Anthraquinone

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was carried out in accordance with internationally valid GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: cca 8 weeks

Route of administration:

oral: gavage

Vehicle:

other: 1% Guar Gum in distilled water

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): not mentioned
- Concentration in vehicle: 1 %
- Amount of vehicle (if gavage): 20 ml/kg

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear daily
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

8 weeks

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
150, 600 and 2400 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

12 animals

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Observations of the animals included clinical signs, body weights, and food consumption. All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily throughout the duration of the study. Daily during treatment (approximately 30 to 90 minutes postdose), each animal was removed from the cage and given a detailed clinical examination. The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, as well as evaluation of respiration.

Oestrous cyclicity (parental animals):

At initiation of test article administration and until evidence of copulation was observed, or the cohabitation period ended, the females were examined daily by vaginal lavage to establish estrous cyclicity. Vaginal lavage of the females was completed daily by 10:00 A.M. Toward the end of the gestation period, females were examined twice daily for signs of parturition.

Litter observations:

Number of implantation scars/dam, Litter size, Viable pups, Pup sex ratio (% viable males/litter), Stillborn pups, Stillborn index, Pup weights and Pup survival (days 0-4) were observed.

Postmortem examinations (parental animals):

The animals were examined carefully for external abnormalities including masses. The skin was reflected from a ventral midline incision and any abnormalities were identified and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were examined for abnormalities and the organs removed, examined, and, where required, placed in fixative. Special emphasis was placed on organs of the reproductive system. Implantation sites (scars) along the uterus were counted and recorded.
Body weights and protocol-designated organ weights were recorded for all surviving adult animals at the scheduled necropsy and appropriate organ weight ratios were calculated (relative to body weight). Paired organs were weighed together. The right and left testis were weighed separately.
Microscopic examination of fixed hematoxylin and eosin-stained paraffin sections was performed on protocol-designated sections of tissues. The slides for the control and 2400 mg/kg/day group were examined by a board-certified veterinary pathologist. A four-step grading system was utilized to define gradable lesions for comparison between dose groups.
The animals were examined carefully for external abnormalities including masses. The skin was reflected from a ventral midline incision and any abnormalities were identified and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were examined for abnormalities and the organs removed, examined, and, where required, placed in fixative. Special emphasis was placed on organs of the reproductive system. Implantation sites (scars) along the uterus were counted and recorded. In males, Epididymides, Prostate gland, Seminal vesicles, and Testes were weighed. In females, Ovaries and Uterus with cervix were weighed. Food and water were provided ad libitum during the study.

Statistics:

Data for each sex within a set were analyzed separately. The raw data were tabulated within each time interval, and the mean and standard deviation were calculated for each endpoint by sex and group. For each endpoint, treatment groups were compared to the control group using Group Pair-wise Comparisons, Fisher's Exact Test, Arcsin-Square Root or Covariate Analysis.

Reproductive indices:

Mating procedures:
After 2 weeks of treatment, the males were randomly cohabited, one male to one female, from the corresponding treatment or control group. Each female was housed in the cage of a male during pairing. Positive evidence of copulation was established by daily inspection for a copulatory plug in the vagina and/or sperm present in the vaginal lavage. The day on which positive evidence of copulation was observed was considered GD 0. After evidence of mating was observed, the female was returned to an individual cage. The maximum mating period was 14 days, at the end of which any females with no confirmed evidence of mating were returned to individual cages until scheduled euthanasia. After the mating period, the males were individually housed and continued on treatment for approximately 2 weeks. At the end of this period, the males were euthanized and necropsied.

Offspring viability indices:

The number of pups born, liveborn, and stillborn, pup sex ratio (% males per litter) in the treated groups was observed. No macroscopic observations were evident in any pups (stillborn, died on study, or LD 4) at necropsy.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Considerable mortality was seen in F at the 600 and 2400 mg/kg/day levels. Several F in the 600 and 2400 mg/kg/day groups died during the gestation and lactation period. Both M and F rats at all treatment levels were observed with discoloured urine.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A treatment-related effect on food consumption was evident in all treated males and females in comparison to controls,although these reductions were not dose-dependent.Decreased body weights were observed in both males and females at all treatment levels.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A treatment-related effect on food consumption was evident in all treated males and females in comparison to controls,although these reductions were not dose-dependent.Decreased body weights were observed in both males and females at all treatment levels.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test article-related microscopic changes were noted in the liver and glandular stomach of F rats at 2400 mg/kg/day, but not in males.

Other effects:

not specified

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Treatment did produce a lengthening of the estrous cycle; mean estrous cycle length (number of days per cycle) was increased in the treated females in comparison to controls, but was only statistically significant at 600 mg/kg/day.

