REACTIVE BLUE FC 75311

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Additional information

No assessment of reproductive hazard has been undertaken at this tonnage threshold. This is due to the following considerations:

 

1) The substance was not found to be harmful to reproductive developmental processes in a developmental study on a closely related analogue. No abnormalities of offspring were noted in this study at the maximum dose tested (1000 mg/kg/day).  Full justification for the use of this study is provided.

 

2) The substance was negative in mutagenicity assays involving animal cells, and did not have any effects at the highest dose thresholds noted.

3) Repeat-dose studies with oral treatment in rats did not reveal any tumorigenic properties which could be related to the administration of the test substance. Histopathological evaluation of reproductive organs from the 28-day study indicated no effects.Consequently, the substance is considered not to have the potential to cause reproductive effects.

4) The substance has been notified originally under the 7th Amendment, and has been in use for a number of years without effects associated with reproductive toxicity being noted.  

As such, on animal welfare grounds it is not considered appropriate to conduct a further developmental screening test at this level of registration, as the evidence indicates that there is no associated potential teratogenic or reproductive effect associated with the substance. 

Short description of key information:
It is considered that assessment of fertility effects is not scientifically justifiable at this level of registration. A developmental study on a structural analogue is available for the substance, and this is offered in section 7.8.2 of the registration dossier, and demonstrated no effects. No effects were seen on reproductive organs in the repeat dose study on the substance, and the category of substance (reactive dye) is not known for reproductive toxicity effects. Also, the substance has been notified originally under the 7th Amendment, and has been in use for a number of years without effects associated with reproductive toxicity being noted. On the basis of animal welfare it is proposed that the developmental study in conjunction with the lack of effects noted in the other toxicity studies is suitable to address this endpoint. An assessment of reproductive effects can be carried out at the next tonnage threshold.

Effects on developmental toxicity

Description of key information

Assessment of developmental toxicity via read across is undertaken; no adverse effects were observed.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

September 14- November 25, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to recognised test guidance in compliance with GLP. The study is read across to an analogous substance; refer to image and further information below.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Remarks:

.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Company's own breeding colony (Hoe:Wiskf(SPF71))
- Age at study initiation: approx. 65 - 75 days
- Weight at study initiation: Mean body weight of 192.0 ± 15.5 g
- Fasting period before study: None
- Housing: Singly, on wood shavings, in plastic cages
- Diet (e.g. ad libitum): Altromin 1310 ad libitum.
- Water (e.g. ad libitum): Tap water, ad libitum.
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22 °C
- Humidity (%):48- 61 %.
- Air changes (per hr): 16 to 20 changes
- Photoperiod (hrs dark / hrs light): Illuminance from 06:00 to 18:00 hours, measured one metre above the floor in front of the cages, was about 450 lux.

IN-LIFE DATES: From: September 14- November 25, 1993

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The study compound was suspended fresh daily in a concentration of 200 g/l in distilled water and administered daily to all the animals within three hours of manufacture, between 08:00 and 12:00 hours. All the animals received the same volume of liquid, 5 ml/kg body weight. After each weighing of the animals, the compound doses were adjusted according to body weight. The stability and homogeneity of the solutions were guaranteed over a period of 4 hours (Letter of Analytical Laboratory, Hoechst AG, dated September 9, 1993).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Historical laboratory use.
- Concentration in vehicle: 200 g/l
- Amount of vehicle (if gavage): 5 ml/kg based on bodyweight.
- Lot/batch no. (if required): Not specified
- Purity: Not specified.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: Prior to the start of the study, the females were mated with fertile males overnight (15:30 - 07:30 hours) during oestrus. On detection of spermatozoa in the vaginal smear the following morning, they were separated and kept singly. The mating ratio was one male to one female. The day on which spermatozoa were detected was taken as Day 1 of pregnancy. Pregnancy was confirmed when implantation sites were detected in the uterus. Only pregnant animals are reported on in the study data itself. Data on the animals which did not become pregnant are, however, included in the appendices.
- Verification of same strain and source of both sexes: yes – sourced from same supplier
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

Dosing from 7th to the 16th day after mating.

Frequency of treatment:

Daily

Duration of test:

20 days from Day 1.

Remarks:

Doses / Concentrations:
1000 mg
Basis:
actual ingested
1000 mg Structural Analogue 01/ kg body weight

No. of animals per sex per dose:

20 - 24 mated female rats per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Groups of 20 - 24 mated female rats received the following oral doses once daily by stomach tube from the 7th to the 16th day after mating:

Group 1: 5 ml Distilled water/kg body weight (control)
Group 2: 1000 mg C.I Reactive Blue 220 / kg body weight.

The dose stated refers to the compound preparation and not the components of the dye contained therein. The allocation of the animals to the various groups took place after detection of spermatozoa in accordance with the study randomisation plan. The groups were chosen simultaneously.

