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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Flexzone 3C, purity: 97.2%

Test animals

Species:
rat
Strain:
Sprague-Dawley

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- M/F ratio per cage: 1:2
Duration of treatment / exposure:
day 6-15 day of gestation
Frequency of treatment:
daily
Duration of test:
study termination on gestation day 20
No. of animals per sex per dose:
24
Control animals:
yes
Details on study design:
Sex: female

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
62.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Mortality Data

There were no deaths.

Clinical Observations

At 125 mg/kg clinical observations were limited to pre-dosing salivation noted in 10/23 animals and soft dark faeces noted for 6/23 animals.

At 62.5 mg/kg transient noisy respiration was noted in one animal.

Body weight

There were no treatment related effects on bodyweight noted during

pregnancy.

Table: Group mean body weight (g)

 Group  Dose level (mg/kg)                                         Day Post Coitum
     0  3  6  7  8  9  10  11  12 13   14  15  18  20
 1  0 269±16.8 284±20.2 300±22.2 305±23.0 311±23.5  316±21.9 323±20.9 330 ±20.9 337±22.5 344 ±22.0 351 ±22.7 360±22.9 402±27.6 436±29.9
 2  12.5 270 ±14.6 287 ±15.6 300 ±14.6  303±14.8 308 ±16.1 317 ±16.6 324 ±16.5 332 ±18.2 338 ±18.9  346±19.0  354±19.2 362 ±19.7 405 ±23.3  436±27.8
 3  62.5  269±17.5 285 ±18.7  299±19.8 302 ±19.4  308±20.8 314 ±19.9 320 ±20.7  329±22.0  337±21.7  344±22.7 353 ±22.6  362±22.9 401 ±28.3 431 ±32.3
 4  125  265±15.2  282±15.6  297±16.5  300±16.1  307±18.5  311±19.9  321±20.2  329±18.8 338 ±20.6  346±20.4  354±21.1  361±22.2  403±25.3  431±28.1

Food Consumption

At 125 mg/kg there was a statistically significant (p < 0.001) reduction in food consumption from day 6 to day 9 of gestation which coincides with the initiation of dosing.

At 62.5 mg/kg there was also a statistically significant reduction in food consumption over this period (p < 0.01).

After day 9 of gestation food consumption in these two groups was comparable with control animals.

At 12.5 mg/kg there were no significant effects on food consumption throughout the study.

Table: Group mean food consumption

Group  Dose level (mg/kg)                    Days Post Coitum
     0 -3  3 -6 6 -9  9 -12  12 -15  15 -18  18 -20  
 1  0  24.7±2.38 27.8 ±2.81 27.7±2.61  30.7 ±2.96  32.3±2.54 35.3 ±2.50 34.1 ±2.42
 2  12.5  24.6±2.39  27.2±2.77  27.1±3.04  30.2±2.99  32.2±3.57  35.3±3.12  33.5±3.77  
 3  62.5  24.3±3.33  27.6±2.75  25.0**±3.27 29.1 ±3.07  31.7±2.63  35.6±3.11  35.5±3.02  
 4  125  24.3±2.44  27.1±2.34  23.8***±3.87 30.1 ±3.21  32.3±2.96  35.4±2.49  35.7±3.18  

**significantly different from control (p<0.01)

***significantly different from control (p<0.001)

Terminal Studies

Adult Necropsy Data

There were no treatment-related macroscopic findings for adult females at necropsy.

Uterine/ Implantation Data

There were no treatment-related effects on uterine/implantation data.

