1-imino-1H-isoindol-3-amine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-02-08 to 2017-06-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-02-08 to 2017-06-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July, 2016

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/ Develop mental Toxicity Screening Test. EPA 712-C-00-368

Version / remarks:

July, 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl: WI(Han) (Full Barrier)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals:
- Species: Wistar rat, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approx. 14-15 weeks old
- Weight at study initiation: males: 327 - 487 g (mean: 397.89 g)
- Fasting period before study: not reported
- Housing: Animals were housed in groups of 5 per sex in type IV polysulphone cages.
During mating period, males and females were housed together in ratio 1:1 (male to female). After the
confirmation of mating, females were kept individually during gestation/lactation period in type III H,
polysulphone cages and males were returned to their original cage.
- In each cage Altromin saw fibre was used as bedding
- Nesting material was provided latest on GD 18 for all mated females
- Diet (ad libitum): free access to Altromin 1324 maintenance diet for rats and mice
- Water (ad libitum): free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking
water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: At least 5 days.

Environmental conditions:
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours artificial light / 12 hours dark

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. None of the animal showed pathological signs before the first administration. Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estorus cycle (4-5 day cycle) were used in the study. The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight.
Before the first administration all animals to be used for the study were weighed and assigned to the experimental groups. Each animal was marked with its identification number by individual ear tattoo marking. The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females. Then in females, treatment was done during the gestation period and up to post-natal day 12. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female) (if possible). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented.

The day of the vaginal plug and/or sperm was considered as day 0 of gestation.

The cages were arranged in such a way that possible effects due to cage placement were minimised.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the treatment period, formulation samples were prepared and analysed for stability and homogeneity of the test item in the selected vehicle.
Study pre-start stability analysis were included on the samples from high dose and low dose group and the investigation was made for 0 h, 6 h (RT), 10 day (RT), 10 day (2-8 °C) and 10 day -15 to -35°C (10 samples). Pre-start homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups (6 samples).

The test item was homogenous (after 60 min without stirring), therefore during the study samples were collected for the investigation of homogeneity and only samples were taken for substance concentration in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all dose groups (12 samples). Each sample taken during the study was retained in duplicate (sample A, sample B, each of at lea st 5 mL).
The A-samples were analysed and until then stored under appropriate conditions based on available stability data.
The B-samples were retained at -15 to -35 °C and discarded after completion of the final study report.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females. Then in females, treatment was done during the gestation period and up to post-natal day 12. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

7 days per week

Details on study schedule:

General clinical observations were made at least once a day, preferably at the same time each day. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

Dose / conc.:

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

Remarks:

control (C) group

Dose / conc.:

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

Remarks:

low dose (LD) group

Dose / conc.:

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

Remarks:

medium dose (MD) group

Dose / conc.:

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

Remarks:

high dose (HD) group

No. of animals per sex per dose:

10 animals per sex per group

Control animals:

yes, concurrent vehicle

Details on study design:

100 animals (40 males and 60 females) were included in the study. All females were screened for regular estrous cycles for 14 days before the treatment initiation and only 40 females (10 females/group) showing regular estrous cycles were continued in the study. Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estorus cycle (4-5 day cycle) were used in the study.
Before the first administration all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.1.2 software).

Dose selection rationale:
According to the results of a previous dose range finding study the doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).

Body weight and food consumption:
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with pups. All animals were weighed directly before termination.
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Positive control:

not applicable

Parental animals: Observations and examinations:

Clinical observations:
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily. Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Functional observations:
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of lactation of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each
group outside the home cage using a functional observational battery of tests. Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Oestrous cyclicity (parental animals):

Estrous cycles were monitored before treatment starts to select females with regular cyclicity (using vaginal smears) for the study. Further on, vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

For the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

The duration of gestation was recorded and is calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring were recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.

