A mixture of: N,N-diethylpropane-1,3-diamine 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2,2-iminodiethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2-methylaminoethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: N,N-diethylpropane-1,3-diamine 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2,2-iminodiethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2-methylaminoethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate

EC Number:

403-410-1

EC Name:

A mixture of: N,N-diethylpropane-1,3-diamine 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2,2-iminodiethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2-methylaminoethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate

Cas Number:

114565-65-0

Molecular formula:

C18 H16 N8 O3 S2 . x C7 H18 N2 . x C4 H11 N O2 . x C3 H9 N O

IUPAC Name:

(3-aminopropyl)diethylamine; 2-(methylamino)ethan-1-ol; 2-[(2-hydroxyethyl)amino]ethan-1-ol; 6-methyl-2-{4-[2-(triaminopyrimidin-5-yl)diazen-1-yl]phenyl}-1,3-benzothiazole-7-sulfonic acid

Details on test material:

- Name of test material (as cited in study report): Direct Yellow 166
- Molecular weight main component: 456.5
- CA Index Name: 7-Benzothiazolesulfonic acid, 6-methyl-2-[4-[(2,4,6-triamino-5-pyrimidinyl)azo]phenyl]-, compd. with N,N-diethyl-1,3-propanediamine and 2,2'-iminobis[ethanol] and 2-(methylamino)ethanol (9CI)
- CAS Number main component: 40497-29-8
- Description: Orange powder (determined at NOTOX)
- Batch No.: 13/2010
- Purity (= content of main component): 87.6 area% (HPLC)
- Test substance storage: At room temperature in the dark
- Stability under storage conditions: stable
- Expiry date: 26 May 2013

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: (P) approx. 11 weeks
- Housing: pre-mating: animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm); mating: females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm); post-mating: males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage, females were individually housed in Macrolon plastic cages (MIII type, height 18 cm); lactation: pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm).
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: tap water ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 40 - 70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% aqueous solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustments were made for the purity of the test substance.
- Storage conditions: At ambient temperature.

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated.
Detection of mating was not confirmed for animal no. 41 which did deliver live offspring. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 postcoitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase, according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under normal
laboratory light conditions was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

PARENTAL ANIMALS: Males were exposed for 30 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females were exposed for 42-55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy).
PUPS: Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

once daily for 7 days per week

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

- MORTALITY/VIABILITY: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).
- CLINICAL SIGNS: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.
- BODY WEIGHTS: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and
20 post-coitum and during lactation on Days 1 and 4. Female no. 69 showed broken upper incisors and was additionally weighed on Day 16 of the post-coitum period in order to monitor the health status.
- FOOD CONSUMPTION: Weekly, except for males and females which were housed together for mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.
- WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
- GENERAL REPRODUCTION DATA: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Litter observations:

- MORTALITY / VIABILITY: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined.
- CLINICAL SIGNS: At least once daily, detailed clinical observations were made for all animals.
- BODY WEIGHTS: Live pups were weighed on Days 1 and 4 of lactation.
- SEX: Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

Necropsy was conducted on the following days:
- Females which delivered: Lactation Days 5-7.
- Females which failed to deliver (nos. 71-80): Post-coitum Days 25-27 (females with evidence of mating).
- Males: Following completion of the mating period (a minimum of 28 days of dose administration).
- Euthanized in extremis (no. 58): When pain, distress or discomfort was considered not transient in nature or was likely to become more severe.

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The numbers of former implantation sites and corpora lutea were recorded for all paired females. Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Cervix, Clitoral gland, Coagulation gland, Epididymides (fixed in modified Davidson's solution and transferred to formalin after fixation), Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes (fixed in modified Davidson's solution and transferred to formalin after fixation), Uterus, Vagina, all gross lesions.
Testes and epididymides weights and terminal body weight were recorded for all parental males on the scheduled day of necropsy.
The following slides were examined by a pathologist:
- The ovaries, testes and epididymides of the animals of Groups 1 and 4.
- The additional slides of the testes of the males of Groups 1 and 4, and all males suspected to be infertile, to examine staging of spermatogenesis.
- The preserved organs and tissues of female no. 58 that was killed in extremis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.

