Salts of 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthenium chloride and 4-{[1-hydroxy-6-({[5-hydroxy-6-(phenyldiazo)-7-sulfonaphthalen-2-yl]carbamoyl}amino)-3-sulfonaphthalen-2-yl]diazo}benzoic acid (2:1)

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:

from 2008-10-22 to 2008-12-08

Reliability:

1 (reliable without restriction)

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: CRL: (WI) BR Wistar rats
Source: Charles River (Europe) Laboratories Inc. TOXI COOP Ltd. Hungary, 1103 Budapest, Cserkesz u. 90.
Hygienic level: SPF at arrival; standard laboratory conditions during the study
Justification of strain: The rat is a suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.
Number of animals: 48 males, 48 females: 12 animals/sex in the control and dose groups (the expected number of at least 8 pregnant female animals per group was achieved); 2 male and 2 female spare animals were transferred to LAB Research Ltd. spare colony at the completion of the study, as their use was not required.
Sex: Male/Nulliparous, non pregnant females
Age of animals: Young adult rats, approximately 10 weeks old at starting and 12 weeks at mating. The age range within the study was kept to the minimum practicable.
Body weight range at onset of treatment: males: 332-401 g; females: 228-285 g
Acclimatisation time: 14 days
Animal health: Only healthy animals were used for the test, as certified by the clinical veterinarian.
Room: 524
Cage type: type II and III polypropylene/polycarbonate
Housing: Before mating: 4 animals of the same sex / cage
Mating: 1 male and 1 female / cage
Pregnant females were housed individually; the bedding material was suitable for nesting. Details on bedding material are presented in Appendix 6.
Bedding: Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (D-73494 Rosenberg, Holzmühle 1, Germany).
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.2 - 23.5 °C
Relative humidity: 33 - 69 %
Ventilation: 8 - 12 air exchanges/hour
Enrichment: Rodents were group housed, except during mating and pregnancy, to allow social interaction and with deep wood sawdust bedding to allow digging and other normal rodent activities.
The temperature and relative humidity values were checked and recorded twice daily during the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

The test item was administered daily by oral gavage, at similar time. The oral route was selected as a possible route of exposure for humans to F 213 Red. Animals were not treated on the day of gross pathology.
Dosing of both sexes began after an appropriate acclimatisation (A) period and two weeks before mating and was continued up to and including the day before necropsy. Mating began soon after the animals attained full sexual maturity. Dosing was continued in both sexes during the mating period.
Males were dosed for 28 days, 14 days pre-mating (PM) and 14 days mating/post-mating period (M), then they were euthanised and subjected to necropsy examination:
Females were dosed for 14 days pre-mating (PM), for up to 7 days mating period (M), through gestation (up to 24 days) (G) and day 3 post-partum (PP/PN) with necropsy the following day, or shortly thereafter. The day of birth (viz. when parturition was complete) was defined as day 0 post-partum:
*All F1 offspring were terminated on day 4 post partum or shortly thereafter (up to day 6 post partum).

Details on mating procedure:

Mating began 2 weeks after the initiation of treatment with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred (up to 7 days). Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution.
The smears were examined with a light microscope, the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421, or gestation day GD 0). Sperm positive females were caged individually. Mating pairs were clearly identified in the data and are presented in the study report; mating of siblings was avoided.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item was formulated in 1 % methylcellulose in concentrations of 5, 35 and 100 mg/mL in the Central Dispensary of LAB Research Ltd. Formulations were prepared as appropriate to allow their use within 72 hours, and were stored refrigerated pending use, according to stability assessment results. Assessment of test item stability in this vehicle indicated up to 4-hour stability at room temperature and up to 72-hour stability when stored refrigerated, at concentrations from approximately 1 mg/mL to 100 mg/mL (LAB study code 08/612-316AN). A separate analytical report provided this information.
Analytical control (concentration, homogeneity) of dose formulations was performed in the Analytical Laboratory of LAB Research Ltd. on 27 October 2008 (first week of treatment) and 05 December 2008 (last week of treatment). The measured concentrations ranged from 94 to 106 % of nominal concentrations. The results of the chemical analysis were considered suitable for the study purposes.