Postmortem examinations (parental animals):

Pathology:
The males were sacrificed any time after the completion of the mating period (after a minimum dosing
period of 28 days) and females were sacrificed on the respective PND 13 by using anesthesia (ketamine/xylazine).
Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear or from the last day of mating period.
All animals were subjected to a detailed gross necropsy which included careful examination of the
external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.
The number of corpora lutea and implantation sites was recorded for any females sacrificed 26 days after the end of the mating period with no evidence of mating and for any females sacrificed on day 26 post-coitum due to non-delivery.


Haematology:
Haematological parameters from 5 randomly selected males and females (only lactating females were evaluated) from each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals.

The following haematological parameters were examined: haematocrit value (Hct), haemoglobin
content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH),
mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet
count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono),
eosinophils (Eos), basophils (Baso), large unstained cells (Luc).

Blood coagulation:
Coagulation parameters from 5 randomly selected males and females (only lactating females were
evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrificeof the animals.
The following coagulation parameters were examined: prothrombin time (PT), activated partial thromboplastin time (aPTT).

Clinical biochemistry:
Parameters of clinical biochemistry from 5 randomly selected males and females (only lactating
females were evaluated) of each group were examined at the end of the treatment prior to or as part
of the sacrifice of the animals.
The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT),
aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP),
albumin (Alb), urea, total bile acids (TBA), total bilirubin (TBIL), total cholesterol (Chol), glucose (Gluc),
sodium (Na), potassium (K).

Urinalysis:
A urinalysis was performed with samples from 5 randomly selected males and females (only lactating
females were evaluated) prior to or as part of the sacrifice of the animals. Additionally, urine colour/
appearance were recorded.
The following parameters were measured: specific gravity, nitrite, ph-value (pH), protein, glucose,
ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leukocytes.

Organ weights at necropsy:
The wet weight of the organs of 5 randomly selected male and female animals (only lactating females
were evaluated) from each group was recorded as soon as possible. Paired organs were weighed to
gether. Organ weights of animals found dead were not recorded. Reproductive organs were weighed
from all animals.
Thyroid/parathyroid glands from all adult males and females were preserved. Weight of thyroid/
parathyroid glands was measured after fixation.

The following tissues/organs were examined: testes (paired weight), epididymides (paired weight),prostate, seminal vesicles and coagulating glands (complete weight), thyroid/parathyroid glands (from
all adult males and females),
liver, kidneys (paired weight), heart.
Further tissues/organs from the same selected animals were preserved.
All animals found dead were subjected to a gross necropsy and the organs preserved for a histopath
ological examination.
Thyroid/parathyroid glands from non-selected adult animals were preserved for potential histopa
thological examination.

Histopathology:
A full histopathology was carried out on the preserved organs and tissues of the selected animals of
the control and high dose groups which were sacrificed at the end of the treatment period.
Evaluation of thyroid/parathyroid glands from the remaining non-selected adult animals was not deemed necessary
as no test item related histopathological findings were observed in thyroid/parathyroid gland of selected animals and there was
also no test item related effect observed on T4 hormone level in males sacrificed on PND 13.
For the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation

A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study.

Any gross lesion macroscopically identified was examined microscopically in all animals.

Postmortem examinations (offspring):

Clinical biochemistry:
From 2 female pups/litter on day 4 after birth and from 2 pups/litter (1 male and 1 female) at termination on day 13 blood samples were collected. Blood samples from the day 13 pups were assessed for serum levels for thyroid hormones (T4). Total T4 hormone levels were measured. Further assessment of T4 in blood samples from the day 4 pups was not deemed necessary. Additionally, assessment of TSH in day 4 pups and day 13 pups were not considered necessary based on the fact that no histopathological findings were observed in T4 hormone levels of day 13 pups. Pup blood was pooled by litter for thyroid hormone analysis. Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible serum hormone assessments. The two pups per litter were female pups to reserve male pups for nipple retention evaluations. No pups were eliminated where litter size dropped below 8 pups. When there was only one pup available above a litter size of 8, only one pup was sacrificed on PND 4.

Pathology:
All surviving pups were killed by cervical dislocation on PND 13. Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle. Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.

