Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-nitro-, disodium salt, reaction products with 4-[(4-aminophenyl)azo]benzenesulfonic acid, sodium salts

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-nitro-, disodium salt, reaction products with 4-[(4-aminophenyl)azo]benzenesulfonic acid, sodium salts

EC Number:

215-397-8

EC Name:

Benzenesulfonic acid, 2,2'-(1,2-ethenediyl)bis[5-nitro-, disodium salt, reaction products with 4-[(4-aminophenyl)azo]benzenesulfonic acid, sodium salts

Cas Number:

1325-54-8

Molecular formula:

Molecular formula of the main constituents: (1) C38H24N8Na4O12S4 (2) C52H32N10Na6O18S6

IUPAC Name:

decasodium 2-[(1E)-2-{2-sulfonato-4-[(1E)-2-{3-sulfonato-4-[(1E)-2-{2-sulfonato-4-[(1E)-2-{4-[(1E)-2-(4-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]phenyl}ethenyl]phenyl}diazen-1-yl]phenyl}ethenyl]-5-[(1E)-2-{4-[(1E)-2-(4-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]benzene-1-sulfonate 2-[(1E)-2-{2-sulfonato-4-[(1E)-2-{4-[(1E)-2-(4-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]phenyl}ethenyl]-5-[(1E)-2-{4-[(1E)-2-(4-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]benzene-1-sulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Lot/Batch No: PAL-022588
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled water

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Wistar Han™:RccHan™:WIST strain rats

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled water. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for four hours. Formulations were therefore prepared daily.

Details on mating procedure:

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test item formulation were taken and analysed for concentration of DIRECT ORANGE 39 (C.I. 40215) at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services: The concentration of Direct Orange 39 (C.I. 40215) in the test item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

1.2 Samples: The test item formulations were diluted with water to give a final, theoretical test item concentration of approximately 0.01 mg/ml.
1.3 Standards: Standard solutions of test item were prepared in water at a nominal concentration of 0.01 mg/ml.
1.4 Procedure:The standard and sample solutions were analysed by HPLC using the following conditions:
HPLC : Agilent Technologies 1200, incorporating autosampler and workstation
Column : POLYMER X 3μ RP-1 (50 x 4 mm id) or equivalent
Mobile phase : Eluent A: water
Eluent B: acetonitrile
Flow-rate : 0.5 ml/min
UV detectorwavelength: 419 nm
Injection volume : 25 μl
Retention time : ~ 2.5 mins
1.5 Homogeneity Determinations: The low dose level formulation was assessed visually. The high dose level formulation was mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.
1.6 Stability Determinations: The test item formulations were sampled and analysed initially and then after storage in ambient conditions for four hours.

The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.

Duration of treatment / exposure:

42 days (including 15 days before mating)

Frequency of treatment:

Daily

Details on study schedule:

i) Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v) On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43.
ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg bw/day
Basis:
nominal conc.
control group

Doses / concentrations 2

Remarks:

Doses / Concentrations:
30 mg/kg bw/day
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
nominal conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
750 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

12 males per dose and 12 females per dose including control group (total 48 males and 48 females)

Control animals:

yes

Details on study design:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for seven days during which time their health status was assessed. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study.

Examinations

Parental animals: Observations and examinations:

1. Clinical observations:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing during the working week. Animals were observed immediately before dosing soon after dosing and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

2. Functional Observations:

2.1.Behavioural Assessments (Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation)
2.2. Functional Performance tests: Motor activiyt, Forelimb/Hindlimb Grip Strength
2.3. Sensory Reactivity ( Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch Blink reflex, Tail pinch Startle reflex, Finger approach)

3. Body Weight
4. Food Consumption
5. Water consumption

6. Reproduction screening:
6.1.Mating behaviour
6.2. Pregnancy and Parturition
6.3 Litter data including number of offspring born, number of offspring alive recorded daily and reported on Days 1 and 4 post partum, sex of offspring on days 1 and 4 post partum, clinical condition of offspring from birth to day 5 post partum, individual offspring weights on days 1 and 4 post partum;
6. 4 Physical Development of offspring