Table: Summary of adult performance

            Dose level mg/kg
   0  12.5  62.5  125
 Number of females in group  24  24  24  24
 Number pregnant  24  24  24  23
 Number died/killed before day 20  0  0  0  0
 Number not pregnant  0  0  0  1
 Number with total litter loss  0  0  0  0

Table: Group mean data uterine/implantation

 Group  Dose level (mg/kg)  Number of animals  Number pregnant Number of Corpora Lutea Number of Live Fetuses
 
% Male Fetuses
 1  0  24  24  18.4  15.0  49.4
 2  12.5  24  24  18.5  15.2  48.1
 3  62.5  24  24 18.6   13.8  46.8
 4  125  24  23  20.1  16.1  45.9

Table: Group mean litter data

   

       Embryonic/Foetal deaths

    Implantation loss%  Total litter weight (g)  Mean foetal weight (g)  
 Group  Dose level (mg/kg)  Early  Late  Total  Pre  Post      
 1  0  0.54  0.00  0.54  14.9  3.4  55.7  3.72  
 2  12.5  0.83  0.00  0.83  12.6  5.2  59.2  3.91  
 3  62.5  0.96  0.00  0.96  20.3  7.5  52.9  3.80  
 4  125  0.48  0.09  0.57  15.7 3.8   62.4  3.88  

Foetal Data

Throughout all groups there were three foetuses with significant structural anomalies.

At 12.5 mg/kg one foetus, (dam 35, foetus 1) had an encephalocele with a further foetus (dam 44, foetus 7) having an interventricular septal defect. At 0 mg/kg one foetus (dam 14, foetus 5) had an interventricular septal defect, an

atretic left atrium and mitral valve, right sided aorta, aortic arch and ductus arteriosus, no brachiocephalic trunk, no pulmonary trunk with a modified ductus arteriosus exiting from the right ventricle.

There were no intergroup differences in the incidence of foetal external findings.

Table: Incidence of foetal external findings

 Dose level (mg/kg)  0  12.5  62.5  125
 Number of foetuses examined  361  364  332  370
 Total Number of fetuses affeted  2  4  7  1
 Total number of litters affected  2  4  5  1
Group mean per litter (%)  0.6  1.2  3.4  0.3

Table: Group summary of foetal skeletal development

Dose level (mg/kg)  0  12.5  62.5  125
 Number of foetuses examined  174  175  159  182
 Number of rips: 13/13  153F/24L/89%  152F/24L/87.2%  143F/24L/90.1%  154F/23L/82.7%
  13/14  16F/9L/8.3%  13F/10L/7.3%  8F/7L/4.3%  16F/12L/9.6%
  14/14  5f/3L/2.5%  10F/7L/5.4%  7F/6L/5.6%  12F/6/L7.7%
 Number of fully ossified sternebrae <4  5F/5L/2.8%  11F/4L/6.8%  11F/9L/8.5%  4F/4L/2.9%
  4  69F/19L/38.0%  54F/18L/32.4%  52F/21L/36.2%  75F/21L/40.7%
  >4  99F/24L/59.2% 107F/23L/60.8%   94F/20L/55.3%  103F/L22/56.4%
 Number of ost lumbar vertebral centra <7  8F/6L/4.5%  8F/5L/4.1%  12F/7L/9.6%  7F/5L/3.6%
 Number of ost lumbar vertebral centra >7  166F/24L/95.5%  167F/24L/95.9%  146F/24L/90.4%  174F/23L/96.4
 Number of post lumbar vertebral arches <5  26F/12L/16.0%  27F/12L/16.7%  33F/13L/19.6%  23F/12L/12.3
 Number of post lumbar vertebral arches >5  148F/24L/84.0%  148F/24L/83.3%  125F/23L/80.4%  158F/23L/87.7%
 Number of metacarpals/ Metatarsals 0/0  0F/0L/-  0F/0L/- 1F/1L/2.1%   0F/0L/-
  5/6  0F/0L/-  0F/0L/-  1F/1L/0.6%  0F/0L/-
  6/6  2F/2L/1.1%  3F/3L/1.4%  2F/2L/2.9%  4F/3L/1.9
  6/7 0F/0L/-   1F/1L/0.6%  0F/0L/-  2F/2l/1.1%
  7/7  0F/0L/-  2F/1L/1.2%  0F/0L/-  0F/0L7-
  4/8  0F/0L/- 1F/1L/0.5%   0F/0L/- 0F/0L/- 
  6/8  132F/23L/77.0%  110F/21L/65.1%  108F/21L/67.6%  114F/23L/62.0%
  7/8  2F/1L/0.9%  3F/3L/1.5%  3F/3L/1.7%  4F/4L/2.3%
  8/8  38F/11L/21.1%  48F/14L/27.0%  41F/14L/24.6%  57F/18L/32.7%
  8/10  0F/0L/-  5F/1L/2.6%  1F/1L/0.6%  0F/0L/-
Fontanelle size: small  7F/5L/4.3%  21F/7L/12.8%  18F/7L/13.7%  8F/3L/4.0%
Fontanelle size: medium  166F/24L/95.2%  150F/23L/85.8%  132F/21L/77.9%  174F/23L/96.0
 Fontanelle size: large  1F/1l/0.5%  3F/2L/1.4%   9F/4L/8.4%  0F/0l/-

