5-(diisopropylamino)-2-[[4-(dimethylamino)phenyl]azo]-3-methyl-1,3,4-thiadiazolium methyl sulphate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No effects on the pregnancy were observed in either the preliminary or the main teratogenicity study in female rats.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

No teratogenic effects were seen in the preliminary and main study at dose levels up to 80 mg/kg bw/day. Maternal effects were seen at 80 mg/kg bw/day (deaths and unspecific toxic effects) leading to reduced foetal and placental weight in the offspring of the one surviving dam. As a consequence, the NOAEL for teratogenic effects could be considered to be 80 mg/kg bw/day and the NOAEL for maternal effects lies between 20 and 80 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Remarks:

preliminary study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1986/1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

The influence of Basic Blue 159 upon organogenesis and the subsequent outcome of pregnancy was assessed in sexually mature female rats of the CD strain.
For the purpose of this preliminary assessment, Basic Blue 159 was administered by gavage at dose levels of 5, 20, or 80 mg/kg body weight/day to groups of 6 pregnant female rats from Day 6 to Day 15 inclusive of gestation. Control animals received the vehicle, distilled water, throughout the same period.
All females were killed on Day 20 of gestation for examination of their uterine contents.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD strain of Sprague Dawley origin

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River U.K. Limited, Margate, Kent, UK
- Age at study initiation: approximately 9-11 weeks; virgin females.
- Weight at study initiation: in weight range 197 g to 231 g.
- Housing: the animals were housed inside a barriered, limited access, rodent facility.
At various stages of the study the maximum number of rats per cage was:
Stage Number of rats Gage type
M F
Acclimatisation - 5 RC1
Mating 1 1 RB3 modified
Gestation - 1 RB3
- Diet: the rats were allowed free access to a commercially-available laboratory animal diet (Labsure Laboratory Animal Diet No. 1). The manufacturers (Labsure, Lavender Mill, Manea, Cambridgeshire, England) supplied a Certificate of analysis with every batch.
- Water: tap water from the local domestic supply was freely available to the animals via polythene bottles and chromium-plated sipper tube
- Acclimation period: 5 days for females, during which were unspected daily to check their physical condition
- Contaminantes: no contaminantes were reasonably expected to be present in water, diet or bedding at levels known to be capable of interfering with the progress and outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: valuated daily; target were 21°C (range 18 - 25 °C)
- Humidity: valuated daily; target were 55 % (range 40 - 79 %)
- Air changes: the animal room had its own supply of filtered air which was passed to the atmosphere without re-circulation, providing approximately 15 room air changes per hour.
- Photoperiod: 12 hrs dark and 12 hrs light

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Basic Blue 159 was prepared freshly each day as a solution in distilled water. Dosages were of the material as supplied.
Volume-dosage of 10 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Information on the homogeneity of mixing, stability and concentration of the experimental compound in the vehicle was determined by the Sponsor. Samples of each concentration of the test mixtures were taken during the first and last weeks of treatment and analysed by the Sponsor for test chemical content.

Details on mating procedure:

Females were paired on a one-to-one basis with stock males of the same strain.
Each morning following pairing, the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each female and examined for the presence of spermatozoa.
The day on which a sperm positive vaginal smear or at least three copulation plugs were found was designated Day 0 of gestation.

Duration of treatment / exposure:

Animals were dosed daily from Day 6 to day 15 of gestation inclusive.

Frequency of treatment:

once per day

Duration of test:

until Day 20 of gestation

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Dose / conc.:

80 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6 animals for each of the four groups

Control animals:

yes, concurrent vehicle

Maternal examinations:

CLINICAL SIGNS and MORTALITY
All animals were examined daily throughout the study and any visible signs of reaction to treatment were recorded, with details of type, severity, time of onset and duration.

BODY WEIGHT
Females were weighed on days 0, 3, 6, to 16 inclusive, 18 and 20 gestation.

FOOD CONSUMPTION
Fodd consumption was recorded for the periods ending days 3, 6, 9, 12, 16, 18 and 20 of gestation.