7. Laboratory investigations:

7.1. Haematological
7.2. Blood chemistry

8. Pathology
8.1. organ weights including prostate, seminal vesicles, epidiymides, testes, ovaries, uterus and pituitary
8.2. Histopathology including ovaries, pituitary, prostate, coagulating gland, seminal vehicles, epididymides, gross lessions, testes, mammary gland, uterus/cervix, vagina)

Litter observations:

Litter data including number of offspring born, number of offspring alive recorded daily and reported on Days 1 and 4 post partum, sex of offspring on days 1 and 4 post partum, clinical condition of offspring from birth to day 5 post partum, individual offspring weights on days 1 and 4 post partum; Physical Development

Postmortem examinations (parental animals):

All adult animals including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Postmortem examinations (offspring):

All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption, during gestation and lactation, Water Consumption during gestation and lactation, Pre- Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module. Data not analysed by the Provantis data capture system were assessed separately using the SPSS statistical package. Initially, the homogeneity of the data was assessed using Levene’s test. Where Levene’s test was shown to be non-significant (p≥0.05), parametric analysis of the data was applied, incorporating analysis of variance (ANOVA). If this data was shown to be significant, this analysis was followed by pair-wise comparisons using Dunnett’s test. Where Levene’s test was significant, non-parametric analysis of the data was analysed incorporating the Kruskal-Wallis test which if significant, was followed by the Mann-Whitney U test. Dose response relationship was also be investigated by linear regression. Where the data was unsuitable for these analyses, then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Due to the preponderance of non-normally distributed data, reproductive parameters (implantation losses, offspring sex ratio and offspring surface righting) were analysed using non-parametric analyses.

Reproductive indices:

3.4.2.1 Mating Performance and Fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
i) Pre-coital Interval. Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii) Fertility Indices. For each group the following were calculated:
Mating Index (%) =(Number of animals mated/Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females/Number of animals mated) x 100

3.4.2.2 Gestation and Parturition Data
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
i) Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii) Parturition Index: The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring /Number of pregnant females)x 100

3.4.2.3 Litter Responses
The standard unit of assessment was considered to be the litter, therefore, values were first calculated for each litter and the group mean was calculated using the individual litter values. Group mean values included all litters reared to termination (Day5 of age)
i) Implantation Losses (%): Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
Pre–implantation loss =(Number of corpora lutea- number of implantation sites/Number of corpora lutea) x100
Post–implantation loss = (Number of implantation sites - Total number of offspring born/Number of implantation sites) x 100

Offspring viability indices:

ii) Live Birth and Viability Indices
Live Birth Index (%) =(Number of offspring alive on Day 1/ Number of offspring born)x 100
Viability Index (%) = (Number of offspring alive on Day 4/Number of offspring alive on Day 1) x 100
iii) Sex Ratio (% males) = (number of male offspring/total number of offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Isolated instances of noisy respiration, pilo-erection and hunched posture (males 750 and 300 mg/kg bw/day) and noisy respiration (females 750, 300 and 30 mg/kg bw/day).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall body weight gains: males, 750 mg/kg bw/day. Females, 750 mg/kg bw/day, body weight losses during Week1 and reduced body weight gains during gestation. Body weight gains for females (750 mg/kg bw/day) during lactation were higher than controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Overall body weight gains: males, 750 mg/kg bw/day. Females, 750 mg/kg bw/day, body weight losses during Week1 and reduced body weight gains during gestation. Body weight gains for females (750 mg/kg bw/day) during lactation were higher than controls.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: Males treated 750 mg/kg bw/day and 300 mg/kg bw/day. Females treated 750 mg/kg bw/day during pre-mating period.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in mating performance

Details on results (P0)

Fertility. No treatment-related effects on fertility were detected.
Gestation Lengths. Females treated with 750 mg/kg bw/day showed generally longer gestation lengths when compared to controls. The effects were attributed to toxicity to the adult female rather than a direct effect upon reproduction.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive toxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Offspring of lower bodyweights were evident for females treated with 750 mg/kg bw/day when compared to controls.

