4,10-dibromodibenzo[def,mno]chrysene-6,12-dione

Endpoint summary

Description of key information

Repeated oral toxicity:
An OECD test guideline (OECD 422) and GLP-compliant Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed with the test item (Pigment Red 168). Groups of 11 male and 11 female Wistar rats received doses of 0, 100, 300 and 1000 mg/kg bw  by daily gavage for 28 days (males) and 49 days (females). No toxicologically significant changes were noted in any of the parameters investigated in this study (such as clinical signs, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). A No Observed Adverse Effect Level (NOAEL) for the test item of 1000 mg/kg/day was established.
Repeated dermal toxicity:
The dermal route was waived.  The substance is considered not to exert adverse effects.
Repeated inhalation toxicity:
The inhalation route was waived. The substance is considered not to exert adverse effects.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or no or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Reason / purpose for cross-reference:

reference to same 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)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

Animals: Rat, RccHanTM: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories, B.V.,Kreuzelweg 53, 5961 NM Horst / Netherlands
Number of Animals: 46 males and 46 females, 11 males and 11 females per dose group, 2 males and 2 females reserve animals
Age (at delivery): 10 weeks
Body Weight Range (at Start of Treatment): 292 to 348 g, mean 323 g (males), 186 to 227 g, mean 207 g (females)
Identification: Parent animals by cage card and individual animal number (ear tattoo), pups were individually tattooed with Indian ink on day 1 post partum.
Randomization: Randomly allocated to groups by body weight.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There was 12-hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, (batch/lot nos. 02105111001, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland), with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey, UK, batch/lot no. 55). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
Diet: Pelleted standard Harlan Teklad 2914C (batch no. 73/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. Results of representative analyses for contaminants were included in the report.
Water: Community tap-water from Itingen was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of representative samples were included in the report.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

5% solution in water

Details on oral exposure:

DOSE FORMULATIONS

The dose formulations were prepared weekly using the test item as supplied by the Sponsor. No correction for purity or salt/base correction was applied. The test substance was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). The mixtures were stirred using a magnetic stirrer and stored at room temperature (15 - 25 °C).
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS

Dose formulations were stored at room temperature (20 ± 5 °C) in glass beakers, protected from light.
Based upon the results of stability analyses performed within the non-GLP Harlan Laboratories study, dose formulations were stable for at least one week when stored in these conditions.

TREATMENT

Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type studies.
Frequency of Administration: Once daily
Target Dose Levels: Group 1: 0 mg/kg/day (control group), Group 2: 100 mg/kg/day, Group 3: 300 mg/kg/day, Group 4: 1000 mg/kg/day
Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Wistar rats.
Dose Volume: 10 mL/kg body weight
Duration of Acclimatization Period: Minimum 5 days
Duration of Treatment Period: Males: Minimum 4 weeks, Females: Approximately 7 weeks

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (8 days). These samples were stored frozen until analysis.
On the day of the second dose formulations preparation, samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing, kept at room temperature until analysis.
The samples were analyzed GC-FID method. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

The linearity of the analytical systems used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. All calibration points used met the acceptance limit of ±20% variation from the calibration curve derived by linear regression analysis. The calculated regression coefficients (R2) exceeded 0.99.
Blank samples showed no significant absorbance and, therefore, it was confirmed that only 0.5% aqueous solution of carboxymethylcellulose was applied within the control experiment. The application formulations investigated during the study were found to comprise the test item in the range of 88.7% to 115.7%, and met the required content limit of ±20% with reference to the nominal content.
Single results of the test item did not deviate more than 10.0% (acceptance criterion: <15%) from the corresponding mean, and were considered to be homogeneously prepared.
The test item was found to be stable in application formulations of group 2 (10 mg/mL) and group 3 (30 mg/mL), when kept four hours or eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.
However, the result of stability in groups 4 (100 mg/mL) exceeded the acceptance criteria, the maximum deviation of time-zero mean was found to be 28.1% (and 25.5% of repeated analysis) for 4 hours stability and 77.8% for eight day stability.

Duration of treatment / exposure:

MALES

Minimum 4 weeks

FEMALES

Approximately 7 weeks

Frequency of treatment:

Once daily.