WATER CONSUMPTION
Water consumption was recorded for the periods ending days 3, 6, 9, 12, 16, 18 and 20 of gestation.

POST-MORTEM EXAMINATIONS
Animals killed prematurely because of reaction to treatment were subjected to a thorough macroscopic examination of the visceral organs. Specimens of any abnormal tissue were retained.
On day 20 of gestation, the surviving females were killed by inhaled carbon dioxide for examination of their uterine contents. Each animal was first examined macroscopically for evidence of disease or adverse reaction to treatment and specimens of abnormal tissues were retained.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: yes
Examinations included:
- Number of corpora lutea: yes
- Number of implantations: yes
- Number of early resorption site: yes
- Number and distribution of live and dead foetuses in each uterine horn: yes
- Individual placental: yes

Fetal examinations:

- External examinations: yes of individual foetuses
- The neck and the thoracic and abdominal cavities of approximately two thirds of each litter were dissected and examined: yes
- Weight and sex of foetuses: yes

Statistics:

The smnall sample size precluded meaningful statistical evaluation. the biological significance of any suggestive inter-group differences was assessed by reference to control data previously recorded in these laboratories.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The general condition of females in Group 2 (5 mg/kg body weight/day) was comparable with that of the Controls. Females in .Group 3 (20 mg/kg body weight/day) showed excessive salivation after dosing, the first signs were recorded after the fourth dose and daily until the end of the treatment period. Blue staining around the mouth was noted for one animal. All Group 4 females (80 mg/kg body weight/day) exhibited excessive salivation post-dosing commencing after the second or third dose. Blue staining was noted around the mouths of the majority of the group. After the third dose, one female showed tremors, respiratory difficulties and became prone shortly after dosing. Subsequently, four females in this group, became lethargic after receiving 6, 7 (2 animals) or 8 doses and showed a variety of other signs including prone posture, shallow respiration, tremor and loss of the eye closure reflex.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 80 mg/kg body weight/day, 5 of 6 females had to be killed for animal welfare reasons

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Bodyweight gain of Group 2 females (5 mg/kg body weight/day) was similar to that of the Controls. Animals in Group 3 (20 mg/kg body weight/day) showed an initial, transient effect upon bodyweight gain, but the subsequent rate of weight gain was comparable with that of the Controls. The single Group 4 female (80 mg/kg body weight/day) that survived to term showed a depressed bodyweight gain during the majority of the dosing period. Following completion of treatment the rate of weight gain was comparable with that of the Controls.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food intake of the Group 2 females (5 mg/kg body weight/day) was unaffected by treatment. In Group 3 (20 mg/kg body weight/day) a marginal depression was recorded between Days 12 and 15 of gestation, but subsequently recovered to control values. The one surviving Group 4 female (80 mg/kg body weight/day) showed a depressed food intake from the commencement of treatment and did not return to control values until Day 18 of gestation.

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Slight intergroup differences were recorded in the water intake of females in Groups 2 and 3 (5 and 20 mg/kg body weight/day), but there were no consistent effects attributable to treatment. Intake by the one female in Group 4 (80 mg/kg body weight/day) was depressed during Days 9 to 15 of gestation, but subsequently recovered.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy of surviving females at Day 20 of gestation revealed two Group 3 females (20 mg/kg body weight/day) with blue-stained salivary glands. No other treatment-related macroscopic abnormalities were observed.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The one animal in Group 4 (80 mg/kg body weight/day) showed an increased pre-implantation loss

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

Changes in number of pregnant:

no effects observed

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

clinical signs

mortality

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

80 mg/kg body weight/day: depressed foetal and placental weights

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

It was concluded from this preliminary investigation that there were no indications to a teratogenic effect of Basic Blue 159. For the main study the highest dose should not exceed 20 mg/kg body weight/day.

Executive summary:

The influence of Basic Blue 159 upon organogenesis and the subsequent outcome of pregnancy was assessed in sexually mature female rats of the CD strain. For the purpose of this preliminary assessment, Basic Blue 159 was administered by gavage at dose levels of 5, 20, or 80 mg/kg body weight/day to groups of 6 pregnant female rats from Day 6 to Day 15 inclusive of gestation. Control animals received the vehicle, distilled water, throughout the same period. All females were killed on Day 20 of gestation for examination of their uterine contents.