Maternal examinations:

During the study, the animals' behaviour and general physical condition were assessed daily and their feed consumption was monitored continually (and recorded at the same intervals as the body weight). Their body weight development was checked once weekly, and additionally one day after the final administration.

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

All the females were killed on the 21st day after mating by a blow to the head and exsanguination and were then delivered by caesarean section. The uterus was opened and the live and dead foetuses, conceptuses undergoing resorption, placentas and corpora lutea in the ovaries were counted and examined macroscopically. The diameter of the conceptuses undergoing resorption and the placental weights were also determined. After initial examination of the foetuses, the presence of iron in the wall of the uterus of the dams was tested using ammonium sulphide. In this way, it was also possible to count the implantation sites which had not previously been visible in the unstained uterus.

Fetal examinations:

After removal from the uterus, the foetuses were examined for signs of life, outward appearance and outwardly detectable anomalies. The body weight of the foetuses was subsequently determined. Their crown-rump length was measured after the killing. The sex of the foetuses was determined at autopsy.

Approximately half of the foetuses from each litter - also called skeleton foetuses - were fixed in alcohol, dissected where possibleunder a magnifying glass, eviscerated and then cleared in potassium hydroxide solution. The skeletons were stained with alizarin red S and examined under a stereomicroscope for their stage of development and for anomalies.

The remaining foetuses - also called body cross-section foetuses- were fixed in Bouin's solution, cut into body cross sections and examined under a stereomicroscope for organ anomalies.

The foetuses were selected alternately for one or the other of the two procedures, according to their position in the uterus.

Statistics:

The statistical evaluation of the study results was based on comparison of the animals of the compound group and those of the simultaneous control group as well as comparisons of both animal groups with control values from previously conducted studies (normal ranges). Dams with no live foetuses were excluded from the calculation of mean values and statistical evaluation. In comparison with the simultaneous control group, a standard MANOVA with sequentially rejective multiple comparisons was employed to evaluate the body and organ weights, and a multivariate rank order test after Puri & Sen( 1985) was used to evaluate the feed consumption.
Implants and corpora lutea were analysed using the Mantei-Haenszel chi-squared test as were the portions of live and dead foetuses and conceptuses undergoing resorption. The litter means of foetal weights, crown-rump lengths and placental weights were evaluated with a multi­variate analysis of variance. With the described methods, a degree of probability of 5% per parameter group was maintained. The findings observed at autopsy, body cross-section examination and skeletal examination of the foetuses were evaluated separately for foetuses and litters using the exact Fisher test.
In order to compare the data with historic controls, normal ranges were calculated for groups. These are determined so as to contain at least 75% of the values of a control group, with 95% probability. The parallelepiped analysis of Wald(1943) was used for calculating normal ranges for the body weight gain and feed consumption of the dams and litter means of foetal weights, crown rump lengths and placental weights. Corpora lutea and implants were compared with univariate normal ranges after Wilks(1942).The triangular analysis after Rosenkranz(1988) was used to determine ranges for the proportions of live/dead foetuses as well as the conceptuses undergoing resorption.

Indices:

As above.

Historical control data:

Historical data bases for reproductive performance and prevalence of spontaneous malformations in control rats are available based on the laboratories own in-house data. This is detailed within the report.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Clinical examinations

The dams of the compound group and the control dams showed no impairment to behaviour and general condition and survived until the scheduled study end.
17 dams in the compound group excreted black-discolored faeces, mainly in the period from day 8 - 19 of pregnancy. In the other three dams, the faeces were blue in colour from day 8 until 17 or 18 of pregnancy. Furthermore, 17 dams also voided red-discolored urine in the period from day 8 -16 of pregnancy. Slight amounts of compound solution escaped on occasion from the mouths of five dams when removing the tube from the oesophagus of the animals. This was due to the dosing method; the amounts, however, were small and without relevance for assessing the test compound. In another dam from the compound group, local hair loss at the neck was noted from day 15 of pregnancy onwards and scabbing at the neck and both forelimbs was observed from day 17 of pregnancy onwards.

The administration of C. I. Reactive Blue 220 had no influence on the feed consumption of the dams. The dams consumed comparable amounts of feed to those of the controls both during and after the treatment period.

The body weight gain of the dams treated with C. I. Reactive Blue 220 was likewise unaffected. The dams treated with the test compound gained in weight to the same extent as the controls.

Examinations after caesarean section

All 20 dams in the compound group and 19 dams in the control group carried live conceptuses to term and were delivered by caesarean section. One control dam had no live foetuses, but merely 8 empty implantation sites in the uterus (described, for technical reasons, as "aborted" in the report), indicating early embryonic death shortly after implantation. In this animal, the corpora lutea were quite small and could not therefore be determined exactly. The number of corpora lutea, which provides information on the number of ovulations prior to start of treatment, and the number of implants and live foetuses in the remaining dams of the compound group did not differ markedly from those of the previous control animals.

