1-amino-4-hydroxy-2-phenoxyanthraquinone

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Combined repeated dose oral toxicity and reproductive/developmental toxicity screening test. No effects on fertility were reported.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

from March 24 to October 15, 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test conducted according to internationally accepted guidelines.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source : Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 9-10 weeks old, females: 9-10 weeks old.
Body weight at the allocation of the animals to the experimental groups (together with study 140271):
males: 253 - 281 g (mean: 268.93 g, ± 20 % = 215.14 – 322.71 g)
females: 171 - 191 g (mean: 180.34 g, ± 20 % = 144.27 – 216.41 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.
The animal study was authorized by local government under file no. 55.2-1-54-2532.2-11-11.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10/hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 131113)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Animals were housed in groups of 2 animals/sex/cage in IVC cages (type III H, polysulphone cages) during the premating period in both males and females and during postmating period in males depending on the mating status. 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 and males were returned to its original cage. Each cage was provided with Altromin saw fibre bedding (lot no. 131113)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period, a detailed clinical observation outside the home cage was made. None of the animals showed any adverse clinical signs before the study initiation. Before the first administration all animals to be used for the study were weighed.
Mean body weight of the group housed animals was used to assign all animals to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively, while ensuring to keep each animal with its initial cage partner if possible. Each animal was marked with its identification number by individual ear tattoo.

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

corn oil

Details on exposure:

Test item was ground to a fine powder with the help of a mortar and pestle. Afterwards it was weighed into a tared plastic vial on a precision balance and suspended in corn oil. The formulation was homogenized using an ultra turrax (homogenizer).
The test item formulations were prepared once every ten days and stored at 2-8 °C. Every day before the dose administration the formulation samples were allowed to reach the room temperature and the homogeneity was ensured by votexing the sample on vortex machine.

The following doses were evaluated:
Control: 0 mg/kg bw/d
Low Dose: 100 mg/kg bw/d
Medium Dose: 300 mg/kg bw/d
High Dose: 1000 mg/kg bw/d

The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. This dose also happens to be a limit dose. A descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received corn oil using the same volume as used for the test dose groups.

Test item formulation and vehicle were administered at a single dose to the animals by oral gavage with disposable polypropylene feeding tubes for rats (78 mm long; 3.0 mm diameter; Instech Laboratories Inc). The application volume for all groups was 5 ml/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the evidence of 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation/lactation) from all groups (16 samples).
Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and 5 (12 samples).
Samples for stability analysis were taken in the first week of the study at 0 hours, 6 hours (at RT) and 10 days after the preparation (stored at 2-8 °C), from high and low dose formulations (6 samples).
All formulation samples were analysed on the day of sample collection and were stored at -20° C. These samples were analysed after the completion of the toxicity study at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study no. 140262.

Duration of treatment / exposure:

The animals were treated with test item formulation or vehicle days per week for up to 54 days, i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Details on study schedule:

Arrival of the test item: 07 January 2014
Study initiation date: 24 March 2014
1st amendment to study plan: 11 April 2014
2nd amendment to study plan: 30 April 2014
Delivery of animals: 01 April 2014
Acclimatisation period: 01 April 2014 – 07 April 2014
Experimental starting date: 07 April 2014
Date of behavioural observation: 07 April 2014- 22 May 2014
Treatment period males: 10 April 2014 – 08 May 2014
Treatment period females: 10 April 2014 – 02 June 2014
Necropsies males: 08 May 2014 and 09 May 2014
Necropsies females: 20 May 2014 – 03 June 2014
Experimental completion date: 03 June 2014
Completion date of delegated phase (histopathology): 25 September 2014
Completion date of delegated phase (formulation analysis): 29 August 2014

No. of animals per sex per dose:

80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Clinical Observations
General clinical observations were made daily and health condition 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 once a week thereafter, detailed clinical observations were made in all animals 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
Many observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and on day 3 of the lactation period in 5 randomly selected females (only lactating females were evaluated) in a standard functional observational battery of tests.
Observations covered: sensory reactivity to different modalities, grip strength and motor activity, 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).

Body Weight and Food Consumption
Body weight was recorded once before the assignment to experimental groups, on the first day of administration and weekly during treatment period 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) as well as day 4 post-partum along with pups.
Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of dose administration. Food consumption was not measured during mating period in males and females and post-mating period in males.

Litter observations:

Duration of gestation was recorded and calculated from day 0 of pregnancy. Each litter was examined as soon as possible after 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 parturition (day 0 post-partum) and on day 4 post-partum. Any abnormal behaviour was recorded.