Females receiving 5 mg/kg body weight/day showed no adverse response to treatment. At 20 and 80 mg/kg body weight/day all females exhibited excessive salivation after dosing. Five females receiving 80 mg/kg body weight/day were terminated before the end of treatment because of marked adverse responses, which included prone posture, lethargy, tremor, irregular respiration and loss of the eye closure reflex. At necropsy three of these females were found to have ulcerated areas in the fundic region of the stomach.

Weight gain of females receiving 5 mg/kg body weight/day was comparable with that of the control group. Females receiving

20 mg/kg body weight/day showed an initial, transient, reduction of weight gain, the subsequent rate of gain was comparable with that of the Controls. Weight gain of the single female that survived at 80 mg/kg body weight/day was depressed during the majority of the treatment period, but subsequeritly improved to the control rate of gain post treatment.

Food intake of females receiving 5 mg/kg body weight/day was unaffected by treatment. Females receiving 20 mg/kg body weight/day showed a marginal depression during the last four days of treatment, but subsequently recovered to control values. At 80 mg/kg body weight/day, food intake of the one surviving animal was reduced from the start of the treatment period.

Water intake of females receiving 5 and 20 mg/kg body weight/day was unaffected by treatment. At 80 mg/kg body weight/day, water intake was depressed during Days 9 to 15 of gestation, but subsequently improved to control values.

Litter responses of females receiving 5 and 20 mg/kg body weight/day were unaffected by treatment. Mean foetal and placental weight was reduced and pre-implantation loss was increased in the single surviving litter at 80 mg/kg body weight/day.

It was concluded from this preliminary investigation that there were no indications to a teratogenic effect of Basic Blue 159. For the main study the highest dose should not exceed 20 mg/kg body weight/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD strain of Sprague Dawley origin

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River U.K. Limited, Margate, Kent, England.
- Age at study initiation: approximately 9 to 11 weeks of age.
- Weight range of animals: 196 to 238 g.
- Housing: housed inside a barriered, limited access, rodent facility.
At various stages of the study the maximum number of rats per cage was:
Stage Number of rats Cage type
M F
Acclimatisation - 5 RC1
Mating 1 1 RB3 modified
Gestation - 1 RB3

- Diet: The rats were allowed free access to a commercially-available laboratory animal diet Labsure Laboratory Animal Diet No. 1 (Labsure, Lavender Mill, Manea, Cambridgeshire, England)
- Water: Tap water from the public supply was freely available
- Acclimation period: 5 days
- Health: no contaminants were reasonably expected to be present in water, diet or bedding at levels known to be capable of interfering with the progress and outcome of this study.

ENVIRONMENTAL CONDITIONS
- Temperature: range 18 - 25 °C, target value 21 °C
- Humidity: range 40 - 70 %, target value 55 %
- Air changes: the animal room had its own supply of filtered air which was passed to the atmosphere without recirculation, providing approximately 15 room air changes per hour.
- Photoperiod: 12-hour light, 12-hour dark.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Basic Blue 159 was prepared freshly each day as a solution in distilled water. Dosages were of the material as supplied.
Volume-dosage of 10 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each concentration of the test mixtures were taken during the first and last weeks of treatment and analysed by the Sponsor for test chemical content.

Details on mating procedure:

- Impregnation procedure: females were paired on a one-to-one basis with stock males of the same strain.
- Proof of pregnancy: Each morning following pairing, the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each female and examined for the presence of spermatozoa. The day on which a sperm positive vaginal smear or at least three copulation plugs were found was designated Day 0 of gestation.
- Other: females showing unequivocal evidence of mating were allocated to group and cage position in sequence, thus ensuring that animals mated on any one day were evenly distributed amongst the groups. Each animal was assigned a number and identified by tail tattoo.