Duration of treatment / exposure:

Males were dosed daily for 28 days, 14 days pre-mating (PM) and 14 days mating/post-mating period (M), then they were euthanised and subjected to necropsy examination:
Females were dosed daily for 14 days pre-mating (PM), for up to 7 days mating period (M), through gestation (up to 24 days) (G) and day 3 post-partum (PP/PN) with necropsy the following day, or shortly thereafter.

Frequency of treatment:

Daily

Details on study schedule:

Mating began 2 weeks after the initiation of treatment with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred (up to 7 days). Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution.



The smears were examined with a light microscope, the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421, or gestation day GD 0). Sperm positive females were caged individually. Mating pairs were clearly identified in the data and are presented in the study report; mating of siblings was avoided.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
analytical conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
350 mg/kg bw/day
Basis:
analytical conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
50 mg/kg bw/day
Basis:
analytical conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
0 mg/kg bw/day
Basis:
analytical conc.

No. of animals per sex per dose:

48 males, 48 females: 12 animals/sex in the control and dose groups (the expected number of at least 8 pregnant female animals per group was achieved); 2 male and 2 female spare animals

Control animals:

yes

Details on study design:

None

Positive control:

None

Examinations

Parental animals: Observations and examinations:

Mortality occurred in 1 female rat at 350 mg/kg bw/day. This female showed no systemic adverse clinical signs prior to parturition. After parturition on day 44, decreased activity and piloerection were noted for 3 days, followed by death on day 47. Based on the lack of additional mortality within the group, or at high dose, a direct test item-related causality of this death is equivocal. Moreover, it should be noted that all the pups of this female died (2/14 pups were still-born, and 12/14 pups died up to PND3, with 18 corpora lutea and 16 implantation sites). Thus, the death of this animal was considered to be related to post-partal changes.
Surviving animals
Day 0 was regarded as the first day of treatment. Red faeces (50, 350, and 1000 mg/kg bw/day) and/or pink urine (350 and 1000 mg/kg bw/day) were observed in the bedding of the animals’ cages at various time points, as detailed below. These changes were ascribed to elimination of F 213 Red or its metabolites through faeces and/or urine, and were not considered adverse effects. In the surviving animals, piloerection, decreased activity and/or body tone and/or hunched back position were seen in 2/12 males and 1/12 females at 1000 mg/kg bw/day and in 1/12 males at 350 mg/kg bw/day. The toxicological significance of the transient observations in the single mid dose male was considered incidental, since even at 1000 mg/kg bw/day, most of the animals showed no similar changes.
1000 mg/kg bw/day (High Dose)
Red faeces and pink urine were observed in animals’ cages for both sexes, in the bedding, as of day 1 or 8 until the completion of the treatment period on day 27 (males) or from day 1 or 8 up to day 46 (females). In addition, male 4002 (1/12 males) showed decreased activity for 12/28 days, and piloerection with hunched back position for 6/28 days, from day 16 and 22, respectively. Female 4505 (1/12 females) displayed decreased activity and body tone from day 23 to day 43 or 31, respectively. Hunched back position and piloerection were noted in this female for the last 2 days of observation (days 42 and 43).
350 mg/kg bw/day (Mid Dose)
Red faeces occurred in all males and females as of day 1, for the whole duration of treatment. Pink urine was observed in the bedding from day 8 (males, 20/28 days) or 11 (females, 30-36 days) onwards. Male 3008 (1/12 males) displayed transiently decreased activity and body tone for 9/28 days, from day 15 to 23, and hunched back position for 8/28 days, from day 16 to 23.
50 mg/kg bw/day (Low Dose)
There were no systemic adverse effects following administration of 50 mg/kg bw/day F 213 Red. Red faeces were noted in the bedding from day 3 until completion of treatment on day 27 (males) or up to day 46 (females).