Statistics:

for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test.
Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically
analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or
a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.
These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software
(p<0.05 was considered as statistically significant).

Reproductive indices:

- Reproductive indices: copulation, viability and delivery indices
- Fertility index: number of females pregnant/ No. of females copulated x 100

Offspring viability indices:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In terminally sacrificed males, predominant clinical signs observed during the treatment period (premating day 1 to mating/post mating day 14) were nasal discharge in one animal of control group (PMD 12-13), severely increased salivation in one animal of HD (PMD 11) and moving the bedding in 2-3 animals of HD group during very few days of premating, mating and postmating period.
In terminally sacrificed females, major clinical signs observed during the treatment period (Premating day 1 to PND 12) were moving the bedding on one day during gestation period in three females of HD group, moderately increased salivation in two females of HD group on one day during gestation period and abnormal breathing on one day in one female of the HD group on gestation day 8.
The clinical signs salivation and moving the bedding on few days were observed immediately after the dose administration and therefore were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance.
During the weekly detailed clinical observation, no relevant differences between the groups were found.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

During the treatment period of this study, one mortality was observed on day of terminal sacrifice (PND 13):
- Female no. 49 (Control) was found dead on post natal day (PND) 13.
No specific clinical signs were observed in this animal before death or during entire study period.
Histopathologically, the cause of death was not evident.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In both males and females, there was no test item treatment related effect observed on body weight and body weight gain in the dose groups during the study period. There were no statistically significant differences observed for body weight and body weight gain between the dose groups and the control group except statistically significantly lower body weight gain in HD males during premating day 1-7 when compared with the controls.
As this difference was marginal and within the biological variation, it was not considered to be adverse effect due to treatment with test item.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In correlation to the body weight and body weight gain, the food consumption in both males and females tended to increase with the progress of the study in the control, the LD, the MD and the HD group.
No test item related or statistically significant effect on food consumption was observed in males and females during the whole study period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males sacrificed at the end of treatment period, no test item related adverse effects were observed for haematological parameters in treatment groups when compared to the control group.
In females sacrificed at the end of treatment period, no test item related effect or statistically significant effect observed on any of the haematology except statistically lower reticulocytes in LD group when compared with the controls.
Due to lack of dose dependency, this effect on reticulocytes in LD group was considered as incidental. No test item related effect was observed on coagulation parameters in males and females when compared with the respective controls.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males and females sacrificed at the end of treatment period, no test item related or statistically significant effect on any clinical biochemistry parameter in treatment groups was observed when compared with the control except statistically significantly lower TBA group mean value in male HD group.
This effect on TBA in male HD group was considered as incidental and toxicologically irrelevant as all individual values were within the historical data range (6.38 - 93.82 μmol/L). All other group mean and most of the individual values for clinical chemistry parameters in male and females were comparable with control group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The urinalysis performed in selected male and female animals sacrificed at the end of treatment period revealed no test item treatment related effect and all urinary parameters were in the normal range of variation. High protein levels were found in the urine of few male and females of all groups including control group. Therefore, this effect on urine parameters was not considered to be test item related.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In males, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. There were no biologically relevant differences observed in body temperature between the groups.
In females, statistically significantly higher not supported rearing count in HD group before initiation of treatment and statistically significantly higher urination count in LD group during last week of treatment was observed. As this type of difference was either marginal, before imitation of treatment or without dose dependency/consistency, it has no toxicological relevance and could be considered as biological variation. There were no biologically relevant differences in body temperature between the groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The test item did not produce morphological or histological evidence of toxicities in the organs and tissues examined in this study. In conclusion, a histopathological NOEL (No Observed Effect Level) could be established at 25 mg/kg bw/day.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No test item related effect of toxicological relevance or statistical significance was observed in male thyroxine hormone (T4) in the treatment groups when compared to the controls.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Test item had no biologically significant effect on the estrous cycle analysed during 2 weeks premating period after the first administration in treatment groups when compared to the controls. There were no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group.

