Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Reaction mass of 3-[(4-amino-6-bromo-5,8-dihydro-1-hydroxy-8-imino-5-oxo-2-naphtyl)amino]-N,N,N-trimethylanilinium chloride and 3-[(8-amino-dibromo-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride and 3-[(bromo-1,4-dihydroxy-8-imino-5-oxo-5,8-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride
EC Number:
916-466-1
IUPAC Name:
Reaction mass of 3-[(4-amino-6-bromo-5,8-dihydro-1-hydroxy-8-imino-5-oxo-2-naphtyl)amino]-N,N,N-trimethylanilinium chloride and 3-[(8-amino-dibromo-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride and 3-[(bromo-1,4-dihydroxy-8-imino-5-oxo-5,8-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride
Details on test material:
Identification : C059 (Basic Blue 99)
Batch number : 74/75
3-[(Bromo-8-amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (main) : 62.8a/a%
3-[(Dibromo-8-amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (E) : 11.8a/a% 3-[(Bromo-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (F) : 8.4a/a%
3-[(Bromo-5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (J) : 4.1a/a% 3-[(8-Amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (A) : 1.7a/a% 3-[(Sulpho-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (B) : 1.8a/a%
3-[(Dibromo-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (G): 2.1a/a%
3-[(Sulpho-8-amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (O) :1.0a/a%
3-[(Bromo-sulpho-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (N) : 0.7a/a%
Tribromo-8-amino-5-hydroxy-1,4-naphthoquinone (L) : 2.6a/a%
Chloride ion : 20.2%
Sulphate ion : 0.5%
Acetate ion : 2.0%
Zinc ion : 6.6%
Water : 7.9%

Test animals

Species:
rat
Strain:
other: HanRcc:WIST

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: purified water
Details on exposure:
Preparation of Dosing Solutions
Dose formulations were prepared in terms of the test item as supplied.
Frequency of dose formulation : Daily
The test item was weighed into a glass beaker on a tared precision balance and the vehicle was added. During the daily administration period homogeneity of the test item in the vehicle was maintained using a magnetic stirrer.
Dose volume : 1% (10 mL/kg). The volume was based on the weight of each animal on the day the test substance was administered.
Administration : The test item was administered orally, by gavage, once daily in the morning from day 6 through to day 20 post coitum (last treatment).
Rationale for selection of doses : Dose levels were selected based on data from previous studies performed in rats (reprotoxicity study with a single dose level resulted in a NOEL of 50mg/kg bw/day and a 90 day study with dose levels of 20, 60 and 180/360mg/kg bw/day resulted in dose related pigmentation of stomach, adrenals and small intestine and slight retardation of body weight gain at the high dose level).
Analytical verification of doses or concentrations:
no
Details on mating procedure:
After acclimatisation, females were housed with sexually mature males (1:1) in special automatic mating cages (ie : with synchronised timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if the daily vaginal smear was sperm positive or a copulation plug was observed. The day of mating was designated day 0 post coitum. Male rats of the same source and strain were used for mating only. The male rats were not considered part of the test system. The fertility of these males was proved and continuously monitored.
Duration of treatment / exposure:
Animals were treated from day 6 through to day 20 post coitum.
Frequency of treatment:
Animals were treated once daily.
Duration of test:
Animals were sacrificed on day 21 post coitum.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Each group consisted of 22 mated female rats.
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for selection of doses : Dose levels were selected based on data from previous studies performed in rats (reprotoxicity study with a single dose level resulted in a NOEL of 50mg/kg bw/day and a 90 day study with dose levels of 20, 60 and 180/360mg/kg bw/day resulted in dose related pigmentation of stomach, adrenals and small intestine and slight retardation of body weight gain at the high dose level).
Method of allocation : Mated rats were assigned to the different groups using a computer generated random algorithm.

Examinations

Maternal examinations:
Cage side observations : All animal were checked at least twice daily for any mortalities. All animals were observed at least twice daily for signs of reaction to treatment and/or symptoms of ill health. Any female sacrificed or found dead during the study was subjected to macroscopic examination with emphasis on the uterus and contents.
Body weight : Body weights were recorded daily from day 0 until day 21 post coitum.
Termination of the study : On day 21 post coitum, females were killed by CO2 asphyxiation and the fetuses removed by Caesarean section.
Ovaries and uterine content:
Post mortem examination, including gross macroscopic examination of all internal organs, with emphasis on the uterus, uterine contents, position of fetuses in the uterus and number of corpora lutea was performed. the uteri and contents of all females with live fetuses were weighed at necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
Fetal examinations:
Fetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, killed by subcutaneous injection and allocated to one of the following procedures. Microdissection technique (sectioning/dissection technique). At least one half of the fetuses from each litter was fixed then examined by a comination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. Descriptions of any abnormalities and variations were recorded. The remaining fetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. Carcasses were processed through a series of solutions for preservation and storage. The skeletons were examined and all abnormal findings and variations were recorded

