

45

39

29

35

n=185

n=156

n=176

n=185

No. of fetuses with skeletal malformations

1

3

0

2

n=185

n=156

n=176

n=185

  

*186 visceral examinations performed, 187 head examinations performed

Applicant's summary and conclusion

Conclusions:
The test substance was not considered embryotoxic under the conditions of this study. Accordingly, the maternal and developmental NOAELs were established at 1000 mg/kg bw/d under the conditions of this study. The results indicate that test substance was not a selective developmental toxicant and was not embryotoxic or teratogenic under the conditions of this study.