Reproductive function: sperm measures:

not specified

Reproductive performance:

effects observed, treatment-related

Key result

Dose descriptor:

NOEL

Effect level:

<= 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Pup survival was poor in the 600 and 2400 mg/kg/day groups. At 150 mg/kg/day, no effect of treatment was evident from parturition data, litter size, or pup survival to LD 4.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Pup body weights were decreased at all treatment levels; mean pup body weights (by sex and sexes combined) in the 150 mg/kg/day group were similar to control at birth (LD 0), but 8-10% lower by LD 4.

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic observations were evident in any pups (stillborn, died on study, or LD 4) at necropsy.

Histopathological findings:

not specified

Key result

Dose descriptor:

NOAEL

Generation:

F1

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

not specified

Conclusions:

Both male and female rats at all treatment levels were observed with discolored urine, and decreased body weights and food consumption; thus a No-
Observed-Effect-Level (NOEL) was not achieved. Mating, fertility, and fecundity indices (male and female) in all treatment groups were comparable to controls. Likewise, the copulatory interval (days to mating) was unaffected by treatment. No test article-related organ weight changes were noted in rats of either sex. The number of pups born, liveborn, and stillborn in the 150 mg/kg/day group was similar to controls and unaffected by treatment. Pup survival (to LD 4) in the 150 mg/kg/day group was 89% and was comparable to the controls at 97%. Pup body weights were decreased at all treatment levels. Pup sex ratio (% males per litter) in the treated groups was considered comparable to controls and unaffected by treatment.
No macroscopic observations were evident in any pups at necropsy.
Pup survival was poor in the 600 and 2400 mg/kg/day groups. Very few pups survived to LD 4 in the 2400 mg/kg/day group, but no clinical observations were noted for these pups.

Executive summary:

The mating, fertility, and fecundity indices (83.3-100%) for both treated males and females were unaffected by treatment with Anthraquinone and were similar to controls (91.7-100.0%). Likewise, the copulatory interval (days to mating) was unaffected by treatment and ranged from 2.4-3.1 days in the treated groups in comparison to 3.0 in the controls.

No macroscopic observations were evident in any pups at necropsy. At 150 mg/kg/day, no effect of treatment was evident from parturition data, litter size, or pup survival to LD 4. No test article-related organ weight changes were noted in rats of either sex.

Samples (2.0 mL) of the dosing formulations at each concentration were collected from Weeks 1, 3, and 8 to assess the concentration of the test article. The samples were collected from the middle of the container, while stirring, using a syringe, and placed into 20 mL scintillation vials. Recoveries at all levels were relatively low throughout the study period. Recoveries ranged from 66.0-73.0% (Week 1), 59.5-84.7% (Week 3), and 25.1-99.5% (Week 8) in the treated groups.

Additional information

The original study provided by the registrant performed in compliance with the OECD Guideline No. 421 – Reproduction/Developmental Toxicity Screening Test for Anthraquinone with the Good Laboratory Practice (GLP) Regulations was used as a key study for the assessment of human health hazards (Cook J. A., 2010b).

Effects on fertility

In an OECD reproduction toxicity screening test (OECD TG 421) Anthraquinone was studied in rats (strain Sprague Dawley Crl:CD(SD)) orally dosed with 0, 150, 600 and 2400mg/kg bw/day. Duration of the test was 8 weeks; number of animals per dose and sex was 12. Males received Anthraquinone from 14 days prior to pairing and continued untileuthanasia. Femalesreceived Anthraquinone from 14 days prior to pairing through lactation day 4.

The mating, fertility, and fecundity indices (83.3-100%) for both treated males and females were unaffected by treatment with Anthraquinone and were similar to controls (91.7-100.0%). Likewise, the copulatory interval (days to mating) was unaffected by treatment and ranged from 2.4-3.1 days in the treated groups in comparison tothe controls. Treatment did produce a lengthening of the estrous cycle; mean estrous cycle length was significantly increased in the treated females (5.6 days in the 600 mg/kg/day groups) in comparison to controls (4.4 days),but was only significant at 600 mg/kg/day.