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Litter data:
There were no test item treatment related or statistically significant effects observed in treatment groups on litter data parameters like group mean total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still birth, runt on PND 0 as well as number of live pups, male pups, number of female pups and sex ratio on PND 4 and PND 13 when compared with the controls.

Precoital interval and duration of gestation:
There were no test item related or statistically significant effects observed on the duration of precoital interval and the duration of gestation in the dose groups when compared to the control group.

Pre- and post-natal data:
There were no test item treatment related effects observed on the number of corpora lutea, implantation sites, live pups on PND 0, 4 and 13, percent preimplantation loss and post implantation loss in treatment groups when compared with the control group.

Reproductive indices:
There were no test item related effects on the reproductive indices (copulation, viability and delivery indices) in the dose groups when compared to the control group. However, a slightly reduced fertility index (number of females pregnant/ No. of females copulated X 100) of 80 % was observed in the LD group as compared to 90 % in control group. Although there was reduction in fertility index in LD group, it was within the standard pregnancy rate of rat i.e. ≥ 80 % and due to lack of dose dependency, this effect on fertility index was considered as biological variation and not related to treatment with the test item administration.

Thyroid hormone (T4) analysis: No test item related effect of toxicological relevance or statistical significance was observed on pup thyroid weight and PND 13 pup thyroxine hormone (T4) in the treatment groups when compared to the controls.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: general reproductive toxicity

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups. Few specific findings like dark head (pup no. 1 from dam 50 of control group on PND 0), crust near right eye (pup no. 5 from dam 69 of MD group on PND 0) and thin thorax (pup no. 1 from dam 73 of HD group on PND 0) were observed. Few pups from dam no. 73 of HD group were found dead/still born on PND 0 and were found to be partly cannibalised.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No test item related effect on mean mortality of pups between PND 0 and PND 4 and during PND 4-13 in treatment groups when compared to the control group. A marginally higher mean mortality of pups between PND 0 and PND 4 was observed in the MD group (2.16%) compared to the control group (0.00%). This outcome did not achieve statistical significance and was attributed to missing one each pup of 2 dams (pup no. 9 of dam no. 62 and pup no. 7 of dam no.69) on PND 4 and 1, respectively. Due to lack of dose dependency, pup mortality was considered as incidental and not related to the treatment with the test item.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no test item related effect on pup mean weight, total litter weight, female litter weight on PND 0, PND 4 and PND 13 observed in treatment groups when compared with the controls. There was no statistically significant change in dose groups compared to corresponding controls.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Anogenital distance and nipple retention: In males, statistically significant lower pup weight and cube root of pup weight on the day of anogenital measurement was observed in male MD group when compared with the controls which was considered as incidental. There was no statistically significant effect observed on absolute and relative anogenital distance in treatment groups when compared to the controls.

In females, statistically significantly lower absolute and relative anogenital distance was observed in LD and HD groups when compared to the controls.
AGD is always correlated with Cube root of pup body weight, litter size and sex ratio (for data normalization to simulate linear measurement).

In male and females, parameters like pup body weight, litter size and sex ratio were not affected in treatment groups and these parameters are correlated with anogenital distance (AGD) although statistically significant group mean pup weight and cube root of pup weight in male MD groups and AGD value in LD and MD group females were observed. In the light of absence of effect on pup weight and cube root of pup weight, effect on AGD in LD and HD group females cannot be considered as test item related.

No statistically significant effect of toxicological relevance was observed on nipple retention in the male pups of any of the groups when compared with the controls.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive toxicity screening

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Conclusions:

The NOAEL for the test item was found to be at least 25 mg/kg bw/day for reproductive toxicity screening.

Executive summary:

The aim of this study performed according to OECD TG 422 and in compliance to GLP, was to assess the possible adverse effects of

Ingrain Blue 2.2on male and female Wistar rats to the reproductive/developmental system after repeated dose administration with dose levels of 5, 10 and 25 mg/kg body weight/day. The treatment period was 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females.

