Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No treatment related effects were observed on the fertility and mating performance of male and females rats when examined in two-generation reproductive toxicity studies at doses of balsalazide disodium of 2000 mg/kg bw/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
reproductive toxicity, other
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Read across is considered possible due to the almost identical structural formulae of the two substances and their treatment in the body.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Read across is proposed between balsalazide disodium salt dihydrate (CAS No.150399-21-6) and balsalazide acid (CAS No. 80573-04-2).

3. ANALOGUE APPROACH JUSTIFICATION
Balsalazide disodium salt dihydrate is a prodrug used as a registered pharmaceutical for the effective treatment of inflammatory bowel diseases and has been used without any major recorded adverse side effects for many years. Toxicological studies are available on balsalazide disodium salt dihydrate and are considered to be directly relevant to balsalazide acid due to the almost identical structural formulae of the two substances and their treatment in the body.
Following ingestion of balsalazide salt dihydrate, in the acidic pH of the stomach (approximately pH 4-5 in rats and pH 2 in humans), the salt will be converted to the free acid. Following transit to the intestinal tract, the more alkaline pH (approximately pH 6.5-8 in rats and pH 5-8 in humans) will result in conversion of the free acid to the salt equivalent. Therefore, the substance available for absorption in the small intestine will be the same regardless of the form ingested originally.
If absorption of either substance into the bloodstream was to occur, the buffering effect of the blood would also ensure that the substances would be in the same form prior to reaching any organs.
Hence, the toxicological studies on balsalazide salt dihydrate can be used to predict the toxicological effects of balsalazide acid.

4. DATA MATRIX
Please see attached justification.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Remarks on result:
not determinable due to absence of adverse toxic effects
Reproductive effects observed:
no
Endpoint:
fertility, other
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
no guideline followed
Principles of method if other than guideline:
No guideline is specified in the report.
GLP compliance:
no
Species:
rat
Strain:
Wistar
Remarks:
Biorex
Sex:
male
Route of administration:
oral: feed
Duration of treatment / exposure:
3, 12 and 27 weeks
Frequency of treatment:
Daily
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
5 male animals per dose were used for the fertility test after 3, 12 and 27 weeks of treatment.
Control animals:
yes, plain diet
Details on study design:
The test was performed by mating each male with 5 females of proven fertility for 4 days then females were killed 14 days later.
Postmortem examinations (parental animals):
SACRIFICE
- Maternal animals: All females were sacrificed 14 days after mating.

GROSS NECROPSY
- The number of pregnant females, corpora lutea, live/ dead foetuses and early/ late resorptions were recorded.
Reproductive performance:
no effects observed
Description (incidence and severity):
Male fertility was not affected by the treatment
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Sex:
male
Basis for effect level:
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Remarks on result:
not measured/tested
Remarks on result:
not measured/tested
Remarks on result:
not measured/tested
Reproductive effects observed:
no

For general toxicity results, please refer to IUCLID Section 7.5.1 (96 week study).

Conclusions:
No treatment related effects on male fertility were observed up to the highest dose tested.
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
no guideline followed
Principles of method if other than guideline:
No guideline is specified in the report.
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Remarks:
CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Male: 6 - 7 weeks; Female: 10 - 11 weeks
- Weight at study initiation: (P) Males: 182 - 225 g; Females: 203 - 248 g
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The volume of administration was fixed at 12 mL/kg bw.
Duration of treatment / exposure:
The male rats were treated from 71 days prior to mating and throughout the mating phase and until they were sacrificed.

Females were treated for 15 days prior to mating and throughout mating, gestation, lactation until they were sacrificed (approximately 24 days after postpartum).
Frequency of treatment:
Daily
Details on study schedule:
At day 35 of post partum, 26 pairs of the animals per group were selected for F1/F2 generation study. At 10 weeks of age, they were continuously mated and the study was repeated as for the first (P0) parental animals except the animals were not treated.
Dose / conc.:
120 mg/kg bw/day
Dose / conc.:
600 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
36 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose selection was based on a preliminary oral study in rats in which doses of 500, 1000 and 2000 mg/kg bw/day were used. The male rats were treated from 15 days prior to mating and throughout the mating, gestation, lactation until they were sacrificed (approximately 4 days after postpartum). Increased incidence of salivation was seen in mid and high dose treated rats. At the highest tested dose, no effects were seen on fertility and general reproductive performance of rat.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily (before and after delivery)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly; Additionally, dams were weighed on days 0, 3, 6, 10, 13, 17 and 20 of gestation and days 1, 4, 7, 11, 14, 18, 21 and 25 of lactation.

