Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch 135214 of Disperse Blue 359 was a dark blue powder with a purity of 99 %
Specific details on test material used for the study:
Batch 135214 of Disperse Blue 359 was a dark blue powder with a purity of 99 %

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Nulliparous and non-pregnant females and untreated animals were used at initiation of the study.
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants
Sex:
male/female
Details on test animals and environmental conditions:
48 females and 40 males. At the end of the pretest phase, 40 females with at least two regular estrous cycles were selected at random and further used in the study. The remaining females were removed from the study.
Environmental controls for the animal room were set to maintain 18 to 24 °C, a relative humidity of 40 to 70%, at least 10 room air changes/hour, and a 12 hour light/12 hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on mating procedure:
Following a minimum of 14 days of treatment for the males and females, the animals were cohabitated on a 1:1 basis within 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 was confirmed, the males and females were separated. A maximum of 14 days was allowed for mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted once during the treatment phase (16th July 2017), stored, then dispatched to test site on dry ice for formulation analysis. A dose control system (DCS) was used as an additional check to verify the dosing procedure according to Standard Operating Procedures.
Samples of formulations for homogeneity and accuracy were stored on dry ice immediately after sampling. Stability samples were kept at room temperature under normal laboratory light conditions for 5 hours, then placed on dry ice. Samples retained on dry ice until receipt at ABL, The Netherlands.
Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 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 85-115% 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:
Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating and up to the day prior to scheduled necropsy. Females that delivered were treated for 50-62 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 42-51 days.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal mil or from exposure to maternal urine/faeces.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males and 10 females for each dose group
Control animals:
yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:
The following observations were recorded for parental animals.
Mortality/viability: At least twice daily.
Clinical signs: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at no specific time point, but within a similar time after treatment for the respective animals. Once prior to start of treatment and at weekly intervals during the treatment period, this was also performed outside the home cage in a standard arena.
The time of onset, grade and duration of any observed sign as 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.
Functional Observations: The following functional observations test were performed on each individual animal of the selected 5 animals/sex/group.
Hearing ability (HEARING), pupillary reflex (PUPIL L/R), and static righting reflex (STATIC R) (score 0=normal/present, score 1=abnormal/absent).
Fore and hind limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
Locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulation represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by te animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
The selected males were tested during week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
Body weights: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.
Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17 and 2o post-coitum and during lactation on PND 1, 4, 7, and 13.
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, cage debris of pregnant females was also examined for evidence of abortion or premature delivery. 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.
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. During pretest, this was done for 48 females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.
Sperm parameters (parental animals):
The reproductive organs were examined from all males that failed to sire. Additional slides of testes were made of the 5 selected males of groups 1 and 4 and all males suspected to be infertile and stained with PAS/hematoxylin to assess spermatogenesis.
Litter observations:
Mortality / Viability: The number of live and dead pups were determined on PND 1 and daily thereafter. Pups 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). The circumstances of any death were recorded in detail. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective tables.
Body weights: Live pups were weighed on PND 1, 4, 7 and 13.
Sex: Sex was determined for all pups on PND 1 and 4. Sex ratio (% male pups / % female pups) was calculated per group.
Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
Areola/nipple retention: On PND 13, all males in each litter were examined for the number of areola/nipples.
Postmortem examinations (parental animals):
At the end of the treatment period all rats from all groups were killed and subjected to complete necropsies. Histopathologic examination was performed on an extensive list oforgans and tissues from five selected group 1 and 4 animals, adrenal glands from all selected males, as well as organs with macroscopic findings from all rats.
Postmortem examinations (offspring):
At the end of the treatment period all rats from all groups were killed and subjected to complete necropsies. Histopathologic examination was performed on an extensive list oforgans and tissues from five selected group 1 and 4 animals, adrenal glands from all selected males, as well as organs with macroscopic findings from all rats.
Offspring viability indices:
Viability index (%)= (Number of live offspring on Day 4 before culling x 100)/(Number live offspring on Day 1 after littering)
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices across the groups were 97-100%.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No toxicologically relevant clinical signs were observed up to 1000 mg/kg.

Salivation seen after dosing among animals of the 1000 mg/kg dose group during the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). In all males and females of the 1000 mg/kg group, general blue discolouration, blue staining of the back and dark faeces were observed. This most likely represented the test item, a dark blue powder.