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

MALES

Acclimatization: 8 days
First Test Item Administration: Day 1 of pre-pairing
Pre-Pairing: 14 days
Pairing: 14 days
Gestation: Approximately 21 days
Treatment Ends: On day before sacrifice
Necropsy: After treatment for at least 28 days, when no longer needed for assessment of reproductive effects

FEMALES

Acclimatization: 8 days
First Test Item Administration: Day 1 of pre-pairing
Pre-Pairing: 14 days
Pairing: 14 days maximum
Gestation: Approximately 21 days
Treatment Ends: On day 4 post partum
Necropsy: On day 5 post partum (pups on day 4 post partum)

Positive control:

Not required

Observations and examinations performed and frequency:

VIABILITY/MORTALITY

Twice daily

CLINICAL SIGNS

Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION

Males: Weekly during pre-pairing and after pairing periods.
Females: Pre-pairing period days 1 - 8 and 8 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and lactation days 1 - 4.
No food consumption was recorded during the pairing period.

BODY WEIGHTS

Recorded daily from treatment start to day of necropsy.

DETAILED CLINICAL OBSERVATIONS

Detailed clinical observations were performed outside the home cage in all animals. In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, it was performed once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.
Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

FUNCTIONAL OBSERVATIONAL BATTERY

At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were performed with five P generation males and five P generation females from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:
- cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
- Hand-held observations: muscle tone, constituation, skin, pupile size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.
- Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
- Reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
- Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.
Any abnormal findings were recorded and, where appropriate, graded in severity.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

CLINICAL LABORATORY INVESTIGATIONS

Blood samples were obtained on the day before or on the day of the scheduled necropsy from five males of each group. Blood samples from five lactating females of each group were obtained at the end of the pre-pairing period. Blood samples were drawn from the retro-orbital plexus of all animals under light isoflurane anesthesia.. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

The following hematology parameters were determined:
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Leukocyte count, total
- Differential leukocyte count:
- Platelet count
- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:
- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Aspartate aminotransferase
- Alanine aminotransferase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Bile acids
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio

Sacrifice and pathology:

TERMINATION AND NECROPSY

Males were sacrificed after treatment of at least 28 days, when no longer needed for the assessment of reproductive effects. Dams were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

All animals sacrificed were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred. For the parent animals, special attention was directed at the organs of the reproductive system.The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS

At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately. In addition, from 5 males and females sacrificed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.
- Adrenal glands (weighed as pairs)
- Brain
- Heart
- Kidneys (weighed as pairs)
- Liver
- Thymus
- Spleen

TISSUE PRESERVATION

The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Prostate
- Seminal vesicles with coagulating gland
- Testes (in Bouin’s fixative)
- Epididymides (in Bouin’s fixative)
The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Ovaries
In addition, from the five males and females per group selected for organ weights and from all animals found dead or killed in extremis, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Gross lesions
- Brain (representative regions including cerebrum, cerebellum and pons)
- Spinal chord
- Small and large intestines (incl. Peyer’s patches)
- Stomach
- Liver
- Kidneys
- Adrenals
- Spleen
- Heart
- Thymus
- Thyroids, and parathyroids if possible
- Trachea and lungs (preserved by inflation with fixative and then immersion)
- Uterus (with vagina)
- Urinary bladder
- Lymph nodes (mesenterial, mandibular)
- Peripheral nerve (sciatic)
- Bone marrow

HISTOTECHNIQUE

All organ and tissue samples to be examined by the principal investigator for histopathology phase were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the principal investigator.

HISTOPATHOLOGY

Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.
A histopathology peer review was performed. A histopathology phase report was provided by the principal investigator and included in the report.

Other examinations:

MATING, GESTATION, LACTATION

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if: the daily vaginal smear was sperm positive or a copulation plug was observed. The day of mating was designated day 0 post coitum. If a female did not mate during the 14-day pairing period, this female was paired with a male of the same group which had already mated successfully. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility. All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

REPRODUCTIVE AND OFFSPRING VIABILITY INDICES

From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices.

LITTER OBSERVATIONS

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

POSTMORTEM EXAMINATION OF OFFSPRING

Pups were sacrificed on day 4 post partum. All animals were sacrificedby by an injection of sodium pentobarbital and subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. Pups dead during the study, except those excessively cannibalized, were examined macroscopically.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Clinical signs:

no effects observed

Description (incidence and severity):

All test item-treated animals had reddish discoloration of the feces which resulted from administration of a dyestuff and had no toxicological relevance.