FOOD CONSUMPTION:
- Time schedule for examinations: Weekly (before and after delivery)

WATER CONSUMPTION: Yes
- Time schedule for examinations: Weekly (before and after delivery)


The offspring were reared by the dams until weaning.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, number of stillbirths, number of live births, growth and differential, developmental parameters (righting reflex, pinna detachment, tooth eruption, eyelid separation, visual and auditory function tests, testes descent, vaginal opening, learning ability test and open field test).
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals sacrificed after mating.
- Maternal animals: About 23 pregnant rats were sacrificed on day 20 of gestation and were examined for the number of corpora lutea, the number of implants, the number of dead or resorbed foetuses and the number of live foetuses. The live foetuses were sexed and weighed. Foetuses were eviscerated and alternate foetuses from each litter were examined for skeletal major/ minor abnormalities, the remaining foetuses were examined for visceral abnormalities and variations. The remaining dams (13/ group) were allowed to deliver spontaeously.
On day 25 of post-partum, all dams were sacrificed and necropsied.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed on day 25 of post partum.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Increased salivation was seen in high dose treated rats.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male from the control group was found dead after vehicle administration during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
By the end of the treatment period, the body weight gains were reduced by 4 - 9% in treated males when compared to the control values while in females body weight gains were decreased by 8% in high dose treated females during the gestation period.
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Throughout the study period significant increases in water intakes were seen in high dose treated rats.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Dams sacrificed at Day 20:
No treatment related gross lesions were seen in female rats of F0 generation. There were no siginifcant changes in pregnancy parameters.
Reproductive function: oestrous cycle:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
92 - 97% of rats mated within the first 4 days of pairing. Mating performance (100%), conception rate (94 - 100%) and fertility index (94 - 100%) were comparable in all groups.

For the dams allowed to deliver, no significant differences in the gestation period between the groups were noted. The implantation sites and litter sizes were lower in the high dose treated groups compared to the controls but it was not statistically significant.
Dose descriptor:
NOEL
Effect level:
> 2 000 mg/kg bw/day
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no significant effect on fertility test and mating performance test of F1 generation rats.
Dose descriptor:
NOAEL
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Foetuses sacrificed at day 20:
No treatment related major malformation was seen in foetuses except increased incidences of bilateral hydronephrosis were seen in treated groups (control = 0.0%, low dose = 0.5%, mid dose = 1.2% and high dose = 2.2% [in 3 litters], historical control: mean 0.3% and range = 0 - 1.7%). Other anomalies found in the study were considered not to be treatment related.

F1 generation:
No treatment related gross lesions were seen in rats of the F1 generation.
From day 1 onwards there were no significant effects on viability indices, postnatal development and differentiation were comparable in all groups.
Phyical development was comparable in all groups.

Increased incidences of bilateral hydronephrosis were seen in the foetuses of treated groups which is considered a developmental effect. This is potentially a treatment related effect but was not observed in the F1 generation from the Dams that were allowed to deliver. The incidence in the mid dose group was within the historical control range and the high dose group used test concentrations above those that are considered relevant for classification, as they are unnecessarily high and significantly above any expected human exposure.
Dose descriptor:
NOAEL
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Physical development was comparable in all groups and no drug related gross lesions were seen in the F2 pups at necropsy.
Dose descriptor:
NOAEL
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
There were no abnormal effects on the fertility and mating performance of the treated male and female rats at doses up to and including 2000 mg/kg bw/day of Balsalazide.
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
no guideline followed
Principles of method if other than guideline:
No guideline is specified in the report.
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Remarks:
CD
Sex:
female
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The volume of administration was fixed at 12 mL/kg bw.
Duration of treatment / exposure:
From day 15 of gestation to day 25 after parturition.
Frequency of treatment:
Daily
Details on study schedule:
All dams were allowed to litter normally and raise their pups to weaning.