Oestrous cyclicity (parental animals):

There were no significant differences between the control and test item treated groups with regard to reproductive ability, and in the mating, fertility and gestation indices. In the females, the mating index was 100 %. The fertility index was 92 % at 0, 50 and 1000 mg/kg bw/day, with a gestation index of 82 % in the controls. As there was no dose-response, and relatively low indices were observed in the control group, these differences were not considered a test item-related effect. Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within 7 days of pairing (cohabitation).

Sperm parameters (parental animals):

There were no significant differences between the control and test item treated groups with regard to reproductive ability, and in the mating, fertility and gestation indices. In the males, the mating index was 100 % in all groups. A slightly lower male fertility index (92%) was noted at 0, 50 and 1000 mg/kg bw/day and was not considered toxicologically significant, or test-item related. No effects were observed at 350 mg/kg bw/day.

Litter observations:

Pups or litter examination did not reveal any effects considered directly test item-related compared to observations noted in the control group. No external abnormalities ascribed to test item administration could be detected at the clinical or macroscopic examinations of the pups. The number of male and female pups, and the sex ratio were similar in the control and treated groups. No significant variations were observed on PN 4 compared to PN 0.
There were no changes that could be ascribed to test item administration in the survival index values at PN 0 or PN 4 for either sex. The survival index on PND 4 was minimally to slightly lower in the treated animals than in controls, without attaining statistical significance. This difference was attributed to a few individual animals, in which most or all foetuses died: 1/11 pregnant low dose females (female 2502), 3/12 pregnant mid dose females (females 3503, 3508, and 3509), and 1/11 pregnant high dose females (female 4505). The other litters within the treated groups showed a normal distribution of mortality. Moreover, no dose-response was noted.
At 50 and 1000 mg/kg bw/day, the mean survival index of the pups was within the expected normal values for the population of Wistar rat. At 350 mg/kg bw/day, the lower mean value was considered attributable to litter 3508, in which all 14 pups and the dam died up to PND 3, and day 47, respectively. The dam’s death was related to post-partal changes and not attributed to treatment.
Based on these observations, the variations noted in pup mortality and mean survival index were considered incidental and not related to test item

Postmortem examinations (parental animals):

F 213 Red administered daily by oral gavage to Wistar rats, for 28 days (males) and up to 46 days (females), at 50, 350 or 1000 mg/kg bw/day, led to clinical signs in the parental (P) generation.
Red faeces and/or pink urine were observed at all dose levels. These changes were ascribed to elimination of F 213 Red or its metabolites through faeces and/or urine, and were not considered adverse effects.
One mid dose female (1/12) died on day 47. In this animal, after parturition on day 44, decreased activity and piloerection were noted for 3 days, followed by death on day 47. At necropsy, red coloured digestive content and dark discoloration/red of the kidneys, stomach, small and large intestine were regarded as test item-related. Small spleen and autolytic changes of the small intestine were also noted. Based on the lack of additional mortality within the same group or at higher dose levels, a direct test item-related cause of death was considered equivocal. Moreover, it should be noted that all 14 pups of this female died. Thus, the death of this animal was considered to be related to post-partal changes.
In the surviving animals, decreased activity and/or body tone, piloerection and/or hunched back position were seen in 2/12 males and 1/12 females at 1000 mg/kg bw/day and in 1/12 males at 350 mg/kg bw/day. The toxicological significance of the observations in the single mid dose male was considered incidental, since at 1000 mg/kg bw/day, most of the animals showed no similar changes. There were no systemic adverse effects following administration of 50 mg/kg bw/day.