Considerable mortality was seen in females at the 600 and 2400 mg/kg/day levels. Several females in the 600 and 2400 mg/kg/day groups died during the gestation and lactation period. At 150 mg/kg/day, no effect of treatment was evident from parturition data. No test article-related organ weight changes were noted in rats of either sex. Test article-related macroscopic changes were noted in female rats, but not in males. Black or red foci were observed in the glandular stomach of females at 600 and 2400 mg/kg/day that died and an erosion/ulcer was noted in the pyloric stomach of one female that died at 2400 mg/kg/day. Also at 2400 mg/kg/day, minimal focal necrosis and moderate periportal vacuolization of hepatocytes were observed in one female, edema was seen in two of seven animals that died, and erosion/ulcer in the glandular stomach was seen in three of these animals. Test article-related microscopic changes were noted in the liver and glandular stomach of female rats at 2400 mg/kg/day, but not in males.

Both male and female rats at all treatment levels were observed with discoloured urine (brown, orange, or red). A treatment-related effect on food consumption was evident in all treated males and females in comparison to controls, although these reductions were not dose-dependent. Decreased body weights were observed in both males and females at all treatment levels. In females, gestation body weight gain was significantly lower than control over most intervals (GD 0-7, 7-14, 14-20, and 0- 20) in the 600 and 2400 mg/kg/day groups. Body weight gain in the 150 mg/kg/day group during lactation was comparable to controls.In addition, decreased activity was observed in four and five females in the 600 and 2400 mg/kg/day groups, respectively, and one male in the 150 mg/kg/day group.

Since both, male and female rats at all treatment levels were observed with discoloured urine and decreased body weights and food consumption; a No-Observed-Effect-Level (NOEL) was not achieved(Cook J. A., 2010b).

Developmental toxicity/teratogenicity

In an OECD reproduction toxicity screening test (OECD TG 421) Anthraquinone was studied in rats (strainSprague Dawley Crl:CD(SD))orally dosed with0, 150, 600 and 2400mg/kg bw/day. Duration of test was 8 weeks. Number of animals per dose and sex was 12. Males received Anthraquinone from 14 days prior to pairing and continued untileuthanasia. Femalesreceived Anthraquinone from 14 days prior to pairing through lactation day 4.

At 150 mg/kg/day, no effect of treatment was evident from litter size, or pup survival to LD 4.The number of pups born, liveborn, and stillborn in the 150 mg/kg/day group was similar to controls and unaffected by treatment. Pup survival (to LD 4) in the 150 mg/kg/day group was 89% and was comparable to the controls at 97%, but itwas poor in the 600 and 2400 mg/kg/day groups.Very few pups survived to LDthe 2400 mg/kg/day group, but no clinical observations were noted for these pups.Pup body weights were decreased at all treatment levels; mean pup body weights in the 150 mg/kg/day group were similar to control at birth (LD 0), but 8-10% lower by LD 4.Pup sex ratio (% males per litter) in the treated groups was considered comparable to controls and unaffected by treatment. No macroscopic observations were evident in any pups at necropsy(Cook J. A., 2010b).

Short description of key information:
The test substance, Anthraquinone was tested for reproduction toxicity using the OECD Test Guideline No. 421 Reproduction/Developmental Toxicity Screening Test.
Based on the study provided by the registrant , NOAEL was not defined due to the fact, that there were health effects regarding general toxicity in both, male and female rats present at all treatment levels.
Mating, fertility, and fecundity indices and copulatory interval in all treatment groups were comparable to controls.
Considering developmental toxicity, pup body weights were decreased at all treatment levels and pup survival was poor in the 600 and 2400 mg/kg/day groups, but no further clinical observations were noted for the pups (Cook J. A., 2010b).

Effects on developmental toxicity

Description of key information

Proposal is Repr. 2 H361d

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Additional information

OECD 414 (2018)

An animal study showed developmental toxicity at the medium and high dose tested. Compared to the control group of animals, an increased frequency of some types of malformations and a significantly increased frequency of variations were observed. These findings were not observed at the lowest dose. Given the observed maternal toxicity at the middle and highest dose, it cannot be excluded that the observed developmental toxicity was not caused by the observed maternal toxicity. Based on the results of the prenatal toxicity study, it was concluded that there are effects that can be unequivocally attributed to maternal toxicity, and then there are effects for which the same is not certain and can be both a consequence of maternal toxicity (and possibly insufficient maternal care) as well as a direct effect of the test substance on development, which means that classification/non-classification cannot be excluded based on apparent maternal toxicity.

The classification criteria of the CLP Regulation include developmental effects in the presence of other toxic effects that are not considered secondary to maternal toxicity for both Category 1B and Category 2.

Justification for classification or non-classification

In view of all of the above, as far as reproductive parameters are concerned, a conclusion was made on the classification of Anthraquinone with inclusion in category 2 for reproductive and developmental toxicity - Repr.2, H361d, because the evidence from these studies considered together is not sufficiently convincing for the inclusion of the substance in the category 1.