Duration of treatment / exposure:

Animals were dosed daily from Day 6 to day 15 of gestation inclusive.

Frequency of treatment:

once per day

Duration of test:

until Day 20 of gestation

Dose / conc.:

1.5 mg/kg bw/day (actual dose received)

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Dose / conc.:

15 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

21 animals for each group of treatment

Control animals:

yes

Details on study design:

Control animals received the vehicle at the same volume-dosage during the treatment period.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS
All animals were examined daily throughout the stay and any visible signs of reaction to treatment were recorded, with details of type, severity, time of onset and duration.

BODY WEIGHT
Females were weighed on Days 0, 3, 6, to 16 inclusive, 18 and 20 of gestation.

FOOD CONSUMPTION
Food consumption was recorded on Days 0, 3, 6, 9, 12, 16, 18 and 20 of gestation.

POST-MORTEM EXAMINATIONS
On day 20 of gestation, the females were killed by inhaled carbon dioxide for examination of their uterine contents. Each animal was first examined macroscopically for evidence of disease or adverse reaction to treatment and specimens of abnormal tissues were retained.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: yes
Examinations included:
- Number of corpora lutea: yes
- Number of implantations: yes
- Number of early resorption site: yes
- Number and distribution of live and dead foetuses in each uterine horn: yes

Fetal examinations:

- External examinations: each foetus was weighed, sexed and examined for any external abnormalities. Individual placental weights and placental abnormalities were recorded.
- Soft tissue examinations: the neck and the thoracic and abdominal cavities of approximately half of each litter were dissected and examined. All foetal abnormalities were recorded and the offspring eviscerated prior to fixation in industrial methylated spirit.
- Skeletal examinations: the eviscerated foetus were processed and stained with Alizarin-red, usind a modification of the Dawson staining technique and the skeletons were examined.
- Internal examinations: by free-hand serial selectioning. The remainder of the foetus in each litter were placed in Bouin's fixative. After period of fixation they were examined by the Wilson free-hand serial sectioning technique.

Statistics:

The significance of suggestive inter-group differences was tested using appropriate statistical tests, each of which has been specified where used.
Differences with an associated probability of P<0.05 were considered to be statistically significant.


Data were expressed as group means with standard deviations (S.D.), where appropriate:

Maternal bodyweight
Group mean values and S.D. were calculated on Days 0, 3, 6 to 16, 18 and 20 of gestation. Weight changes were plotted graphically with respect to Day 6 of gestation.

Maternal food intake
For each animal, mean daily intakes were calculated for periods of Days 0-2, 3-5, 6-8, 9-11, 12-15, 16-17 and 18-19 of gestation. Group mean daily intakes and S.D. were calculated for these periods.

Litter data group mean values and S.D. were calculated for numbers of corpora lutea, implantations, resorptions (early, late and total) and live young (male, female and total) at Day 20 post coitum.

Group mean foetal and placental weights
Foetal observations

Indices:

Pre-implantation loss included losses due to non-fertilisation of ova and very early post-implantation deaths (i.e. those occurring during the first 2-3 days post-implantation), in addition to true pre-implantation loss.
Post-implantation loss did not include the first 2 to 3 days post-implantation as any deaths that occurred in this phase would leave no remains visible at Day 20.

Group mean foetal and placental weights
Foetal observations - Group values for foetal observations at necropsy and at free-hand serial sectioning or skeletal evaluation
The percentage incidence of observations on the sex organs was expressed relative to the number of foetuses of the appropriate sex.
The number of litters in which an observation occurred was also reported for each group.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Gross pathological findings:

no effects observed

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

Changes in number of pregnant:

no effects observed

Dose descriptor:

NOEL

Effect level:

>= 15 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no effects were observed

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Skeletal malformations:

effects observed, non-treatment-related

Visceral malformations:

effects observed, non-treatment-related

Details on embryotoxic / teratogenic effects:

Examination of foetuses at necropsy and following free-hand serial sectioning or skeletal processing revealed a small number of anomalies in all groups, the majority of which were of types and incidences previously recorded in control foetuses of this strain of rat in these laboratories and showed no association with treatment.
Two grossly abnormal foetuses were seen at necropsy, one in Group 2 (1.5 mg/kg/day) with no lumbar spine, agenesis of the tail and imperforate anus and one in Group 4 (15 mg/kg/day) with exencephaly, open eye and spina bifida occulta; the underlying skeletal defects were confirmed at subsequent skeletal examination.
Examination following free-hand serial sectioning also revealed a small number of foetuses with more complex morphological changes, viz.
Group 1 (Control) One foetus with slight hydrocephaly.
Group 2 (1.5 mg/kg/day) One foetus with a small diaphragmatic hernia.
Group 3 (5 mg/kg/day) One foetus with a retro-oesophageal right subclavian artery
Because of the isolated and disparate nature of these observations they were considered to be unrelated to treatment.
Examination of foetuses after skeletal processing showed that the incidence of incomplete ossification of the supra-occipital bone in Group 4 foetuses (15 mg/kg/day) was significantly higher than in the concurrent control group (P<0.01) and was also considerably above the background control range. The incidences in Groups 2 and 3 (1.5 and 5.0 mg/kg/day) were slightly above the background control range but were not significantly different from the concurrent control value. The incidences of incomplete ossification of the interparietal and hyoid bones in Groups 3 and 4 (5.0 and 15.0 mg/kg/day) were marginally increased compared with the concurrent controls, but all values were well within the background control ranges. Other ossification parameters showed slight intergroup differences, but there were no indications of any consistent effects of treatment.

Dose descriptor:

NOAEL

Effect level:

>= 15 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

NOAEL (maternal toxicity and development): 15 mg/kg/day

Executive summary:

The influence of Basic Blue 159 upon the progress and outcome of pregnancy was assessed in sexually mature rats of the CD strain.

For this purpose Basic Blue 159 was administered by gavage at dose levels of 1.5, 5.0 or 15.0 mg/kg body weight/day to groups of 21 pregnant rats from Day 6 to Day 15 of gestation inclusive. Control animals received the vehicle, distilled water, throughout the same period.

All females were killed on Day 20 of gestation for examination of their uterine contents.

The general condition of the dams was unaffected by treatment, and no deaths occurred.

Bodyweight gain of females in all treated groups was essentially similar to that of the controls.

Food intake was unaffected by treatment with Basic Blue 159.

There were no adverse effects of treatment upon litter size, post-implantation survival or foetal and placental weights.

At 15 mg/kg body weight/day there was a statistically significant increase in the incidence of foetuses with incomplete ossification of the supra-occipital bone and there was a lesser, non-significant increase at 5.0 and 1.5 mg/kg body weight/day. There were marginal increases in the incidences of incomplete ossification of the interparietal and hyoid bones at 5 and 15 mg/kg body weight/day, but these values remained within the background control ranges. There were no morphological changes that were considered to be related to treatment.

It was concluded from these investigations that oral administration of Basic Blue 159 at dosages of 1.5, 5.0 and 15.0 mg/kg body weight/day to pregnant rats during organogenesis was without significant adverse effects upon maternal performance, litter parameters and growth and survival in utero. Ossification of the foetal posterior cranial bones was slightly reduced, most obviously at 15.0 mg/kg body weight/day, but there were no adverse morphological changes that were considered to be related to treatment.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

80 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The influence of Basic Blue 159 upon organogenesis and the subsequent outcome of pregnancy was assessed in sexually mature female rats of the CD strain. For the purpose of this preliminary assessment, Basic Blue 159 was administered by gavage at dose levels of 5, 20, or 80 mg/kg body weight/day to groups of 6 pregnant female rats from Day 6 to Day 15 inclusive of gestation. Control animals received the vehicle, distilled water, throughout the same period. All females were killed on Day 20 of gestation for examination of their uterine contents.

Females receiving 5 mg/kg body weight/day showed no adverse response to treatment. At 20 and 80 mg/kg body weight/day all females exhibited excessive salivation after dosing. Five females receiving 80 mg/kg body weight/day were terminated before the end of treatment because of marked adverse responses, which included prone posture, lethargy, tremor, irregular respiration and loss of the eye closure reflex. At necropsy three of these females were found to have ulcerated areas in the fundic region of the stomach.