F:number of foetuses in category

L:number of litters in category

%:group mean per litter

Skeletal findings:

At 125 mg/kg there were statistically significant effects on the incidence of skeletal findings. At this dose level there was an increased incidence of irregularly and incompletely ossified cranial and facial bones (p < 0.001 - P < 0.05). There was also an increased incidence of no ossification of hyoid (p < 0.001), unilaterall bilateral wavy ribs (p < 0.05) and semi bipartite vertebral centra (p < 0.01). At 62.5 mg/kg there was a statistically significant increase in incomplete ossification of more than one cranial bone (p < 0.05).

At 12.5 mg/kg there was a statistically significant increase in incomplete ossification of more than one facial bone (p < 0.05).

remarks: The only adverse effect noted in the foetuses and related to treatment was a retardation of ossification in the high dose group (125 mg/kg bw/d); and a low percentage of wavy ribs, 3.8% compared with 0.6% in the controls. Wavy ribs are a commonly observed effect in studies, especially in presence of maternal toxicity, and normally disappears post-natal and is not considered as a malformation but as a variation. There are no adverse effects in low and mid dose pups.

Table:Group incidence of foetal skeletal findings

Dose level (mg/kg)  0  12.5  62.5  125
 Number of foetuses examined  174  175  159  182
 Total Number of fetuses affeted  72  98  86  124
 Total number of litters affected  22 23  22  23
Group mean per litter (%) 40.3  53.3  51.3  69.5
 Incomplete ossification of more than one cranial bone  12F/10L/ 6.9%  13F/9L/ 7.5%  25*F/11L/15.2 %  35**F/16L/17.8%
 incomplete ossification of more than one facial bone  2F/2L/ 1.1%  10*F/5L/5.3%  6F/6L/5.1%  19**F/10L/10%
 irregular ossification of one cranial bone  1F/1L/0.5%  2F/2L/1.0% 3F/3L/1.7%   8*F/7L/ 4.8%
 irregular ossification of one facial bone  0F/0F/-  4F/4L/ 1.9%  3F/3L/ 1.5%  9**F/6L/ 5.0%
 irregular ossification of more than one facial bone  3F/3L/ 1.6%  4F/4L/ 2.1%  7F/5L/ 3.8%  19**F/8L/9.7%
 no ossification of hyoid  16F/11L/9.4%  27F/13L/14.4%  24F/12L/ 13.8%  43***F/16L/22.3%
 unilateral/bilateral wavy ribs  1F/1L/0.6%  0F/0L/-  2F/2L/ 1.1%  7*F/5L/3.8%
 one thoracic vertebral centrum semi-bipartite  10F/6L/5.6%  14F/7L/ 7.3%  18F/9L/9.7%  30**F/14L/16.4%

*: significantly different from control (p<0.05)

**: significantly different from control (p<0.01)

***: significantly different from control (p<0.001)

F:number of foetuses in category

L:number of litters in category

%:group mean per litter

Visceral Findings:

The overall incidence of visceral findings for foetuses was considered comparable for all groups. Intergroup variations for specific visceral findings were not considered to be significant.

Table: Group incidence of foetal visceral findings

Dose level (mg/kg)  0  12.5  62.5  125
 Number of foetuses examined  187  189 172 188
 Total Number of fetuses affeted  61 64 66  75
 Total number of litters affected  23  23  23 19
Group mean per litter (%) 33.0   34.7  39.6  37.2

Applicant's summary and conclusion