No adverse effects of test item were found on estrous cyclicity, litter data, litter weight data, precoital interval and duration of gestation, pre and post-natal data, reproductive indices, pup survival data, anogenital distance and nipple retention, pup thyroid weight and thyroid hormone analysis in pups sacrificed on PND 13 and pup external findings in all treatment groups.

There were no test item-related adverse effects to the reproductive/developmental system up to 25 mg/kg bw/day. Therefore, the No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity is considered to be at least 25 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July, 2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/ Develop mental Toxicity Screening Test. EPA 712-C-00-368

Version / remarks:

July, 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-imino-1H-isoindol-3-amine

EC Number:

222-426-8

EC Name:

1-imino-1H-isoindol-3-amine

Cas Number:

3468-11-9

Molecular formula:

C8H7N3

IUPAC Name:

1-imino-1H-isoindol-3-amine

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl: WI(Han) (Full Barrier)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals:
Species: Wistar rat, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approx. 14-15 weeks old
- Weight at study initiation: males: 327 - 487 g (mean: 397.89 g), females: 213 - 277 g (mean: 240.33 g)
- Fasting period before study: not reported
- Housing: Animals were housed in groups of 5 per sex in type IV polysulphone cages
- In each cage Altromin saw fibre was used as bedding
- Diet (ad libitum): free access to Altromin 1324 maintenance diet for rats and mice
- Water (ad libitum): free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: At least 5 days.

Environmental conditions:
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours artificial light / 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Prior to the start of the treatment period a detailed clinical observation outside the home cage was
made. None of the animal showed pathological signs before the first administration.
Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10
females/ group) with regular estorus cycle (4-5 day cycle) were used in the study.
The test item and vehicle were administered at a single dose to the animals by oral gavage. The appli
cation volume for all groups was 5 mL/kg body weight.
Before the first administration all animals to be used for the study were weighed and assigned to the
experimental groups
Each animal was marked with its identification number by individual ear tattoo marking

Vehicle:

water

Details on oral exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females. Then in females, treatment was done during the gestation period and up to post-natal day 12. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the treatment period, formulation samples were prepared and analysed for stability and homogeneity of the test item in the selected vehicle.

Study pre-start stability analysis were included on the samples from high dose and low dose group and the investigation was made for 0 h, 6 h (RT), 10 day (RT), 10 day (2-8 °C) and 10 day -15 to -35 °C (10 samples). Pre-start homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups (6 samples).

The test item was homogenous (after 60 min without stirring), therefore during the study samples were collected for the investigation of homogeneity and only samples were taken for substance concentration in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all dose groups (12 samples).

Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich and until then stored under appropriate conditions based on available stability data. The B-samples were retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion of the final study report.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females.

Then in females, treatment was done during the gestation period and up to post-natal day 12. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

control (C) group

Doses / concentrations 2

Dose / conc.:

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

Remarks:

low dose (LD) group

Doses / concentrations 3

Dose / conc.:

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

Remarks:

medium dose (MD) group

Doses / concentrations 4

Dose / conc.:

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

Remarks:

high dose (HD) group

No. of animals per sex per dose:

10 animals per sex per group

Control animals:

yes, concurrent vehicle

Details on study design:

100 animals (40 males and 60 females) were included in the study.

Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. None of the animal showed pathological signs before the first administration.

Before the first administration all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.1.2
software).

Dose selection rationale: According to the results of a previous dose range finding study the doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).

Body weight and food consumption: The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. All animals were weighed directly before termination. Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

Clinical observations:
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once
daily.Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge),piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Functional observations:
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of lactation of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery of tests. Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Haematology:
Haematological parameters were examined in 5 randomly selected males and females (only lactating females were evaluated) from each group at the end of the treatment prior to or as part of the sacrifice of the animals.
The following haematological parameters were examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono),eosinophils (Eos), basophils (Baso), large unstained cells (Luc).

Blood coagulation:
Coagulation parameters from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. The following coagulation parameters were examined: prothrombin time (PT), activated partial thromboplastin time (aPTT).