Weight gain of females receiving 5 mg/kg body weight/day was comparable with that of the control group. Females receiving

20 mg/kg body weight/day showed an initial, transient, reduction of weight gain, the subsequent rate of gain was comparable with that of the Controls. Weight gain of the single female that survived at 80 mg/kg body weight/day was depressed during the majority of the treatment period, but subsequeritly improved to the control rate of gain post treatment.

Food intake of females receiving 5 mg/kg body weight/day was unaffected by treatment. Females receiving 20 mg/kg body weight/day showed a marginal depression during the last four days of treatment, but subsequently recovered to control values. At 80 mg/kg body weight/day, food intake of the one surviving animal was reduced from the start of the treatment period.

Water intake of females receiving 5 and 20 mg/kg body weight/day was unaffected by treatment. At 80 mg/kg body weight/day, water intake was depressed during Days 9 to 15 of gestation, but subsequently improved to control values.

Litter responses of females receiving 5 and 20 mg/kg body weight/day were unaffected by treatment. Mean foetal and placental weight was reduced and pre-implantation loss was increased in the single surviving litter at 80 mg/kg body weight/day.

It was concluded from this preliminary investigation that there were no indications to a teratogenic effect of Basic Blue 159. For the main study the highest dose should not exceed 20 mg/kg body weight/day.

In the main study, Basic Blue 159 was administered by gavage at dose levels of 1.5, 5.0 or 15.0 mg/kg body weight/day to groups of 21 pregnant rats from Day 6 to Day 15 of gestation inclusive. Control animals received the vehicle, distilled water, throughout the same period.

All females were killed on Day 20 of gestation for examination of their uterine contents.

The general condition of the dams was unaffected by treatment, and no deaths occurred.

Bodyweight gain of females in all treated groups was essentially similar to that of the controls.

Food intake was unaffected by treatment with Basic Blue 159.

There were no adverse effects of treatment upon litter size, post-implantation survival or foetal and placental weights.

At 15 mg/kg body weight/day there was a statistically significant increase in the incidence of foetuses with incomplete ossification of the supra-occipital bone and there was a lesser, non-significant increase at 5.0 and 1.5 mg/kg body weight/day. There were marginal increases in the incidences of incomplete ossification of the interparietal and hyoid bones at 5 and 15 mg/kg body weight/day, but these values remained within the background control ranges. There were no morphological changes that were considered to be related to treatment.

It was concluded from these investigations that oral administration of Basic Blue 159 at dosages of 1.5, 5.0 and 15.0 mg/kg body weight/day to pregnant rats during organogenesis was without significant adverse effects upon maternal performance, litter parameters and growth and survival in utero. Ossification of the foetal posterior cranial bones was slightly reduced, most obviously at 15.0 mg/kg body weight/day, but there were no adverse morphological changes that were considered to be related to treatment.

Hence, no teratogenic effects were seen in the preliminary and main study at dose levels up to 80 mg/kg bw/day. Maternal effects were seen at 80 mg/kg bw/day (deaths and unspecific toxic effects) leading to reduced fetal and placental weight in the offspring of the one surviving dam. As a consequence, the NOAEL for teratogenic effects could be considered to be 80 mg/kg bw/day and the NOAEL for maternal effects lies between 20 and 80 mg/kg bw/day.

Justification for classification or non-classification

No developmental or teratogenic effects were seen in the preliminary and main teratogenicity study. The only effects seen, were secondary due to the general toxicity of the test item at the highest dose level tested in the preliminary study of 80 mg/kg bw/day. This general toxic effect is taken into consideration with the classification of STOT RE 2 (H373) in the repeat-dose section of this dossier, which is based on a NOAEL of 40 mg/kg bw/day for the 28-day repeat-dose study in male and female rats.

No classification for reproductive effects was deemed necessary based on the results of the teratogenicity studies.