Postmortem examinations (offspring):

Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores). Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

The following calculations were performed (if possible):
Mating index (%): Number of females mated / Number of females paired x 100
Fertility index (%): Number of pregnant females / Number of females paired x 100
Conception index (%): Number of pregnant females / Number of females mated x 100
Gestation index (%): Number of females bearing live pups / Number of pregnant females x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

The following calculations were performed (if possible):
Percentage live males at First Litter Check: Number of live male pups at First Litter Check / Number of live pups at First Litter Check x 100
Percentage live females at First Litter Check: Number of live female pups at First Litter Check / Number of live pups at First Litter Check x 100
Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation / Number of live pups at First Litter Check x 100
Viability index: Number of live pups on Day 4 post partum / Number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
No mortality occurred during the study period that was considered to be related to treatment with the test substance.
One female at 100 mg/kg bw/d was killed in extremis on Day 23 post-coitum due to a difficult/prolonged parturition. This was considered to be an incidental finding and unrelated to treatment with the test substance.
One female at 1000 mg/kg bw/d showed hunched posture and/or piloerection, on two days during the pre-mating period. Piloerection and pale appearance was shown by one female at 1000 mg/kg bw/d during the last week of the post-coitum period. A relationship of these findings to treatment cannot be excluded.
Yellow faeces seen among all groups treated with the test substance was considered to be related to staining properties of the test substance (an orange formulation).
One female at 300 mg/kg bw/d showed broken upper incisors, hunched posture and piloerection during the post-coitum phase. These findings were not considered to be related to treatment.
The occurrence of alopecia and scabs noted among the dose groups was within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Treatment-related effects on body weight (gain) at 1000 mg/kg bw/d can not be excluded.
Body weight (gain) of males at 1000 mg/kg bw/d was lower than for male controls primarily towards the end of the mating period (statistically significant on several occasions).
Three females at 1000 mg/kg bw/d showed weight loss (up to 5% of Day 1 values) over Days 1 to 8 of the premating period. Body weights of these females had recovered at commencement of the mating period. Since 8/10 females at 1000 mg/kg bw/d did not deliver offspring, lower body weights were recorded at 1000 mg/kg from Day 7 post-coitum onwards.
Body weights and body weight gain of animals at 100 and 300 mg/kg bw/d remained in the same range as controls over the treatment period.
The food consumption of (largely non-pregnant) females at 1000 mg/kg bw/d during the last week of the post-coitum period was lower than in other groups.
Food consumption before or after correction for body weight of all treated male groups and female groups at 100 and 300 mg/kg bw/d was similar to that of control animals.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Two females at 1000 mg/kg bw/d became pregnant out of ten mated/paired females, resulting in a significantly reduced fertility and conception index. None of these pregnant females delivered live offspring, resulting in a gestation index of 0%. No corpora lutea were found in the non-pregnant females at 1000 mg/kg. One of the pregnant females had only one corpora luteum. The other pregnant female showed 11 corpora lutea. Both pregnant females had only one implantation site.
Mating index and precoital time were normal at all dose levels. Fertility and conception indices and number of corpora lutea and implantation sites were normal at 100 and 300 mg/kg bw/d.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Testes and epididymides weights of treated males were similar to those of control animals.
Terminal body weights of males at 1000 mg/kg bw/d were slightly lower than those of male controls (the difference were not significant in statistical terms).

GROSS PATHOLOGY (PARENTAL ANIMALS)
Treatment-related macroscopic changes at 1000 mg/kg bw/d can not be excluded.
One female at 1000 mg/kg bw/d showed (soft) black−brown contents in the left horn of the uterus and vagina. Another female at this dose level showed enlargement of the placenta.
A female at 100 mg/kg bw/d experienced a difficult/prolonged parturition and was killed. This animal showed general pale discolouration and the placenta was found to be stuck in the cervix. These were considered to be incidental findings and unrelated to treatment with the test substance. This animal had delivered only one live pup and there were still 18 live f
oetuses in the uterus.
The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings included pelvic dilation of the kidneys, nodules and/or foci on the epididymides, nodules on the clitoral glands, scab formation on the skin, alopecia, foci on the preputial glands, reduced size of the preputial glands and fluid in the uterus.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The two pregnant females at 1000 mg/kg bw/d showed placental tissues and/or fluid and cell debris in the uterus. These findings corresponded to necropsy observations of soft blackbrown contents in uterus of one female and an enlarged placenta in another female; in the first of these 2 females, also moderate mucification of the vaginal epithelium and fluid with cell debris in the vagina were seen.
The spermatogenic staging profiles were normal for all Group 1 and Group 4 males and for Group 2 (100 mg/kg bw/d) male no. 18.
The remainder of the microscopic findings recorded were within the normal range of background pathology encountered in Wistar rats of this age and strain.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: at 1000 mg/kg/d clinical signs, food consumption and body weight changes