Postmortem examinations (offspring):

A total of 56 pups were found dead between days 0 - 4 post partum. These pups were either still born, or died, after they did not suck milk, and/or were cold, on occasion being cannibalised. Three litters (3/11) were predominantly affected at 50 mg/kg bw/day, 2/12 at 350 mg/kg bw/day and 1/11 at 1000 mg/kg bw/day. At macroscopic observation, autolysis was observed on occasion. Dark red discoloration in the lungs was found in 3 mid dose pups. The positive floating test of the lungs was recorded in 34/44 pups examined. Dilatation of the stomach, dark discoloration of the stomach and/or small intestine and/or yellow gelatinous material in the gastro-intestinal system were seen in pups at all the dose levels tested, with no dose-response. No deaths or observations in pups were likely related to treatment. There were no clinical or behavioural changes noted in the surviving offspring.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Red coloured digestive content and/or dark discoloration/red of the stomach, small and large intestine and/or kidneys were seen at dose levels from 50 to 1000 mg/kg bw/day.

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Reproduction parameters examined.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

None.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Sex:

male/female

Basis for effect level:

other: Pup growth.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

None.

Applicant's summary and conclusion

Conclusions:

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for F 213 Red for parental effects was 350 mg/kg bw/day. For reproduction parameters, no effects were noted in any dose level resulting in a NOAEL of 1000 mg/kg bw/day. For pup growth rate, the NOAEL was 350 mg/kg bw/day.

Executive summary:

The objective of this study was the Reproduction/Developmental Toxicity Screening Test with the test item F 213 Red in the Rat according to OECD 421 and the draft OECD guidance document 43. The guideline is designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing.

The purpose of this study was to obtain initial information on the possible effects of the test item, F 213 Red, on reproduction and development when administered orally (by gavage) to CRL:(WI)BR rats at repeated doses of 50, 350 or 1000 mg/kg bw/day in 1 % methylcellulose, compared to control animals. As a screening test, it was intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and on development of the F1 offspring from conception to day 4 post-partum associated with administration of repeated doses.

Stability and homogeneity of the test item in the vehicle, 1% methylcellulose, was analytically proven. Assessment of test item stability in this vehicle, in the conditions employed on the study (LAB study code 08/612 316AN) indicated up to 4 hours stability at room temperature and up to 72 hours stability when stored refrigerated, at concentrations from approximately 1 mg/mL to 100 mg/mL in 1 % methylcellulose formulations, with a recovery within the acceptable range of 100 ± 10 % (actual range: 105 - 93 % of nominal). Analysis of dosing solutions was performed at all concentrations on samples collected during the first and last week of treatment. The measured concentrations ranged from 94 106 % of nominal concentrations. The analytical results were considered suitable for the study purposes.

Clinical observations for signs of ill health or reaction to treatment were made once daily. Special attention was paid to evaluation of the mating, pregnancy, parturition and post-partal periods, and relevant parameters and/or indices were measured and/or calculated. Body weight and food consumption were measured at least weekly. Gross necropsy was conducted at the end of the treatment period. The absolute and relative organ weights of selected organs and tissues were determined. A histopathological examination was performed on the selected preserved organs and tissues of the animals of the control and high dose groups, and on abnormal tissues from low and mid dose groups.

Results and Conclusion

F 213 Red administered daily by oral gavage to Wistar rats, for 28 days (males) and up to 46 days (females), at 50, 350 or 1000 mg/kg bw/day, led to clinical signs in the parental (P) generation.

Red faeces and/or pink urine were observed at all dose levels. These changes were ascribed to elimination of F 213 Red or its metabolites through faeces and/or urine, and were not considered adverse effects.

One mid dose female (1/12) died on day 47. In this animal, after parturition on day 44, decreased activity and piloerection were noted for 3 days, followed by death on day 47. At necropsy, red coloured digestive content and dark discoloration/red of the kidneys, stomach, small and large intestine were regarded as test item-related. Small spleen and autolytic changes of the small intestine were also noted. Based on the lack of additional mortality within the same group or at higher dose levels, a direct test item-related cause of death was considered equivocal. Moreover, it should be noted that all 14 pups of this female died. Thus, the death of this animal was considered to be related to post-partal changes.