Sexual maturation:

no effects observed

Gross pathological findings:

no effects observed

Details on results (F1)

The effects seen on litter size, sex ratio and viability were attributed to toxicity to the adult female rather than a direct effect of the test item upon these reproductive parameters.
The effects seen on offspring growth and development of the litters were attributed to toxicity to the adult female rather than a direct effect of the test item to the offspring.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

750 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Offspring development

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

TABULAR SUMMARY REPORT OF EFFECTS ON REPRODUCTION/DEVELOPMENT

Observations		Dose level (mg/kg bw/day)
		0 (control)	30	300	750
Paired females	number	12	12	10	12
Females showing evidence of copulation	number	12	12	10	12
Pregnant females	number	12	11	10	10
Conception days 1 -4	number	12	11	10	10
Gestation = 22 days	number	1	2	0	0
Gestation = 23 days	number	1	0	0	1
Gestation = 23 1/2 days	number	7	5	6	1
Gestation = 24 1/2 days	number	3	4	4	8
Dams with live young born	number	12	11	10	10
Dams with live young at Day 4 post partum	number	12	11	10	9
Corpora lutea/dam	mean	13.8	13.0	14.2	12.6
Implants/dam	mean	12.4	11.9	12.5	11.0
Live offspring/dam at Day 1 post partum	mean	11.3	11.2	11.9	9.0
Live offspring/dam at Day 4 post partum	mean	11.1	10.8	11.9	8.9
Sex ratio: % males at Day 1 post partum	mean	53.3	55.4	47.6	67.6
Sex ratio: % males at Day 4 post partum	mean	52.5	59.5	47.6	67.3
Litter weight (g) at Day 1 post partum	mean	68.30	68.05	72.12	52.50
Litter weight (g) at Day 4 post partum	mean	97.32	91.40	101.73	73.18
Male offspring weight (g) at Day 1 post partum	mean	6.31	6.22	6.31	6.10
Male offspring weight (g) at Day 4 post partum	mean	9.17	8.49	8.97	8.74
Female offspring weight (g) at Day 1 post partum	mean	6.07	5.94	5.99	5.67
Female offspring weight (g) at Day 4 post partum	mean	8.96	8.35	8.54	8.13
LOSS OF OFFSPRING/DAM
Pre-implantation (corpora lutea minus implantations)
0	number	8	4	3	4
1	number	2	5	4	1
2	number	1	1	1	1
3	number	0	0	0	1
4	number	0	0	1	1
5	number	0	1	0	1
7	number	0	0	1	0
12	number	1	0	0	0
Pre-natal (implantations minus live births)
0	number	4	5	5	2
1	number	5	4	4	5
2	number	3	2	1	0
3	number	0	0	0	2
Post natal (live births minus offspring alive on Day 4 postpartum)
0	number	9	8	10	7
1	number	2	2	0	1
2	number	0	1	0	1
3	number	1	0	0	0
16	number	0	0	0	1

Applicant's summary and conclusion

Conclusions:

The oral administration of Direct Orange 39 (C.I. 40215) to rats by gavage, at dose levels of 750, 300 and 30 mg/kg bw/day, resulted in treatment-related changes at 750 and 300 mg/kg bw/day. Effects at 300 mg/kg bw/day were considered not to represent an adverse effect. Therefore a ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 300 mg/kg bw/day.

There were no treatment-related effects detected on the reproductive parameters investigated, therefore the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 750 mg/kg bw/day.