Mortality:

no mortality observed

Description (incidence):

All test item-treated animals had reddish discoloration of the feces which resulted from administration of a dyestuff and had no toxicological relevance.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

1. IN-LIVE DATA OF PARENTAL ANIMALS

VIABILITY / MORTALITY

In males and females, there were no unplanned deaths during the acclimatization, pre-pairing or pairing periods. All females survived the gestation and lactation periods.

DAILY CLINICAL SIGNS OR OBSERVATIONS

No test item-related clinical signs of systemic toxicity were noted in males or females at any dose level.

All male rats which received the test item had reddish discolored feces. Seen during the pre-pairing and pairing periods, the onset and severity of this finding was dose-related and began within the first few days of administration. All test item-treated females also showed similar reddish discoloration of the feces during the pre-pairing, pairing, gestation and lactation periods. This finding is considered to be a typical passive effect resulting from oral administration of a dyestuff and is without toxicological relevance.

Additional, isolated findings noted in a small number of test item-treated females included kinked tail apex, transient ruffled fur or dystocia. The low incidence of these findings was considered to indicate that these were background changes without systemic toxicity.

No further clinical signs or observations were noted in males or females at any dose level.

FINDINGS AT DETAILED WEEKLY CLINICAL OBSERVATIONS

No findings were noted at any phase during detailed weekly clinical observations in males or females at any dose level.

FUNCTIONAL OBSERVATIONAL BATTERY

The mean fore-and hind limb grip strength values, the landing foot splay and body temperature of the control rats were similar to those of the test item-treated groups.

No findings were noted during the functional observational battery in males or females at any dose level.

LOCOMOTOR ACTIVITY

Locomotor activity was not affected by the treatment with the test item in males or females at any dose level.

FOOD CONSUMPTION OF MALES

No effects on food consumption of males were observed at any dose level.

At the dose levels of 100, 300 and 1000 mg/kg bw/day, differences in mean food consumption during the pre-pairing period were respectively: -0.4%, -6.3% and ±0.0% (percentages refer to the respective values in the control group).

FOOD CONSUMPTION OF FEMALES

No effects on food consumption of females were observed at any dose level.

The overall differences in mean food consumption at the dose levels of 100, 300 and 1000 mg/kg bw/day were -1.1%, +1.7% and +1.1%, respectively, during the pre-pairing period, +3.4%, +1.7% and -0.4%, respectively, during the gestation period and +17.2%, +13.0% and +11.9%, respectively, during the lactation period (percentages refer to the respective values in the control group).

BODY WEIGHTS OF MALES

The mean body weights and body weight gain of the test item-treated males were similar to those of the control males.

BODY WEIGHTS OF FEMALES

No effects on body weights and body weight gain of test item-treated females were observed at any dose level.

2. CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

HEMATOLOGY

No test item-related effects on hematology parameters were noted in males at any dose level.

In females at all dose levels, reductions of the absolute leukocyte population were noted. Although the differences attained statistical significance (p<0.05 or p<0.01) and all exceeded the lower limit of the historical control data, there was no dose-response relationship.

These differences were due to the respective absolute lymphocyte population fractions, which were reduced significantly (p<0.01) at all dose levels, but without any dose-response relationship. Only the difference noted at 300 mg/kg/day exceeded the lower limits of the historical control data. In contrast, the relative lymphocyte counts were not significantly different from those of the control females, remained within the limits of the historical control data and were not dose-dependent.

In the absence of similar changes in the males, these isolated differences are considered to be without toxicological relevance.

CLINICAL BIOCHEMISTRY

No test item-related effects on clinical biochemistry parameters were noted in males at any dose level.

At 1000 mg/kg/day, the mean creatinine levels of males were significantly higher (p<0.05) than those of the control males. The mean chloride level of these males was significantly elevated (p<0.01) when compared with the control males. Both differences, however, remained within the respective ranges of the historical control data. The values of both parameters recorded for the control males were, in comparison with the respective mean values of the historical control data, slightly low and this was considered to result in the statistically significant difference.

At 300 m/kg/day, the clinical biochemistry parameters were similar to the control males.

At 100 mg/kg/day, the mean albumin level was significantly reduced (p<0.05) when compared with the control males. There was no dose-response relationship and the value remained within the range of the historical control data. No toxicological relevance was associated with this finding.

No test item-related effects on clinical biochemistry parameters were noted in females at any dose level.

At 1000 and 300 mg/kg/day, the mean glucose levels were significantly elevated when compared with the control females (which were considered to be slightly low; therefore the differences seen in the treated groups were considered to be inconsequential). These differences were within the ranges of the historical control data. All other parameters compared favorably with those of the controls.