Clinical biochemistry:
Parameters of clinical biochemistry from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bile acids (TBA), total bilirubin (TBIL), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K).

Urinalysis:
A urinalysis was performed with samples from 5 randomly selected males and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals. Additionally, urine colour/appearance were recorded. The following parameters were measured: specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leukocytes.

Sacrifice and pathology:

The males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) and females were sacrificed on the respective PND 13 by using anesthesia (ketamine/xylazine). Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal
smear or from the last day of mating period.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ weights at necropsy:
The wet weight of the organs of 5 randomly selected male and female animals (only lactating females were evaluated) from each group was recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead were not recorded. Reproductive organs were
weighed from all animals. Thyroid/parathyroid glands from all adult males and females were preserved. Weight of thyroid/para thyroid glands was measured after fixation.

The following tissues/organs were examined: testes (paired weight), epididymides (paired weight), prostate, seminal vesicles and coagulating glands (complete weight), thyroid/parathyroid glands (from all adult males and females), liver, kidneys (paired weight), heart. Further tissues/organs from the same selected animals were preserved. All animals found dead were subjected to a gross necropsy and the organs preserved for a hist
opathological examination. Thyroid/parathyroid glands from non-selected adult animals were preserved for potential histopathological examination.

Histopathology:
A full histopathology was carried out on the preserved organs and tissues of the selected animals of the control and high dose groups which were sacrificed at the end of the treatment period. Evaluation of thyroid/parathyroid glands from the remaining non-selected adult animals was not deemed necessary as no test item related histopathological findings were observed in thyroid/parathyroid gland of selected animals and there was also no test item related effect observed on T4 hormone level in males sacrificed on PND 13.
A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study. Any gross lesion macroscopically identified was examined microscopically in all animals.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and
clinical biochemistry were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In terminally sacrificed males, predominant clinical signs observed during the treatment period (premating day 1 to mating/post mating day 14) were nasal discharge in one animal of control group (PMD 12-13), severely increased salivation in one animal of HD (PMD 11) and moving the bedding in 2-3 animals of HD group during very few days of premating, mating and postmating period.
In terminally sacrificed females, major clinical signs observed during the treatment period (Premating day 1 to PND 12) were moving the bedding on one day during gestation period in three females of HD group, moderately increased salivation in two females of HD group on one day during gestation period and abnormal breathing on one day in one female of the HD group on gestation day 8.

The clinical signs salivation and moving the bedding on few days were observed immediately after the dose administration and therefore were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance.

During the weekly detailed clinical observation, no relevant differences between the groups were found.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

During the treatment period of this study, one mortality was observed on day of terminal sacrifice (PND 13):
- Female no. 49 (Control) was found dead on post natal day (PND) 13.
No specific clinical signs were observed in this animal before death or during entire study period.
Histopathologically, the cause of death was not evident.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In both males and females, there was no test item treatment related effect observed on body weight and body weight gain in the dose groups during the study period. There were no statistically significant differences observed for body weight and body weight gain between the dose groups and the control group except statistically significantly lower body weight gain in HD males during premating day 1-7 when compared with the controls. As this difference was marginal and within the biological variation, it was not considered to be adverse effect due to treatment with test item.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In correlation to the body weight and body weight gain, the food consumption in both males and females tended to increase with the progress of the study in the control, the LD, the MD and the HD group. No test item related or statistically significant effect on food consumption was observed in males and females during the whole study period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males sacrificed at the end of treatment period, no test item related adverse effects were observed for haematological parameters in treatment groups when compared to the control group.

In females sacrificed at the end of treatment period, no test item related effect or statistically significant effect observed on any of the haematology except statistically lower reticulocytes in LD group when compared with the controls. Due to lack of dose dependency, this effect on reticulocytes in LD group was considered as incidental.