At day 35 of post partum, 20 pairs of the animals per gtoup were selected for F1/F2 generation study. At 10 weeks of age they were continuously mated and caesarean section was performed on the F1 dams on day 20 of gestation. Uterine contents and foetuses were examined and preserved in acetone and Bouin's fluid.
Dose / conc.:
120 mg/kg bw/day
Dose / conc.:
600 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
22 pregnant rats per group
Control animals:
yes, concurrent vehicle
Details on study design:
The dose selection was based on a preliminary oral study in rats in which the highest tested dose (2000 mg/kg bw/day) had no effect on fertility and general reproductive performance of rat. The Sponsor selected 2000 mg/kg bw/day as the highest dose for the main study.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6, 12 and 15 of post coitum and daily thereafter until termination of the study

FOOD CONSUMPTION: Yes
- Time schedule: Days 0-2, 3-5, 6-8, 9-11, 12-14, 15-17 and 18-19 of gestation and 1-3, 4-6, 7-10, 11-13, 14-17, 18-20 and 21-24 of lactation
Litter observations:
The number of live/ dead pups were recorded and the live pups were weighed and sexed. Postnatal body weight changes of the pups were recorded until the age of 25 days.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, number of stillbirths, number of live births, growth and differential, developmental parameters (righting reflex, pinna detachment, tooth eruption, eyelid separation, visual and auditory function tests, testes descent, vaginal opening, learning ability test and open field test)
Postmortem examinations (parental animals):
SACRIFICE
- Maternal animals: All dams were sacrificed and necropsied on day 25 of post partum.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Gross pathological findings:
no effects observed
Reproductive performance:
no effects observed
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Critical effects observed:
no
Reproductive performance:
no effects observed
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Critical effects observed:
no
Gross pathological findings:
no effects observed
Description (incidence and severity):
No external abnormalities were observed in the F2 foetuses.
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
No adverse effects were seen in rats during the perinatal and postnatal period following oral doses of up to 2000 mg/kg bw/day of Balsalazide.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
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 evidence of developmental toxicity in rats and rabbits was observed when tested up to dose levels of 2000 mg/kg bw/day and 1200 mg/kg bw/day, respectively.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Read across is considered possible due to the almost identical structural formulae of the two substances and their treatment in the body.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Read across is proposed between balsalazide disodium salt dihydrate (CAS No.150399-21-6) and balsalazide acid (CAS No. 80573-04-2).

3. ANALOGUE APPROACH JUSTIFICATION
Balsalazide disodium salt dihydrate is a prodrug used as a registered pharmaceutical for the effective treatment of inflammatory bowel diseases and has been used without any major recorded adverse side effects for many years. Toxicological studies are available on balsalazide disodium salt dihydrate and are considered to be directly relevant to balsalazide acid due to the almost identical structural formulae of the two substances and their treatment in the body.
Following ingestion of balsalazide salt dihydrate, in the acidic pH of the stomach (approximately pH 4-5 in rats and pH 2 in humans), the salt will be converted to the free acid. Following transit to the intestinal tract, the more alkaline pH (approximately pH 6.5-8 in rats and pH 5-8 in humans) will result in conversion of the free acid to the salt equivalent. Therefore, the substance available for absorption in the small intestine will be the same regardless of the form ingested originally.
If absorption of either substance into the bloodstream was to occur, the buffering effect of the blood would also ensure that the substances would be in the same form prior to reaching any organs.
Hence, the toxicological studies on balsalazide salt dihydrate can be used to predict the toxicological effects of balsalazide acid.