3. TERMINAL FINDINGS

ORGAN WEIGHTS

There were no test item-related differences in the mean absolute or relative organ weights in males.

At 1000 mg/kg/day, no differences in mean absolute or relative organ weights were noted when compared with the controls.

At 300 mg/kg/day, significantly lower mean absolute heart weights (p<0.01) and significantly higher mean absolute kidney weights (p<0.05) were noted in males when compared with the control males. The mean relative heart weights of these males were significantly lower (p<0.05) when compared with the control males. Insofar as no differences were seen in the high dose, these findings were considered to be incidental.

At 100 mg/kg/day, no differences in mean absolute or relative organ weights were noted when compared with the controls.

In females at 1000, 300 and 100 mg/kg/day, no differences in mean absolute or relative organ weights were noted when compared with the controls.

MACROSCOPICAL FINDINGS

There were no gross lesions that could be attributed to treatment with the test item. All gross lesions recorded were considered to be within the range of normal background alterations.

HISTOPATHOLOGY FINDINGS

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

Testes - Detailed Examination Including Sperm Staging: the stages were checked on completeness of cell populations, completeness of stages and degenerative changes. The histopathologic evaluation of the testes did not reveal any test-item related changes in males treated with 1000 mg/kg/day.

Unilateral tubular degeneration/atrophy was recorded at minimal severity in animal nos. 6 and 7 of the control group, in no. 24 at 100 mg/kg/day and in no. 40 at 1000 mg/kg/day. Among them, spermatic retention was observed in animal no. 24. In this animal (no. 24), there were completeness of stages and cell populations, and spermatic retention was not observed in animals treated with 1000 mg/kg/day. Therefore, spermatic retention recorded in this animal was considered not to be related to the treatment with the test item.

No specific morphological changes were noted that were related to the infertility observed in the male reproductive organs of animal no. 30.

4. REPRODUCTION, BREEDING AND PUP DATA
 
MATING PERFORMANCE AND FERTILITY
 
No effect on mating performance or fertility was observed at any dose level.
 
At 1000 mg/kg/day, the mean precoital time was marginally (but not significantly) longer when compared to the controls. The precoital times at 100 mg/kg/day and 300 mg/kg/day compared favorably with those of the control group.
 
One female (no. 86) at 1000 mg/kg/day and one female (no. 72) at 300 mg/kg/day mated during a second pairing period. The former female mated after three days; the latter after 11 additional days. All other females mated during the first pairing period.
 
Three females were found to be not pregnant: female nos. 49 and 50 of the control group and female no 58 treated with 100 mg/kg/day. Evidence of mating (sperm plug or positive smear) was not found in females 49 and 50, whereas both were observed in female no 58. The two control females were found to have gained weight after the pairing period and were therefore assumed to be pregnant.
 
The fertility index and conception rate (number of females achieving pregnancy as a percentage of females paired and mated, respectively) were identical. The values recorded for females treated with 300 mg/kg/day and 1000 mg/kg/day exceeded those of the control females as well as those treated with 100 mg/kg/day.
 
All pregnant females of the control group and all females treated with 1000 mg/kg/day gave birth to living pups, and the gestation indices (number of females with living pups as a percentage of females pregnant) were 100%. The gestation indices of the females treated with 100 mg/kg/day and 300 mg/kg/day were 90.0% and 90.9%, respectively.
 
CORPORA LUTEA COUNT
 
No effects on corpora lutea count were observed at any dose level.
 
Comparison of the mean number of corpora lutea noted in test item-treated dams did not show any toxicologically relevant differences when compared with those of the control females. Females treated with 100 mg/kg/day had more corpora lutea than the controls, but this difference was not seen at higher doses and therefore considered to be incidental.
 
DURATION OF GESTATION
 
No effects on duration of gestation were observed at any dose level.
 
The respective durations of gestation were 21.3, 21.8 and 22.0 days for females treated with 100, 300 and 1000 mg/kg/day, which compared favorably with 21.6 days noted for the control females.
 
IMPLANTATION RATE AND POST-IMPLANTATION LOSS
 
No effects on implantation rate or post-implantation loss were observed at any dose level.
 
The mean number of implantations per dam was generally similar in control and test item-treated groups with slightly elevated values noted in females treated with 100 mg/kg/day.
 
The post-implantation loss of control females and females treated with 1000 mg/kg/day were generally similar, but markedly higher in females treated with 100 or 300 mg/kg/day. In the absence of a dose response relationship, these differences were considered to be incidental.
 