Postmortem examinations (parental animals):

Pathology
All male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29 or 30, while female animals were sacrificed on post-natal day 4 using an anaesthesia.
All surviving pups were killed by decapitation on post natal day 4.
Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Non-pregnant females (females 42 and 71) were sacrificed on day 26 from day of sperm positive vaginal smear or from the last day of mating period. All animals were subjected to a detailed gross necropsy which includes 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), and all organs showing macroscopic lesions of all adult animals were preserved in 10 % neutral buffered formalin, except for testes and epididymides which were fixed in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Organ Weights
The wet weight of the organs (liver, uterus with cervix, kidneys, thymus, adrenals, thyroid/parathyroid glands, testes, spleen, epididymides, brain, prostate, seminal vesicles and coagulating glands, pituitary gland, ovaries, heart) of 5 sacrificed males and 5 females (only lactating females were evaluated) randomly selected from each group was recorded as soon as possible. Paired organs were weighed together. In addition reproductive organs of all animals were weighed.
The following tissues (brain (cerebrum, cerebellum and pons), heart, spinal cord, ovaries (females), liver, uterus with cervix (females), kidneys, vagina (females), adrenal glands, testes (males), stomach, epididymides (males), small and large intestines (including Peyer´s patches), prostate and seminal vesicles with coagulating glands as a whole (males), thymus, urinary bladder, thyroid, lymph nodes (mesentric and axillary), spleen, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, lung and trachea, bone with bone marrow (sternum), mammary glands, pituitary gland, gross lesions, oesophagus, skin) of the same selected animals from each group were preserved in 10 % neutral buffered formalin except for testes and epididymides that were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol.

Histopathology
A full histopathology was carried out on the preserved organs and tissues (see above) of 5 randomly selected male and female animals (only lactating females were evaluated) of the control and high dose groups which were sacrificed at the end of the treatment period.
All gross lesions from all groups, were examined by light microscopy. Furthermore, testes, epididymides, prostate, seminal vesicles with coagulating glands, ovaries, uterus with cervix and vagina were examined in all animals, and, on the testes, special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Because of possible treatment-related findings noted in the high dose group, spleen and bone marrow (sternum) from 5 selected male and female animals of the low and medium dose groups (groups 2 and 3, respectively) were examined to establish a no-effect level.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin and were examined in all animals.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle for the evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Postmortem examinations (offspring):

Pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each sex by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).

Reproductive indices:

There were no effect on copulation, fertility and delivery indices of dose groups. Fertility indices (number of pregnant females/number of copulated females × 100) observed in the control and the dose groups were C 90 %, LD 100 %, MD 100 % and HD 100 %.

Offspring viability indices:

There was no effect on viability index of dose groups. The slightly lower viability index in the LD group was considerd as incidental due to the lack of dose related response.

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicological relevance. However, red feces in HD group, due to test item colour, were noted. Other clinical signs (e.g. alopecia, slight abnormal breathing, nasal discharge, salivation and regurgitation) in a few animals of control and/ or dose groups, were considered incidental.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No statistically significant differences.

Food efficiency:

not specified

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):

In MD and HD groups. In males, there was statistically significantly lower mean RBC, HCT and Hb in MD and HD groups; statistically significantly higher mean MCV and reticulocyte count in MD and HD group; statistically significantly higher mean MCH in HD group. In females, there was statistically significantly lower mean RBC, Hb, HCT value in HD group and statistically significantly higher mean MCV, MCH and reticulocyte count in HD group.
Statistically significantly lower MCHC value in male MD group, higher eosinophil and lower LUC count in female MD group were no dose- or tretment-related. In males and females, no effect on the blood coagulation parameters.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, the treatment-related findings were recorded in the spleen and bone marrow. The increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen was recorded in both sexes of MD and HD groups, along with an increase in incidence and/or severity of hemosiderin deposition in the spleen and erythropoiesis in the bone marrow. These findings are most likely to be the histomorphologic indicator of hemolytic anemia and were considered to be adverse event attributable to treatment with the test item.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Description (incidence and severity):

Except for confirmation of mating.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Detailed examination of testes revealed no abnormalities on the completeness of stages and cell populations, except for male no. 31 (HD group). In testis of male no. 31, it was impossible to evaluate the stages and cell population because of complete tubular atrophy, considered a spontaneous change.
No dose response relationship in any findings recorded in the male reproductive organs including testes, epididymides, prostate glands, coagulating glands and seminal vesicles which were examined in this study, and therefore, all histologic findings were considered to be spontaneous changes which may be recorded in animals of this strain and age.

Reproductive performance:

no effects observed

Description (incidence and severity):

Precoital Interval and Duration of Gestation:
No effect in dose group animals when compared to the controls.

Pre- and Post-Natal Data:
No effect on the pre and post natal data including the no. of corpora lutea, no. of implantation sites, no. of live pups (PND 0 and 4), percent pre and post implantation loss.

