Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD guideline 414 in compliance to GLP.

Data source

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

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:
Test item : HC Blue No 2 (B037)
EC number : 251-410-3
Batch number : 3I394
Assay : 99.5% (titration)

Test animals

Species:
rat
Strain:
Sprague-Dawley

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% carboxymethylcellulose in water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A satisfactory agreement was observed between the nominal and actual concentrations of the test item in the administered dosage forms since the deviations from nominal concentration remained within an acceptable range of +/-10%.
Duration of treatment / exposure:
Animals were treated from day 6 to day 19 post coitum inclusive.
Frequency of treatment:
Animals were treated once daily.
Duration of test:
Animals were sacrificed on day 20 post coitum.
No. of animals per sex per dose:
Twenty four
Control animals:
yes, concurrent vehicle

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
Mortality
No premature deaths occurred during the study.

Clinical signs
All females given HC Blue No2 had blue coloured urine from approximately day 7 post coitum until necropsy and blue coloured extremities (except in one female at 100 mg/kg bw/day) from approximately day 14 post coitum until necropsy. This finding was associated with blue coloured fur in the urogenital area of one female given 300 mg/kg bw/day and of 12/24 females given 1000 mg/kg bw/day towards the end of gestation. Excessive salivation (ptyalism) was observed in 10/24 females given 300 mg/kg bw/day and 17/24 females given 1000 mg/kg bw/day for a few days after start of dosing until the end of the treatment period. Other clinical signs (nodosities on the abdomen, masses on the mammary glands, abnormal growth of teeth) were randomly distributed within the groups and are commonly noted in rats of this strain and age.

Body weight
There was a slight, but statistically significant lower mean body weight gain from day 6 to day 9 post coitum for the group receiving 1000 mg/kg bw/day when compared to controls. Body weight gain for the remainder of the treatment period was similar to that of the controls. There were no effects on group mean maternal body weight or body weight gain for the groups receiving 100 or 300 mg/kg bw/day.The net body weight change was considered to be unaffected by treatment with HC Blue No 2.

Food consumption
Group mean food consumption was significantly lower from day 6 to day 9 post coitum for the group given 1000 mg/kg bw/day when compared to the controls. For the remainder of the treatment period, food intake for this group was similar to that of controls. There were no effects on group mean maternal food consumption at 100 or 300 mg/kg bw/day.

Necropsy findings
No findings recorded at maternal necropsy were considered to be related to treatment.

Gravid uterus weight
The differences noted for gravid uterus weight in the treated groups, when compared to controls were minor, not statistically significant and not dose related. They were not considered to be treatment related.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity : lower body weight gain and food consumption observed at the highest dose
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Litter data
The mean number of corpora lutea, implantation sites and the percentage of pre-implantation loss were similar between treated and control groups. There were no effects on the group mean numbers of implantations or live fetuses or on the extent of post implantation loss for any group. There were no effects of treatment with HC Blue No 2 on group mean fetal weights or on the percentage of male fetuses; values were comparable with controls in all groups.

Soft tissue observation
No soft tissue malformations were observed in any group. The fetal soft tissue variations observed (hematoma in the cranial cavity, dilated renal pelvis, dilated ureters, enlarged atrial chamber, hemorrhage in abdominal cavity) were randomly distributed within the groups including the control.

Skeletal malformation
The only malformations observed (two fetuses with supernumerary lumbar vertebra at 100 mg/kg bw/day and one fetus with an absence of lumbar vertebra at 300 mg/kg bw/day) were not considered to be treatment related as they were not recorded in the high dose group.

Skeletal variation
At 1000 mg/kg bw/day there was a statistically significant increase in the fetal incidence of incomplete ossification of the interparietal when compared to controls (22 vs 11 in the control group). However this difference was not statistically significant when expressed as a mean percentage of affected fetuses and fell within the background control data range. Consequently it was not considered to be treatment related. At 1000 mg/kg bw/day the fetal incidence of incomplete ossification of the parietal was significantly greater when compared with the controls (6 vs 0 in the control group). This finding was not significant when expressed as a mean number of affected fetuses and fell within the background control data range. It was also not considered to be treatment related.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Administration of HC Blue No 2 (B037) to pregnant female Sprague-Dawley rats from days 6 to 19 post-coitum inclusive at 100, 300 and 1000 mg/kg bw/day caused blue colouration of urine and extremities in all females. In addition, blue coloured fur was observed in many females given 1000 mg/kg bw/day. Ptyalism was also observed in females given 300 and 1000 mg/kg bw/day. Dosing at 1000 mg/kg bw/day caused maternal toxicity, shown by significantly lower body weight gains and food consumption, while there were no indications of any effects on the pregnancy parameters or embryo-fetal development. The NOAEL for maternal toxicity was 300 mg/kg bw/day and the NOEL for developmental toxicity was 1000 mg/kg bw/day.