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Occurrence of black faeces was observed in all animals of 200 and 500mg/kg bw/day from day 7 post coitum onwards and was considered to be due to the content of test item. The bedding material was discoloured (slight blue) in all groups treated with the test item. This was considered to be due to the excretion of the test item in the urine. Occurrence of hair loss in two animals from the 60mg/kg bw/day dose group was considered incidental.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item related deaths occurred in this study. One dam from the 500mg/kg bw/day dose group was found dead in the morning of day 11 post coitum. In the absence of any other relevant test-item related finding in this dose group and as isolated occurrence, the death was considered to be not related to the treatment with the test item.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight development was similar in all groups and not influenced by treatment with the test item. Corrected mean body weight gains were also similar in all groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption was similar in all groups. No test item related effect was noted.
Details on results:
No test item related deaths occurred in the study. Occurrence of black feaces, due to the content of the test item, were observed in all animals of the 200 and 500 mg/kg bw/day dose groups from day 7 post coitum onwards. The bedding material was bluish in colour in all groups treated with the test item, most likely due to the excreted test item in the urine. One dam from the 500 mg/kg bw/day dose group was found dead in the morning of day 11 post coitum. In the absence of any other relevant test item related finding in this dose group and as an isolated occurrence, its death was considered to be not related to the treatment with the test item. The mean food consumption and body weight development were similar in all groups. No test item related effect was observed. All animals from the 500 mg/kg bw/day dose group and individual animals from the 200 mg/kg bw/day dose group were found to have bluish discoloured stomach wall and intestine wall. Kidneys and placentae were bluish discoloured in all dams from the 500 mg/kg bw/day group. The discolouration was considered to be due to the colour of the test item and considered to be not adverse.

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Numbers of post implantation loss were similar in all groups and not influenced by treatment with the test item.
Dead fetuses:
no effects observed
Description (incidence and severity):
Numbers of live and dead fetuses were similar in all groups and not influenced by treatment with the test item.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Of 22 female rats mated in each of the groups, 22, 22, 22 and 21 survived to the scheduled day of sacrifice in the 0, 60, 200, 500mg/kg bw/day dose groups. Of these, 22, 21, 21 and 21 were pregnant and used for calculation of the reproduction data.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
At necropsy all animals from the 500mg/kg bw/day dose group and individual animals from the 200mg/kg bw/day were found to have bluish discoloured stomach wall and intestine wall. In addition, kidneys and placentae were bluish discoloured in all dams from the 500mg/kg bw/day. The discolouration was considered to be due to the colour of the test item and considered to be not adverse.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
> 500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed at highest dose

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly increased mean body weight values were calculated for the 500mg/kg bw/day dose group male and female fetuses. On an individual basis, statistical significant values were also calculated for the 60mg/kg bw/day dose group total fetuses and 200mg/kg bw/day dose group female fetuses. As the values were only minimally above the control group values, the results were considered to reflect the normal range of biological variability and to be incidental.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratios were close to 50/50 in all groups and not influenced by treatment with the test item. Ratio values were (% males/%females) 47/53, 48/52, 52/48 and 48/52 in the 0, 60, 200, 500mg/kg bw/day dose groups.
External malformations:
no effects observed
Description (incidence and severity):
External examination did not reveal any test item related findings.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
During skeletal examination of the fetuses abnormal findings of bones were noted in 15/129 examined fetuses (in 7 litters) of 0mg/kg bw/day group, 21/121 examined fetuses (in 12 litters) of 60mg/kg bw/day group, 15/122 examined fetuses (in 9 litters) of 200mg/kg bw/day group, 23/127 examined fetuses (in 14 litters) of 500mg/kg bw/day group. Neither the types nor the incidences of findings did indicate any effect related to the treatment with the test item. Occurrence of rudimentary cervical ribs was found in 4 fetuses out of 3 litters in each of the 200 and 500mg/kg bw/day dose groups and was considered of incidental nature due to the low incidence and in absence of an obvious dose dependency.

On both individual and litter basis, statistically significant decreased incidences of non ossified talus and non ossified cervical vertebral body-1 was found in the 500mg/kg bw/day group. On an individual basis decreased incidences of non ossified talus were found in the 200mg/kg bw/day group. However the values were well within the range of historical reference data of the laboratory and thus considered to reflect the range of normal biological variability. Other statistical significances occurred without obvious dose dependency and were considered to be incidental.