Reproductive Indices:
No effect on the copulation, fertility and delivery indices of dose groups when compared to controls. A fertility index (number of pregnant females/ number of copulated females X 100) observed in the control and the dose groups were C 90 %, LD 100 %, MD 100 % and HD 100 %.
No effect on viability index of dose groups when compared to control. However, there was slightly lower viability index in the LD group, but in the absence of dose related response, the finding was considered incidental.

Sperm stages of testes were checked on completeness of cell populations, completeness of stages and degenerative changes. Nothing particular was observed in testes of animal no. 2 (control), which was mated with female no. 42 not giving birth. Slight spermatic granulomas and minimal mononuclear cell foci in epididymides and minimal alveolitis in lungs were recorded in male no. 2. However, similar minimum to slight lesions are often observed in male animals that can produce pregnancy to the pairing females, and therefore, it is unlikely that these findings were related to infertility.
In male no. 31 (HD group), marked tubular atrophy (ie. so-called sertoli cell-only tubules) in testes and marked oligospermia (ie. azoospermia) in epididymides were observed, causing the paired female no. 71 not to give birth. Complete tubular atrophy sometimes happens spontaneously in the male rats of this strain and age, and sporadic occurrence with only one case is unlikely to be related to treatment with the test item.
Nothing particular was observed in female reproductive organs (ovaries, uterus with cervix and vagina) of the paired female nos. 42 (control) and 71 (HD group) which did not give birth.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no effects of toxicological relevance on litter weight data including pup mean weight, total litter weight, male litter weight and female litter weight measured on PND 0 and PND 4.

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:

no effects observed

Description (incidence and severity):

There were no gross external findings of toxicological relevance in any of the dose groups when compared to controls.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Litter Data:
No effect of toxicological relevance on litter including no. of pups born, no. of live pups, no. of still birth and no. of runts on PND 0 and no. of male and female pups and sex ratio on PND 0 and PND 4.

Pre- and Post-Natal Data
There were no effect on the pre and post natal data including the no. of corpora lutea, no. of implantation sites, no. of live pups (PND 0 and 4), percent pre and post implantation loss.

Pup Survival Data:
There were no effect on the survival of the pups from PND 0 to PND 4 was observed in any dose group when compared to controls. One pup (pup no. 4) from female no. 59 of LD group was missing on PND , likely cannibalised by dam. This finding was considered incidental.

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:

viability

mortality

body weight and weight gain

gross pathology

Reproductive effects observed:

not specified

Conclusions:

Based on results in male and female Wistar rats exposed to dose levels of 100, 300, and 1000 mg/kg bw/d of test substance for at least 28 days, no observed adverse effect level (NOAEL) is 100 mg/kg bw/d for systemic toxicity in males and females and 1000 mg/kg bw/d for reproduction/developmental toxicity.

Executive summary:

Method

Combined repeated dose toxicity and reproductive/developmental toxicity study in male and female Wistar rats.

Test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period up to 54 days, i.e. 14 days pre-mating and maximum 14 days of mating in both males and females, and during the gestation period up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 -29 days. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study. Each group comprised 10 male and 10 female Wistar rats, dosed as follows:

Control (C):                  0        mg/kg/d

Low Dose (LD):            100     mg/kg/d

Medium Dose (MD):     300     mg/kg/d

High Dose (HD):           1000   mg/kg/d

Test item formulation was prepared once in every ten days and stored at 2-8°C. Test item was suspended in corn oil and dose volumes were adjusted individually based on weekly body weight measurements administered as 5 ml/kg bw/d.

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

Changes in body weight, food consumption, haematological and clinical biochemistry, urinalysis, functional signs including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were recorded during the study.

After 14 days of treatment, animals were mated (1:1) for a maximum of 14 days. Vaginal smears of females were checked daily to see evidence of mating. After confirmation of mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.

Males were sacrificed on treatment days 29 and 30 and females along with their pups were sacrificed on post natal day 4. Non-pregnant females (females 42 and 71) were sacrificed on day 26 from day of sperm positive vaginal smear or from the last day of mating period.

Pups sacrificed on postnatal day 4 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups as well as all gross lesions from all groups, were examined by light microscopy. Furthermore, testes, epididymides, prostate, seminal vesicles with coagulating glands, ovaries, uterus with cervix and vagina were examined in all animals, and, on the testes, special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Because of possible treatment-related findings noted in the high dose group, spleen and bone marrow (sternum) from 5 selected male and female animals of the low and medium dose groups were examined to establish a no-effect level.

Results

No mortality occurred in the control or any dose group during study period.

No clinical signs of toxicological relevance in dose groups when compared to control.

During the weekly detailed clinical observation, no significant changes or differences between the groups were found. No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.

In males and female, no adverse changes of toxicological relevance on body weight, body weight gain and food consumption in dose groups when compared to the concurrent control. 