In one female of the 1000 mg/kg red discolouration of urine was noted on 3 consecutive days during the premating phase and on 5 consecutive days during the mating phase. As this concerned only one female that delivered a healthy litter, this was considered not to be a sign of toxicological relevance. Incidental findings that were noted included salivation in Group 3, rales, alopecia, scales and focal erythema. These findings occurred 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 not to be signs of toxicological relevance. No additional findings were noted during the arena observations in this study.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No treatment- related mortality occurred during the study period.
One female at 1000 mg/kg was sacrificed at Day 24 post-coitum due to suspected total littler loss. However, at necropsy one fetus was found and it was concluded that this female had delivery difficulties. The fetus was found in the left horn of the uterus and was malformed. Macroscopic findings of the fetus consisted of a misshapen head and edema of the right-hand leg. Based on the single occurrence of these findings this was considered to be unrelated to treatment. Moreover, these findings could be related to the extended duration of gestation in the female (24 days compared to on average 21.4 days in the control group).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A treatment-related increase in body weight of male and female PND 13-15 pups at 1000 mg/kg was noted (not statistically significant, relative difference from control: 9%). This increase might be related to the increased glucose levels in the dams. However, as the increase remained within normal limits, this effect was considered not adverse.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematology findings consisted of a treatment relate decrease in lymphocytes (relative difference from control: 30 %) in males treated at 1000 mg/kg group. A non-significant decrease was seen in white blood cells (WBC) (relative difference from control: 20 %) for males at 1000 mg/kg. The decrease seen in WBC is likely the result of the decrease in lymphocytes. As the value of lymphocytes in males treated at 1000 mg/kg was within normal limits and based on the absence of corroborative changes in other endpoints, the findings were considered not adverse.
Haematological parameters of treated females were considered not to have been affected by treatment. Any statistically significant changes in males at 100 or 300 mg/kg were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, a few statistically significant differences were noted between the 1000 mg/kg group and the control group:

A decrease in total bilirubin concentration was noted in treated males (relative difference from control: 40%). A similar, but not statistically significant, decrease was observed in 300 mg/kg treated males (relative difference from control: 15%) and 1000 mg/kg treated females (relative difference from control: 20%). The mean total bilirubin concentration of males treated at 1000 mg/kg was below historical control data.

An increase in glucose level was noted in 1000 mg/kg treated females (relative difference from control: 18%). The glucose levels remained within normal limits. Any statistically significant changes at 100 or 300 mg/kg were not considered to be toxicologically significant as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

Thyroid hormone analyses: Serum levels of T4 in F0 males were not considered to be affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test item related histopathological effects were observed during the course of the study.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No test item related histopathological effects were observed during the course of the study.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not considered to have been affected by treatment. Most females had regular cycles of 4 days. Extended di-estrus occurred in female no.43 (Control) which had a normal litter, and in female no. 74 (1000 mg/kg) which had delivery difficulties on Day 24 post-coitum. Given their incidental nature and absence of a doserelated incidence, these findings did not indicate a relationwith treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.
Reproductive performance:
no effects observed
Description (incidence and severity):
There was one control treated couple (male 7 and female 47), one couple treated at 100 mg/kg /day (male 15 and female 55) without offspring. No abnormalities were seen in the reproductive organs, which could account for their lack of offspring.
One female at 1000 mg/kg/day (female 74) was euthanized due to delivery difficulties. No abnormalities were recorded in the reproductive organs that could explain this. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
histopathology: non-neoplastic
histopathology: neoplastic

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
coagulating gland
gonad
preputial gland
seminal vesicle
testes
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
female reproductive system
Organ:
cervix
clitoral gland
coagulating gland
ovary
preputial gland
uterus
vagina

Results: F1 generation

General toxicity (F1)

Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
treatment-related increase in body weight of male and female PND 13-15 pups at 1000 mg/kg was noted (not statistically significant, relative difference from control: 9%). This increase might be related to the increased glucose levels in the dams. However, as the increase remained within normal limits, this effect was considered not adverse.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
A treatment-related increase in T4 of PND 13-15 pups at 1000 mg/kg was noted (not statistically significant, relative difference from control: 17% male and 19% female pups).
The increase remained within normal limits and no other corroborative changes were seen, therefore this effect was considered not adverse.

Details on results (F1)

Developmental NOAEL: at least 1000 mg/kg.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
endocrine system
Organ:
pituitary gland
thyroid gland

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, based on these results the following No Observed Adverse Effect Levels (NOAELs) were derived:
Parental NOAEL: at least 1000 mg/kg.
Reproduction NOAEL: at least 1000 mg/kg.
Developmental NOAEL: at least 1000 mg/kg.
Executive summary:

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg). A treatment-related increase in body weight of male and female PND 13-15 pups at 1000 mg/kg was noted (not statistically significant, relative difference from control: 9%).This increase might be related to the increased glucose levels in the dams. However, as the increase remained within normal limits, this effect was considered not adverse. A treatment-related increase in T4 of PND 13 -15 pups at 1000 mg/kg was noted (not statistically significant, relative difference from control: 17% male and 19% female pups). The increase remained within normal limits and no other corroborative changes were seen,therefore, this effect was considered not adverse.