No test item related effect was observed on coagulation parameters in males and females when compared with the respective controls.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males and females sacrificed at the end of treatment period, no test item related or statistically significant effect on any clinical biochemistry parameter in treatment groups was observed when compared with the control except statistically significantly lower TBA group mean value in male HD group. This effect on TBA in male HD group was considered as incidental and toxicologically irrelevant as all individual values were within the historical data range (6.38 - 93.82 µmol/L). All other group mean and most of the individual values for clinical chemistry parameters in male and females were comparable with control group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The urinalysis performed in selected male and female animals sacrificed at the end of treatment period revealed no test item treatment related effect and all urinary parameters were in the normal range of variation. High protein levels were found in the urine of few male and females of all groups including control group. Therefore, this effect on urine parameters was not considered to be test item related.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In males, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. There were no biologically relevant differences observed in body temperature between the groups.

In females, statistically significantly higher not supported rearing count in HD group before initiation of treatment and statistically significantly higher urination count in LD group during last week of treatment was observed. As this type of difference was either marginal, before imitation of treatment or without dose dependency/consistency, it has no toxicological relevance and could be considered as biological variation.

There were no biologically relevant differences in body temperature between the groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In males and females, there were no statistically significant differences in the absolute and relative organ weights of the dose groups when compared to the control group except statistically significantly higher absolute liver weights were observed in HD group females when compared with the controls. In the light of fact that no test item related histopathological findings and effects on liver enzymes were observed, this marginal increase in female liver weight was not considered to be adverse.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Few specific macroscopic changes were recorded for the male and female animals, which based on microscopic examination were not considered to be of test item treatment relevance.
The predominant macroscopic changes observed were brown soft mass observed at the head of the epididymis (male no. 15 of LD group), small right thyroid gland (male no. 12 of LD group), liver- diaphragmal herniation (female no. 79 of HD group), cyst on ovary (Female no. 46 of control group), red mass and red fluid filled uterus and red vaginal discharge (female no. 59 of LD group) and enlarged mandibular lymph nodes (female no. 53 of LD group). Few organs like gastrointestinal tract, lung, kidney and spleen from female no. 49 of control group were autolytic.
Macroscopic findings correlating with histopathological observations were observed in following animals:
- animal no. 15 the brown, soft mass observed at the head of the epididymis histologically correlated with severe diffuse necrosis of the epididymis head
- animal no. 46 the cyst observed at necropsy histologically correlated with an ovarian follicular cyst
The above mentioned findings were deemed incidental. All other observed gross lesions reflected the animal physiology or, in absence of corresponding histopathological findings, were considered of no toxicological relevance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Ingrain Blue 2.2 did not produce morphological or histological evidence of toxicities in the organs and tissues examined in this study.

In conclusion, a histopathological NOEL (No Observed Effect Level) could be established at 25 mg//kg bw/day.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No test item related effect of toxicological relevance or statistical significance was observed in male thyroxine hormone (T4) in the treatment groups when compared to the controls.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: general toxicity

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The NOAEL for the test item in this study is considered to be at least 25 mg/kg bw/day for general toxicity.

Executive summary:

The aim of this study performed according to OECD TG 422 and in compliance to GLP, was to assess the possible adverse effects of Ingrain Blue 2.2 on male and female Wistar rats after repeated dose administration with dose levels of 5, 10, and 25 mg/kg body weight/day.

The test item was administered daily in graduated doses to 4 groups of test animals (each comprising 10 male and 10 female rats), one dose level per group for a treatment of 63 days i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. The doses applied were: control, low dose (5 mg/kg bw/day), medium dose (10 mg/kg bw/day) and high dose (25 mg/kg bw/day).

During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, all animals were sacrificed and observed macroscopically.

In conclusion:

- There was one mortality observed in the study on day of terminal sacrifice (PND 13). Histopathologically cause of the death was not evident. 

-  No adverse effects of test item were found on male and female clinical observations, functional observations, body weight development, food consumption, thyroid hormone analysis in parental males, haematology and coagulation, clinical biochemistry, urinalysis, gross macroscopic findings at necropsy, organ weights and histopathology in all treatment groups. 

The NOAEL of the test item in this study for general toxicity is considered to be at least 25 mg/kg bw/day.