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The live foetuses delivered in the compound group were normally developed. Their body weights and lengths did not differ from those of the control foetuses and lay in the range of our previous control values. The number of live foetuses weighing less than 3.0g and thus qualifying according to our definition as slightly retarded or retarded corresponded to that of the control group. The sex ratio was relatively balanced in both groups, the males predominated slightly in the control group and the females predominated slightly in the compound group.

Dead embryofoetal primordia were found in both groups. They consisted exclusively of conceptuses undergoing resorption (referred to in the appendices, for technical reasons, as "early intrauterine deaths"). These had died at a relatively early stage and exhibited diameters of up to 4.6mm (compound group) or 6.6mm (control group). Dead foetuses were not found in either of the two groups. The number of dead embryofoetal primordia in the dams of the compound group was low and did not differ from that of the control dams.

The placentas of the foetuses of most dams in the compound group were unremarkable macroscopically and in respect of their weights, which corresponded to those of the control group. The placenta of one foetus in the control group was relatively heavy, weighing 0.82 g. The findings do show that no malformations appeared in any foetus in the compound group. One control foetus exhibited a right-sided microphthalmia.

The autopsy and body cross-section examinations revealed in individual or some foetuses in both groups: blood in the abdominal cavity and a distention of one renal pelvis or of one or both ureters. The following findings were displayed by one foetus each from the compound group: blood in the right cerebral hemisphere, the vicinity of the stomach or in the vicinity of the right kidney and the analogous ureter; a caudalward displacement of both kidneys. Findings confined to individual foetuses in the control group were: blood in the thoracic cavity, pericardium of right kidney; a haematoma in one liver lobe; a relatively large stomach, taut with fluid; a bipartitionof the left pulmonary lobe.

The skeletons of the foetuses in the compound group were at roughly the same stage of development as those of the control foetuses. Their stage of ossification corresponded to the 21st day of pregnancy. The number of foetuses in the 1000 mg/kg group displaying a poor or missing ossification of sternebrae and 5th metacarpal bone and the ossification of fewer than two caudal vertebral centres did not differ markedly from that of the control foetuses. The number of foetuses exhibiting individual cranial bones which were not yet or not completely ossified was significantly higher than the simultaneous control group, but lay within the range of previous control values (maximum frequency 61.5 %). In one compound foetus, the second metacarpal bone of both forelimbs and in four foetuses the third phalanx of the digits of both hind limbs were not ossified.

Apart from these findings, individual or several foetuses in both groups exhibited longitudinally-displaced, dislocated, fragmented sternebrae, wavy and/or thickened ribs, uni- or bilaterally; and numerous foetuses, an additional short rib anlage, uni-or bilaterally, at the 1st lumbar vertebra. The number of foetuses exhibiting an additional rib at the first lumbar vertebra was significantly higher than that of the simultaneous control group but lay within the range of the previous control group (maximum frequency 62.4 %). One foetus each from the compound group exhibited a fragmented 13th thoracic vertebral centre and a 14th thoracic vertebra with analogous short rib pair. Both humeri were short and both scapulae were short and bent costalwards in one foetus of the compound group. In another foetus from the same litter, the right scapula was short and bent costalwards. One finding confined to individual foetuses from the control group was a short additional rib anlage, left- or both sided, at the 7th cervical vertebra.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

This study is read across to the structural analogue C.I. Reactive Blue 220, which is considered structually equivalent to the substance to be registered. Justification for this read across is detailed below under "attached background information".

The "no observable adverse effect level" (NOAEL) of C.I. Reactive Blue 220 in rats was therefore at least 1000 mg/kg/day for both maternal and developmental toxicity under the conditions of this study.

Executive summary:

This study is read across to the Structural Analogue 01, which is considered structurally equivalent to the substance to be registered. Justification for this read across is detailed below under "attached background information".

As can be seen from the results reported in the preceding section, the oral administration of the Structural Analogue 01 at a dose of 1000 mg/kg did not lead to any impairment of feed consumption or body-weight gain of the dams. The black or blue discoloration of the faeces in all dams of and the red discoloration of the urine in most of the dams of the compound group is due to the excretion of the dye. Likewise, the grey-green discoloration of the renal cortex detected at autopsy in 13 dams is compound-related. The renal pelvic dilatation noted in one dam of the compound group is likewise spontaneous (this disorder is known to occur spontaneously in the rat strain used here; compare the finding in one control dam).

The intrauterine development of the foetuses was likewise unaffected by the administration of the Structural Analogue 01. The foetuses delivered in the dose group were normally developed and did not differ from the control foetuses in terms of body weight and length. The placentas of the foetuses were also largely unremarkable.