In males and females, there were findings of toxicological relevance on haematological parameters in MD and HD groups. In males, there was statistically significantly lower mean RBC, HCT and Hb in MD and HD groups; statistically significantly higher mean MCV and reticulocyte count in MD and HD group; statistically significantly higher mean MCH in HD group. In females, there was statistically significantly lower mean RBC, Hb, HCT value in HD group and statistically significantly higher mean MCV, MCH and reticulocyte count in HD group.

In males and females, there was no effect on the blood coagulation, clinical biochemistry and urinary parameters. Enlarged spleen was recorded in 3/10 males of MD group, in 4/10 males and one female of HD group, was considered to be treatment-related change.

All other gross lesions recorded were within the range of normal background alterations which may be recorded in animals of this strain and age.

In males and females, there was statistically significantly higher absolute and relative (to terminal body weight) spleen weight in MD and HD groups, considered to be adverse events attributable to treatment with the test item.

Micoscopically, treatment-related findings in MD and HD groups were recorded in the spleen and bone marrow. Increase in mean severity of erythrocytic extramedullary hemopoiesis in the spleen along with an increase in incidence and/or severity of hemosiderin deposition in the spleen and erythropoiesis in the bone marrow were seen. These findings are histomorphologic indicator of hemolytic anemia and are attributed to treatment with the test item.

In all dose groups, no effects on duration of precoital interval, duration of gestation, pre and post natal data including the no. of corpora lutea, no. of implantation sites, no. of live pups (PND 0 and 4), percent pre and post implantation loss, copulation, fertility, delivery and viability indices were noted.

Fertility indices (number of pregnant females/ number of copulated females X 100) observed in the control and the dose groups were C 90 %, LD 100 %, MD 100% and HD 100%.

There were no effects of toxicological relevance on litter in terms of: no. of pups born, no. of live pups, no. of still birth and no. of runts on PND 0, no. of male and female pups and sex ratio on PND 0 and PND 4.

Moreover, no effects were seen on pup mean weight, total litter weight, male litter weight and female litter weight measured on PND 0 and PND 4, survival of the pups from PND 0 to PND 4 and gross external findings.

Conclusion

NOAEL of 1000 mg/kg/d was identified for reproduction/developmental toxicity.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

As no data on toxicity to reproduction of Disperse Red 060 was available, a read across approach was followed. Details on this process are reported in section 13.

A reproduction/developmental toxicity screening test was performed in male and female Wistar rats exposed to Similar Substance 02 at oral doses of 100, 300, and 1000 mg/kg bw/d. Haemolytic anaemia was observed in both sexes at 300 and 1000 mg/kg/d, microscopically seen as extramedullary haematopoiesis in bone marrow and spleen. This also correlated with dose-dependently lower levels of red blood cell parameters (RBC, Hb and Hct) and a concurrent increase in reticulocytes and mean cell haemoglobin and mean cell volume.

There were no effect on the reproduction/ developmental parameters. Accordingly, the no observed adverse effect level (NOAEL) is 100 mg/kg bw/d for systemic toxicity in males and females and 1000 mg/kg bw/d for reproductive/developmental toxicity.

Effects on developmental toxicity

Description of key information

Combined repeated dose oral toxicity and reproductive/developmental toxicity screening test. No effects on developmental toxicity were reported. 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A reproduction/developmental toxicity screening test was performed in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg/d. Haemolytic anaemia was observed in both sexes at 300 and 1000 mg/kg/d, microscopically seen as extramedullary haematopoiesis in bone marrow and spleen. This also correlated with dose-dependently lower levels of red blood cell parameters (RBC, Hb and Hct) and a concurrent increase in reticulocytes and mean cell haemoglobin and mean cell volume.

There were no effect on the reproduction/ developmental parameters. Accordingly, the no observed adverse effect level (NOAEL) is 100 mg/kg bw/d for systemic toxicity in males and females and 1000 mg/kg bw/d for reproductive/developmental toxicity.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), reproductive toxicity includes adverse effects on sexual function and fertility in adult males and females, adverse effects on development of the offspring and adverse effects on or via lactation.

Substances are classified in Category 1 for reproductive toxicity when they are known to have produced an adverse effect on sexual function and fertility, or on development in humans or when there is evidence from animal studies, possibly supplemented with other information, to provide a strong presumption that the substance has the capacity to interfere with reproduction in humans.

Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1.

Test substance did not show any evidence of reproductive toxicity in a combined repeated dose toxicity and reproductive/developmental toxicity (OECD 422) covering fertility, development and post-partum up to day 4. Accordingly, a NOAEL was established at the high dose of 1000 mg/kg bw/d.

On these bases, the substance is considered as non toxic for reproduction.