4. DATA MATRIX
Please see attached justification.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
effects observed, non-treatment-related
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
no guideline followed
Principles of method if other than guideline:
No guideline is specified in the report
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 18 - 26 weeks old
- Weight at study initiation: 3.31 - 5.04 kg
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The volume of administration was fixed at 10 mL/kg bw.
Duration of treatment / exposure:
Day 6 - 19 of gestation
Frequency of treatment:
Daily
Duration of test:
Up to day 29 of gestation
Dose / conc.:
120 mg/kg bw/day
Dose / conc.:
600 mg/kg bw/day
Dose / conc.:
1 200 mg/kg bw/day
No. of animals per sex per dose:
15 pregnant females per group
Control animals:
yes, concurrent vehicle
Details on study design:
The selection of the doses were based on a preliminary teratology study in rabbits in which oral doses of 500, 1000 and 2000 mg/kg bw/day were used. Pregnant rabbits were treated from day 6 - 19 of gestation. Increased incidence of reduced or loose faeces were seen in mid and high dose treated rabbits. One high dose treated rabbit was found dead and the cause of death was most likely drug related. Additionally, one high dose treated female aborted during the study period. During the treatment period, body weights were decreased by 82% along with about 42% reduction in food consumptions in high dose treated females. No treatment related effects were seen at necropsy (day 29 of gestation). Based on these results the Sponsor selected 1200 mg/kg bw/day as the highest dose for the main study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION: Yes
- Time schedule: Days 1-5, 6-12, 13-19, 20-23 and 24-28

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:

OTHER: Histopathology was performed on all the rabbits that died during the study.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: No data
- Skeletal examinations: Yes: two-thirds of the foetuses
- Head examinations: No data
- Visceral examination: Yes: one-third of the foetuses
Mortality:
mortality observed, treatment-related
Description (incidence):
One female from the low dose group was killed in extremis and the cause of death could not be established with certainty.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains were decreased by 14% and 28% during the gestation period 0-20 at 600 and 1200 mg/kg bw/day, respectively when compared to the control values. However, during the post dosing period, the body weight gains were increased in animals treated with mid and high dose levels such that by termination day 29 their body weights were comparable to that of the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intakes were also decreased by 11% during the dosing period at 1200 mg/kg bw/day dose levels.
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Three females (1 from the control group, 1 from mid dose group and 1 from high dose group) aborted during the study period. These abortions were considered not to be treatment related.
Pre- and post-implantation loss:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
THe overall rate of pregnancies significantly decreased in mid and high dose treated rabbits (control = 100%, low dose = 100%, mid dose = 60% and high dose 67%).
Other effects:
no effects observed
Description (incidence and severity):
The number of corpora lutea showed no differences between the treated and control groups.
Dose descriptor:
LOEL
Effect level:
600 mg/kg bw/day
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
changes in number of pregnant
Abnormalities:
effects observed, treatment-related
Localisation:
not specified
Fetal body weight changes:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Abnormalities:
no effects observed
Developmental effects observed:
no

Due to the apparent effect on the maintenance of pregnancy in females receiving 600 mg/kg bw/day and above, the sponsor repeared part of the experiment. In this repeat test three groups were included in which pregnant rabbits were given oral doses of 0 (vehicle), 300 and 600 mg/kg from day 6 to 19 of gestation. This experiment had a few problems, such as older rabbits were used and were not in an active growth phase (even control rabbits lost their weights during the gestation period). Hence, no conclusion could be made from this study.

Conclusions:
Maternal toxicity was seen at 600 and 1200 mg/kg bw/day dose levels. Additionally, mid and high dose levels adversely affected the pregnancy rates (reason unknown). However, there was no evidence of a teratogenic potential.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
no guideline followed
Principles of method if other than guideline:
No guideline is specified in the report.
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The volume of administration was fixed at 12 mL/kg bw.
Duration of treatment / exposure:
Day 6 - 17 of gestation
Frequency of treatment:
Daily
Duration of test:
Up to day 25 post partum