The morphological examination of the offspring of the dams treated with the Structural Analogue 01 did not reveal any malformations. The following changes, which were detected in individual or some foetuses from the compound group at autopsy or body cross-section examinations, are to be regarded as minor anomalies: haemorrhages in the brain or in various parts of the body; caudalward displacement of both kidneys; dilatation of renal pelves or ureters. These changes also occur in control foetuses, so a connection with the administration of the compound is unlikely. Other minor anomalies were: longitudinally displaced, dislocated, fragmented sternebrae, wavy or thickened ribs (detected in several foetuses); fragmented 13th thoracic vertebral centre and short length of both humeri (one foetus each); flexion and short length of one or both scapulae (two foetuses). However, the anlage of a 14th thoracic vertebra with a short analogous rib pair in one foetus and the short rib anlage, uni- or bilaterally, at the 1st lumbar vertebra detected in numerous foetuses are to be classified as variations. The number of foetuses affected by the stated alterations lay in the range of the spontaneous rate, with the exception of the blood accumulation in the vicinity of the stomach and the displacement of both kidneys. However, these change only affected one foetus each so that a causal connection with the administration of Structural Analogue 01 is unlikely here also.

The skeletons of the foetuses of the compound group did not reveal any delayed ossification which could be rated as a retardation. The poor or missing ossification of cranial bones, sternebrae, 2nd or 5th metacarpal bone, third phalanx of the digits of the two hind limbs and the ossification of fewer than two caudal vertebrae may be classified as a retardation, but in this study these findings reflect the normal stage of ossification of the skeleton in this phase of development.

In summing up, it can be seen from these findings that Structural Analogue 01 administered to rats in oral doses of 1000 mg/kg under the chosen study conditions is not toxic to dams or their conceptuses. Likewise, no indication of any teratogenic effect of the test compound was found.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

This study is read across to the Structural Analogue 01, which is considered structurally equivalent to the substance to be registered. Justification for this read across is detailed below under "attached background information".

As can be seen from the results reported in the preceding section, the oral administration of the Structural Analogue 01 at a dose of 1000 mg/kg did not lead to any impairment of feed consumption or body-weight gain of the dams. The black or blue discoloration of the faeces in all dams of and the red discoloration of the urine in most of the dams of the compound group is due to the excretion of the dye. Likewise, the grey-green discoloration of the renal cortex detected at autopsy in 13 dams is compound-related. The renal pelvic dilatation noted in one dam of the compound group is likewise spontaneous (this disorder is known to occur spontaneously in the rat strain used here; compare the finding in one control dam).

The intrauterine development of the foetuses was likewise unaffected by the administration of the Structural Analogue 01. The foetuses delivered in the dose group were normally developed and did not differ from the control foetuses in terms of body weight and length. The placentas of the foetuses were also largely unremarkable.

The morphological examination of the offspring of the dams treated with Structural analogue 01 did not reveal any malformations. The following changes, which were detected in individual or some foetuses from the compound group at autopsy or body cross-section examinations, are to be regarded as minor anomalies: haemorrhages in the brain or in various parts of the body; caudalward displacement of both kidneys; dilatation of renal pelves or ureters. These changes also occur in control foetuses, so a connection with the administration of the compound is unlikely. Other minor anomalies were: longitudinally displaced, dislocated, fragmented sternebrae, wavy or thickened ribs (detected in several foetuses); fragmented 13th thoracic vertebral centre and short length of both humeri (one foetus each); flexion and short length of one or both scapulae (two foetuses). However, the anlage of a 14th thoracic vertebra with a short analogous rib pair in one foetus and the short rib anlage, uni- or bilaterally, at the 1st lumbar vertebra detected in numerous foetuses are to be classified as variations. The number of foetuses affected by the stated alterations lay in the range of the spontaneous rate, with the exception of the blood accumulation in the vicinity of the stomach and the displacement of both kidneys. However, these change only affected one foetus each so that a causal connection with the administration of Atructural Analogue 01 is unlikely here also.

The skeletons of the foetuses of the compound group did not reveal any delayed ossification which could be rated as a retardation. The poor or missing ossification of cranial bones, sternebrae, 2nd or 5th metacarpal bone, third phalanx of the digits of the two hind limbs and the ossification of fewer than two caudal vertebrae may be classified as a retardation, but in this study these findings reflect the normal stage of ossification of the skeleton in this phase of development.

In summing up, it can be seen from these findings that the Structural Analogue 01 administered to rats in oral doses of 1000 mg/kg under the chosen study conditions is not toxic to dams or their conceptuses. Likewise, no indication of any teratogenic effect of the test compound was found.

Justification for classification or non-classification

The above study has been ranked reliability 1 according to the Klimish et al system. This ranking was deemed appropriate because the study was conducted to GLP and in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008). No classification for reproductive effects is therefore required.