Examination of cartilage did not reveal any test item related findings. During cartilage examination of the fetuses abnormal findings were noted in 1/129 examined fetuses (in 1 litter) of 0mg/kg bw/day dose group, 1/120 examined fetuses (in 1 litter) of 60mg/kg bw/day dose group, 3/120 examined fetuses (in 3 litters) of 200mg/kg bw/day dose group, 2/127 examined fetuses (in 2 litters) of 500mg/kg bw/day dose group. Neither the types nor the incidences of cartilage findings indicated any effect related to the treatment with the test item.
Visceral malformations:
no effects observed
Description (incidence and severity):
Visceral examination did not reveal any test item related findings. During visceral examination of fixed fetuses findings were noted in 66/137 examined fetuses (in 21 litters) of 0mg/kg bw/day group, 81/131 examined fetuses (in 21 litters) of 60mg/kg bw/day group, 72/131 examined fetuses (in 20 litters) of 200mg/kg bw/day group, 69/138 examined fetuses (in 21 litters) of 500mg/kg bw/day group. One individual case of diaphragmatic hernia in 500mg/kg bw/day group was found and considered to be incidental due to its isolated occurrence. All other findings were of common nature in this strain of rat and neither incidences nor distribution over the groups did indicate any effect due to the treatment with the test item.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
At Caesarean necropsy the mean fetal weights were significantly increased in both sexes at 500 mg/kg bw/day. Skeletal examinations revealed decreased incidences of non-ossified cervical vertebral body-1 and of non-ossified talus at 500 mg/kg bw/day.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

The relevant reproduction parameters (numbers of post implantation loss, numbers of live and dead fetuses) were similar in all groups and not influenced by treatment with the test item.

The sex ratios were close to 50:50 in all groups and not influenced by treatment with the test item. Statistically significantly increased mean body weight values were calculated for the 500 mg/kg bw/day dose group. External examination did not reveal any test item related findings. Visceral examination did not reveal any test item related findings. On both individual and litter basis, statistically significant decreased incidences of non-ossified talus and non ossified cervical vertebral body-1 were found in the 500 mg/kg bw/day dose group.

Applicant's summary and conclusion

Conclusions:
The treatment with the test item was well tolerated by the pregnant rats. There were no test item related effects on post implantation loss or number of fetuses. At Caesarean necropsy the mean fetal weights were significantly increased in both sexes at 500 mg/kg bw/day. Skeletal examinations revealed decreased incidences of non-ossified cervical vertebral body-1 and of non-ossified talus at 500 mg/kg bw/day, indicative of advanced ossification. A NOAEL for embryo/fetal toxicity was at the dose level of 200 mg/kg bw/day. The NOEL for embryo/fetal toxicity was at the dose level of 60 mg/kg bw/day. The study did not reveal any teratological or embryo lethal effect up to the high dose level of 500 mg/kg bw/day.
Executive summary:

The purpose of this study was to assess the effects of Basic Blue 99 on the pregnant female rat and development of the embryo and fetus when administered orally by gavage once daily to mated female rats from day 6 through to day 20 post coitum, inclusive. Each group consisted of 22 mated female rats. Basic Blue 99 was administered once daily at dose levels of 0 (vehicle control), 60, 200, 500 mg/kg bw/day. A standard dose volume of 10 mL/kg bw with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (purified water). All females were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean section. Examination of dams and fetuses was performed in accordance with international recommendations. Maternal data : No test item related deaths occurred in this study. Occurrence of black faeces, due to the content of the test item, were observed in all animals of the 200mg/kg bw/day and 500mg/kg bw/day dose groups from day 7 post coitum onwards. The bedding material was discoloured (slight blue) in all groups treated with the test item, most likely due to the excreted test item in the urine. One dam from the 500mg/kg bw/day dose group was found dead on the morning of day 11 post coitum. In the absence of any other relevant test item related finding in this dose group, the death was considered to be not related to the treatment with the test item. The mean food consumption and body weight development were similar in all dose groups. No test item related effect was noted. The relevant reproduction parameters (number of post-implantation loss, number of live and dead fetuses) were similar in all groups and not influenced by treatment with the test item. All animals from the 500mg/kg bw/day group and individual animals from the 200mg/kg bw/day group were found to have bluish discoloured stomach wall and intestine wall. In addition, kidneys and placentae were discoloured (bluish) in all dams from the 500mg/kg bw/day dose group. The discolouration was considered to be due to the colour of the test item and considered to be not adverse. Fetal data : The sex ratios were close to 50/50 in all groups and not influenced by treatment with the test item. Statistically significantly increasedmean body weight values were calculated for male and female fetuses of the 500mg/kg bw/day dose group. External examination did not reveal any test item related findings. Visceral examination did not reveal any test item related findings. No test item related abnormalities were noted. On both individual and litter basis, statistically significant decreased incidences of non-ossified talus and non-ossified cervical vertebral body-1 were found in the 500mg/kg bw/day dose group.