In the surviving animals, decreased activity and/or body tone, piloerection and/or hunched back position were seen in 2/12 males and 1/12 females at 1000 mg/kg bw/day and in 1/12 males at 350 mg/kg bw/day. The toxicological significance of the observations in the single mid dose male was considered incidental, since at 1000 mg/kg bw/day, most of the animals showed no similar changes. There were no systemic adverse effects following administration of 50 mg/kg bw/day.

A total of 56 pups were found dead between days 0 - 4 post partum. These pups were either still born, or died, after they did not suck milk, and/or were cold, on occasion being cannibalised. Three litters (3/11) were predominantly affected at 50 mg/kg bw/day, 2/12 at 350 mg/kg bw/day and 1/11 at 1000 mg/kg bw/day. At macroscopic observation, autolysis was observed on occasion. Dark red discoloration in the lungs was found in 3 mid dose pups. The positive floating test of the lungs was recorded in 34/44 pups examined. Dilatation of the stomach, dark discoloration of the stomach and/or small intestine and/or yellow gelatinous material in the gastro-intestinal system were seen in pups at all the dose levels tested, with no dose-response. No deaths or observations in pups were likely related to treatment. There were no clinical or behavioural changes noted in the surviving offspring.

In the 1000 mg/kg bw/day adult animals, slightly lower body weight and body weight gain values than control animals were observed in both sexes. The overall lower body weight gain in males throughout the study was 28 %. In females, body weight effects were noted predominantly towards the end of the gestation period, with an overall lower body weight gain from day 0 to parturition of -31 %. These variations were not generally statistically significant, with the exception of the male body weight gain during the first week of treatment.

In the pups, slight but not statistically significant lower group mean litter weights or mean litter weight gain were noted at 1000 mg/kg bw/day. Mean pup-weight-per-litter and pup-weight-gain-per litter when evaluated as all individual pups/group on PND 0 and 4, were statistically lower than controls at all the dose levels tested, with a difference in the body weight gain of up to 27 % at 1000 mg/kg bw/day.

The offspring body weight changes at 1000 mg/kg bw/day were considered a possible treatment-related effect.

Slightly lower food consumption occurred in parental males and females at 1000 mg/kg bw/day throughout the study, correlated with the body weight changes at this dose level and was considered a possible effect of the treatment. In the males, the changes attained statistical significance during the first week of treatment. In the females, statistically significant lower values were observed from days 0 to 7 and GD 14 to 21.

In the 350 and 50 mg/kg bw/day animals, there were no variations in body weight, body weight gain or food consumption that could be ascribed to test item administration, or were considered toxicologically significant.

The parental animals displayed no effects related to treatment with regard to the reproductive ability and mating, fertility, gestation, parturition or post-partal period. Test item administration did not impact the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within 7 days of pairing (cohabitation).

The number of corpora lutea and implantations, as well as pre-implantation, intrauterine and post-natal mortality showed minor variations in the treated animals compared to the control, with no statistical significance. Thus, these variations were not considered related to test item administration, but to biological variability. No test item effect was observed in the duration of pregnancy. All females littered in 21 to 24 days. The number of pups born (mean and total) and live pups born (total) was generally higher in the treated animals than in the control group.

At necropsy of the parental animals, test item-related red coloured digestive content and/or dark discoloration/red of the stomach, small and large intestine and/or kidneys were seen at dose levels from 50 to 1000 mg/kg bw/day. Affected animals included 7/24, 22/23, 24/24 rats at dose levels of 50, 350, and 1000 mg/kg bw/day, respectively. No test item-related microscopic findings were noted, and there were no toxicologically significant effects in the organ weight values, absolute or relative to body or brain weights.

In the F1 generation, there were no pups with gross abnormalities. No effect on pup survival was noted. Gross necropsy of dead pups showed no effects of treatment.