LITTER SIZE AT FIST LITTER CHECK
 
No effects on litter size were observed at any dose level.
 
The mean number of living pups at first litter check noted in the control groups was generally similar to those recorded at 100, 300 and 1000 mg/kg/day. One, two and three pups at the dose levels of 100, 300 and 1000 mg/kg bw/day, respectively, were found dead at first litter check.
 
The mean birth index (defined as the number of live pups at first litter check expressed as a percentage of the number of implantations) recorded at 1000 mg/kg/day compared favorably to that of the control group.
 
POSTNATAL LOSS DAYS 0 - 4 POST PARTUM
 
No effects on postnatal loss were observed at any dose level. At 100 mg/kg/day, litter no 65 had five of the six total male losses and one of the two total female losses. This single high incidence noted in this litter was considered to be incidental.
 
EXTERNAL EXAMINATION OF PUPS AT FIRST LITTER CHECK
 
No test item-related findings were noted in pups at any dose level.
 
One female pup (litter no. 65 at 100 mg/kg/day) was partially cannibalized. One female pup (litter no 73 at 300 mg/kg/day) had no milk in the stomach during external examination at first litter check.
 
No further findings were noted at first litter check or during the first 4 days post partum in pups at any dose level.
 
SEX RATIO
 
The sex ratio of the pups was not affected by exposure to the test item at any dose level.
 
BODY WEIGHTS TO DAY 4 POST PARTUM
 
No test item-related effects on the body weights of the pups were noted at any dose level.
 
MACROSCOPICAL FINDINGS
 
There were no macroscopical findings evident in any pup.

Dose descriptor:

NOAEL

Remarks:

and NOEL (for general toxicity)

Effect level:

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

Sex:

male/female

Basis for effect level:

other: No test item-related effects were observed in males or females up to and including 1000 mg/kg bw/day, the highest dose level administered.

Dose descriptor:

NOAEL

Remarks:

and NOEL (for reproduction and development)

Effect level:

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

Sex:

male/female

Basis for effect level:

other: No test item-related effects on reproduction or development were observed up to and including 1000 mg/kg bw/day, the highest dose level administered.

Critical effects observed:

not specified

SUMMARY OF PERFORMANCE 

P Animals Breeding for F1 Litters 

Group	1	2	3	4
(mg/kg/day)	0	100	300	1000
Female numbers	45-55	56-66	67-77	78-88
Number of females paired	11	11	11	11
Number of females mated	9	11	11	11
Number of females not pregnant (A)	2	1	0	0
Number of females with implantation sites only (B)	0	1	0	0
Number of females which reared their pups until day 4 post partum	9	9	11	11

(A): Female nos. 49, 50 and 58 were not pregnant.

(B): Female no. 64 had only implantation sites.

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats over approximately 28 days. The test item was administered in 0.5% aqueous solution of Carboxymethycellulose as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test item was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

All animals survived the scheduled study period. No test item-related clinical signs or findings during functional observational battery or locomotor activity were noted in males or females at any dose level. Food consumption, body weights and body weight gain were not affected in males or females up to and including the dose level of 1000 mg/kg bw/day. The investigated parameters of hematology and clinical biochemistry did not show changes commensurate with typical toxicological relevance. The mean absolute and relative organ weights of control and test item-treated rats compared favorably. There were neither macroscopical nor microscopical changes that could be related to the treatment with the test item. The evaluation of sperm staging (completeness of cell populations, completeness of stages and degenerative changes) revealed no remarkable changes. The pathomorphological evaluation of the testes likewise showed no changes of toxicological relevance.

No effects on mating performance, fertility, corpora lutea count, duration of gestation, implantation rate and post-implantation loss as well as litter size and post-natal loss were noted at any dose level. No test item-related findings in pups or effects on pups body weights were noted at any dose level.

The NOAEL (No Observed Adverse Effect Level) and NOEL (No Observed Effect Level) for general toxicity in males and females was considered to be 1000 mg/kg bw/day. The NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 1000 mg/kg/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the test item on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

The test item was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

The following dose levels were applied:

 

Group 1:                        0 mg/kg body weight/day (control group)

Group 2:                    100 mg/kg body weight/day

Group 3:                    300 mg/kg body weight/day

Group 4:                   1000 mg/kg body weight/day

 

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (0.4% aqueous solution of carboxymethylcellulose).