Two/ thirds of the pregnant rats were sacrificed on gestation day 20. The remaining third of the dams were allowed to deliver spontaneously.
Dose / conc.:
120 mg/kg bw/day
Dose / conc.:
600 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
32 pregnant rats per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The selection of the doses was based on a preliminary teratology study in rats in which oral doses of 500, 1000 and 2000 mg/kg bw/day were used. Pregnant rats were treated from day 6 - 15 of gestation. Increased incidence of salivation was seen in high dose treated rats. During gestation period body weights were decreased by 12 - 16% in treated rats. No treatment related effects were seen at necropsy (day 20 of gestation). Based on these results, the sponsor selected 2000 mg/kg bw/day as the highest dose for the main study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: On days 0, 3, 6, 18 and 20 of gestation and 1, 4, 7, 11, 14, 18, 21 and 25 of lactation

FOOD CONSUMPTION: Yes
- Time schedule: On days 0-2, 3-5, 6-8, 9-11, 12-15, 16-17 and 18-19 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Two/ thirds of the pregnant rats were sacrificed on gestation day 20. The remaining third of the dams were sacrificed on day 25 post partum.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Other: number of live foetuses
Fetal examinations:
The live foetuses were weighed and sexed.

- External examinations: Yes
- Soft tissue examinations: Yes: half of the foetuses eviscerated
- Skeletal examinations: Yes: half of the foetuses eviscerated
- Head examinations: No data
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Pre- and post-implantation loss:
no effects observed
Changes in pregnancy duration:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
The number of corpora lutea, impants and pre and post implantation loss, weights of foetuses and sex ratio did not show any significant differences between the treated groups and the control group.

In the dams allowed to deliver, the litter size and survival rates were comparable in all groups.
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Abnormalities:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
External examinations were normal except 3 foetuses from one litter of the high dose group had rounded heads and shortened snout. No such finding was reported in the historical control data.
Skeletal malformations:
no effects observed
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral examination of the F1 foetuses revelaed no treatment related effects except in the high dose group one foetus had unilateral slight microphthalmia and another foetus innominate artery was very reduced. These findings were not reported in the historical control data. The Sponsor considered this finding to not to be treatment related because it happened only in one litter.
Description (incidence and severity):
Body weight gains of the pups were decreased by 7% in the high dose group. There were no significant effects on postnatal development and differentiation.

At necropsy of F1-generation at termination there were no abnormalities.

There was no significant effect on fertility test and mating performance test of F1 generation rats. External examination of F2 foetuses revealed no abnormalities.
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
At dosages up to 2000 mg/kg bw/day, no development effects in rats were observed during the study. The postnatal development and the fertility of the offspring were comparable in all groups.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

The mode of action of toxicity and its relevance to humans has not been assessed. However, the sodium salt of balsalazide acid has been authorised for use as a pharmaceutical for the treatment of inflammatory bowel disease.

Justification for classification or non-classification

Increased incidences of bilateral hydronephrosis were seen in the foetuses of treated groups (low dose – 120 mg/kg bw/day = 0.5%, mid dose - 600 mg/kg bw/day = 1.2% and high dose - 2000 mg/kg bw/day = 2.2%; historical control: mean 0.3% and range = 0 - 1.7%) in a fertility and general reproductive performance study in rats (IUCLID entry REACH 8.7.3 - Center for Drug Evaluation and Research (1997) – rats).

CLP allows the use of limit tests for animal studies (CLP Regulation (EC) No. 1272/2008 sections 3.7.2.5.7, 3.7.2.5.9) and importantly refers to the OECD Test Guidelines. In guidelines which are considered relevant for this endpoint, such as Prenatal Developmental Toxicity Study (OECD 414) and the Two-Generation Reproduction Toxicity (OECD 416), 1000 mg/kg is specified in both as a limit test in cases where effects are not expected at that dose. There were no treatment-related biological effects in the 27 week repeat dose up to 1000 mg/kg. Therefore, in the reproductive toxicity test, consideration of the effects seen up to the 1000 mg/kg (mid-dose) must be seen together with the historical control data. Specifically, the effect up to 1000 mg/kg was within the historical control range and not considered significant enough to trigger classification. Any effect seen at doses higher than this are not considered relevant for classification, as they are unnecessarily high and significantly above any expected human exposure. In addition the FDA concluded in 2000, that there is no significant evidence of reproductive toxicity of Balsalazide disodium salt, thus further supporting the argument that no classification is needed.