Executive summary:

The oral administration of Direct Orange 39 (C.I. 40215) to rats for a period of up to eight weeks (including two weeks pre-pairing, gestation and early lactation period for females) at dose levels of 750, 300 and 30 mg/kg bw/day resulted in treatment-related changes at 750 and 300 mg/kg bw/day.

There were three unscheduled deaths during the study. One death was due to an open wound and therefore unrelated to treatment with the test item. One animal was found dead and another was killed in extremis due to emaciation. Histopathological examinations showed the deaths of these two animals to be attributable to inappropriate dosing technique rather than a toxic effect of treatment. The instances of noisy respiration were also considered to be due to dosing technique rather than a toxic effect of treatment.

Males treated with 750 mg/kg bw/day showed a reduction in haemoglobin, erythrocytes and haematocrit counts, with corresponding increases in mean cell haemoglobin and mean cell volume. Increases in mean cell volume and mean cell haemoglobin were also higher for males treated with 300 mg/kg bw/day when compared to controls. These findings are consistent with an anaemic response, however, there were no differences in reticulocyte counts and in the absence of extramedullary haemopoiesis observed following histopathological of the spleen and/or bone marrow, this finding was considered not to represent an adverse effect of treatment. Blood chemical analysis revealed a reduction in total protein level and an increase in A/G ratio when compared to controls for males treated at the highest dose level. In the absence of any structural defects in systemic organs, it is likely that this change may be of limited toxicological significance and related to metabolic stimulation.

An increase in water intake was evident for animals of either sex treated with 750 mg/kg bw/day and organ weight assessments revealed an increase in kidney weights, both absolute and relative to terminal body weights, for animals of either sex treated with 750 and 300 mg/kg bw/day. In the absence of any histopathological renal changes, these increases were considered not to represent an adverse effect of treatment.

Furthermore, an increase in absolute and body weight-relative liver weights when compared to control values, was evident for animals of either sex treated with 750 mg/kg bw/day and for females treated with 300 mg/kg bw/day. The increases in liver weights

were considered to represent and adaptive response to test item administration and in the absence of any histopathological correlates, these increases were considered not to represent an adverse health effect.

The most significant findings detected during the study were the lower overall body weight gains and a reduction in dietary intake detected for males treated with the highest dose level when compared to controls. Actual body weight losses were also evident for

females treated with 750 mg/kg bw/day, together with a reduction in dietary intake, with reductions in body weight gains detected during gestation when compared to controls.

Reduced dietary intake was also detected during the first two weeks of gestation when compared to controls. Slightly smaller litter sizes were evident at 750 mg/kg bw/day when compared to controls. Sex ratio or offspring viability was not affected. Offspring of

slightly lower bodyweights were evident for females treated with 750 mg/kg bw/day when compared to controls. Slightly lower numbers of corpora lutea and implantation sites were noted for females treated with 750 mg/kg bw/day when compared to controls.

Slightly higher pre- and post- implantation losses were also evident at 750 mg/kg bw/day when compared to controls. Furthermore, slightly longer gestation lengths were evident at the highest dose level when compared to controls. These intergroup differences were

most likely attributable to maternal stress. The reduced bodyweight gains and dietary intake observed at the highest dose level, and did not represent true reproductive toxicity. Due to the effect on litter size, the reductions in body weight gains and dietary intake at 750 mg/kg bw/day were considered to represent an adverse effect of treatment and contributed to the minor fluctuations in litter values. The lack of specificity of reproductive effect was concluded to be more likely to be a consequence of an impaired health of adult females that contributed to slightly reduced reproductive performance. Due to the treatment-related effects observed in this study, a ‘No Observed Adverse Effect Level’ (NOAEL) was established at 300 mg/kg bw/day and a ‘No Observed Effect Level’ (NOEL) was established at 30 mg/kg bw/day for systemic toxicity. A true reproductive effect was not observed during the study, therefore a ‘No Observed Effect Level (NOEL) was established at 750 mg/kg bw/day for reproduction and offspring development.