 

The following results were obtained:

 

MORTALITY AND GENERAL TOLERABILITY OF PARENT ANIMALS

 

All animals survived the scheduled study period. No test item-related clinical signs were noted in males or females at any dose level.

 

FUNCTIONAL OBSERVATIONALBATTERYAND LOCOMOTOR ACTIVITY OF PARENT ANIMALS

 

No test item-related effects were noted during functional observational battery or locomotor activity in males or females at any dose level.

 

FOOD CONSUMPTION OFPARENT ANIMALS

 

No effects on food consumption were noted in males or females at any dose level.

 

BODY WEIGHTS OF PARENT ANIMALS

 

No effects on body weights or body weight gain were noted in males or females at any dose level.

 

CLINICAL LABORATORY INVESTIGATIONS IN PARENT ANIMALS

 

No effects on hematology or clinical biochemistry parameters, which were considered to be test item-related, were noted in males or females at any dose level.

 

No effects on mating performance, fertility, corpora lutea count, duration of gestation, implantation rate and post-implantation loss as well as litter size and post-natal loss were noted at any dose level.

 

ORGAN WEIGHTS OF PARENT ANIMALS

 

There were no test item-related differences in the mean absolute or relative organ weights.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENT ANIMALS

 

There were no gross lesions noted in males or females that could be attributed to treatment with the test item. All gross lesions recorded were considered to be within the range of normal background alterations.

 

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

 

The sperm stages were checked on completeness of cell populations, completeness of stages and degenerative changes.The histopathologic evaluation of the testes did not reveal any test-item related changes in males treated with 1000 mg/kg/day.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK UND DURING LACTATION

 

No test item-related findings were noted in pups at any dose level.

 

The sex ratio of the pups was unaffected by exposure to the test item.

 

PUP WEIGHTS TO DAY POST PARTUM

 

No test item-related effects on body weights of pups were noted at any dose level.

 

MACROSCOPICAL FINDINGS IN PUPS

 

No findings considered to be test item-related were found in pups at any dose level.

 

CONCLUSION

 

The NOAEL (No Observed Adverse Effect Level) and NOEL (No Observed Effect Level) for general toxicity in males and females was considered to be 1000 mg/kg bw/day. The NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Quality of whole database:

1 (reliable without restriction)
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or no or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only one study available

Justification for classification or non-classification

Pigment Red 168 does not have to be classified regarding systemic and target organ toxicity after repeated exposure according to the criteria laid down in Directive 67/548/EEC and in Regulation (EC) No 1272/2008, because:

- Pigment Red 168 caused no relevant systemic effects and revealed a NOAEL of 1000 mg/kg/day in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test after oral application.

 

It can reasonably be deduced that Pigment Red 168 does not exert systemic toxic effects after repeated dermal application and thus does not have to be classified according to the criteria laid down in Directive 67/548/EEC and in Regulation (EC) No 1272/2008 and that testing is not scientifically necessary, because:

- Pigment Red 168 caused no relevant systemic effects and revealed a NOAEL of 1000 mg/kg/day in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test,

- it is unlikely that Pigment Red 168 becomes systemically bioavailable to a relevant extend after dermal exposure due to its extremely low solubility in water and n-octanol,

- the repeated dose toxicity after dermal application is not considered to be higher than after oral administration,

- testing for acute dermal toxicity revealed no signs of bioavailability or toxicity.

- Pigment Red 168 does not have to be classified as skin sensitizing or as skin or eye irritating, indicating that its chemical inertness and extremely low solubility in water and n-octanol largely prevent interaction with living cells and tissues.

 

It can reasonably be deduced that Pigment Red 168 does not exert systemic toxic effects after repeated inhalation exposure and thus does not have to be classified according to the criteria laid down in Directive 67/548/EEC and in Regulation (EC) No 1272/2008 and that testing is not scientifically necessary, because:

- Pigment Red 168 caused no relevant systemic effects and revealed a NOAEL of 1000 mg/kg/day in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

- it is unlikely that Pigment Red 168 becomes systemically bioavailable to a relevant extend after inhalation due to its extremely low solubility in water and in n-octanol,

- Pigment Red 168 does not have to be classified as skin sensitizing or as skin or eye irritating, indicating that its chemical inertness and extremely low solubility in water and in n-octanol largely prevent interaction with living cells and tissues,

- Pigment Red 168, when aerosolized in respirable form, is likely to behave like an inert dust.