2

Dose descriptor:

NOAEL

Remarks:

reproduction toxicity

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: at 1000 mg/kg/d significantly reduced fertility and conception index, gestation index of 0%

Results: F1 generation

Details on results (F1)

DEVELOPMENTAL DATA:
At 100 and 300 mg/kg bw/d, gestation index and duration were normal, and no toxicologically relevant effects on parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed at these dose levels.
At 1000 mg/kg bw/d, no developmental data were available since no pups were delivered.
- Gestation: Gestation index and duration of gestation were normal.
- Parturition/maternal care: One female at 100 mg/kg bw/d was sacrificed due to a difficult/prolonged parturition. This was considered to be an incidental finding and unrelated to treatment with the test substance. No signs of difficult or prolonged parturition were noted among the other pregnant females at 100 and 300 mg/kg bw/d. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
- Early postnatal pup development: Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.
Mortality: One pup at 100 mg/kg bw/d was found missing at necropsy, and was most likely cannibalised. No toxicological relevance was attributed to this missing pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. The female at 100 mg/kg bw/d that was sacrificed due to difficult/prolonged parturition had 18 live foetuses in the uterus and had delivered one live pup.
Clinical signs: Scabbing of the head noted for one pup at 100 mg/kg bw/d was within the range considered normal for pups of this age.
Body weights: Body weights of pups were considered to have been unaffected by treatment.
Macroscopy: Scabbing of the head noted for one pup at 100 mg/kg bw/d was within the range of necropsy findings considered normal for pups of this age.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: at 1000 mg/kg, no developmental data were available since no pups were delivered.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The following No Observed Adverse Effect Levels (NOAEL) were derived: Parental NOAEL = 300 mg/kg bw/day; Reproduction NOAEL = 300 mg/kg bw/day; Developmental NOAEL = 300 mg/kg bw/day (at 1000 mg/kg bw/d, no pups were delivered).

Executive summary:

The study was performed according to OECD guideline 421 in compliance with GLP.

Direct Yellow 166 was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 30 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42-55 days).

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Parental results: Clinical signs, food consumption and body weight changes were observed at the high dose level. A relationship to treatment can not be excluded.

 

Reproductive results:

Histopathologically, a normal cyclic activity in the reproductive system of all females at 1000 mg/kg bw/d was observed. However, at 1000 mg/kg bw/d, eight out of ten females did not become pregnant. Neither implantation sites nor macroscopically visible corpora lutea were noted in these females. Two out of ten females at 1000 mg/kg bw/d showed some evidence of pregnancy but delivered no live offspring. One of the pregnant females had only one corpora luteum. The other pregnant female had a near normal number of 11 corpora lutea. Both pregnant females had only one implantation site but showed placental tissues and/or fluid and cell debris in the uterus. One pregnant female showed moderate mucification of the vaginal epithelium and fluid with cell debris in the vagina as well. No reproduction toxicity was observed at 100 and 300 mg/kg bw/d (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites were normal).

 

Developmental results:

No developmental toxicity was observed at 100 and 300 mg/kg bw/d (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development (consisting of mortality, clinical signs, body weight and macroscopy) were normal. At 1000 mg/kg bw/d, no pups were delivered and hence no assessment of developmental toxicity could be made.

 

Conclusion:

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: 300 mg/kg bw/day

Reproduction NOAEL: 300 mg/kg bw/day

Developmental NOAEL: 300 mg/kg bw/day (at 1000 mg/kg bw/d, no pups were delivered)