1,4-bis(mesitylamino)anthraquinone

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed GLP compliant OECD guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 421

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis(mesitylamino)anthraquinone

EC Number:

204-155-7

EC Name:

1,4-bis(mesitylamino)anthraquinone

Cas Number:

116-75-6

Molecular formula:

C32H30N2O2

IUPAC Name:

1,4-bis(mesitylamino)anthraquinone

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River Italy S.p.A., Calco (Lecco), Italy
- Age at study initiation: 8-9 weeks
- Weight at study initiation: (P) Males: 317-322g; Females: 210-216 g;
- Housing:
From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulphone solid bottomed cages measuring approximately 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During the mating period, animals were housed on the basis of one male to one female in clear polycarbonate cages measuring approxiamtely 43x27x18 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
The males were re-caged after mating as they were before mating.
After mating, the females were transferred to individual clear polycarbonate solid bottomed cages measuring approxiamtely 43x27x18 cm
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-24
- Humidity (%):40-70
- Air changes (per hr):15-20
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: To:05 July 2012-05 September 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The required amount of the test item was suspended in the vehicle (corn oil), brought to the final volume appropriate for each concentration (concentrations of 20, 60 and 200 mg/mL) and kept under magnetic stirrer at room temperature prior to use and until the time of dosing of the last animal.
Concentrations were calculated and expressed in terms of test item as supplied

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (content check and homogeneity) and the stability of formulation was satisfactory. Results of the analyses were within the limits of acceptance.
The stability was found to be 24 hours at room temperature in the concentration range of 20 to 200 mg/mL.
Samples of the formulations prepared on Day 1 and on Week 4 of the study were also analysed to check the concentration and homogeneity. Results of the analyses were within the limits of acceptance.
Chemical analyses were carried out by the Analytical Chemistry Department at RTC, using a validated spectrophotometric detection in the range from 1.0 to 250 mg/mL.
The software used for this activity was Empower Probuild No. 2154.

Details on mating procedure:

Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
The female was paired with the same male until positive identification occurred.

Duration of treatment / exposure:

Males were dosed for 32 days (including 2 weeks prior to pairing during the pre pairing period and approximately 2 weeks post pairing). Females were dosed 2 weeks before pairing, during the pairing period, gestation period and up to Day 3 post partum.

Frequency of treatment:

Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before
necropsy.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Duration of test:

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 32 days.
Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum or until the day before necropsy.

Doses / concentrations

Remarks:

Doses / Concentrations:
Doses: 0, 100, 300, 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:Dose levels had been selected in consultation with the Sponsor based on information from a previous non GLP compliant dose range finding study.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:daily

DETAILED CLINICAL OBSERVATIONS: NO

BODY WEIGHT: Yes
- Time schedule: weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on post partum day 4
- Organs examined:Ovaries and uterus

Ovaries and uterine content:

a) number of visible implantation sites;
b) number of corpora lutea (pregnant animals).

Fetal examinations:

- External examinations of pups at day 4 post partum: Yes:
- Soft tissue examinations: : No
- Skeletal examinations: No
- Head examinations: No

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.

The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test.

The criterion for statistical significance was p<0.05.

Indices:

Group mean values were calculated for all parameters. Data from females not pregnant were excluded from group mean calculations as considered appropriate by the Study Director.

The following reproductive indices were calculated:

Males

Copulatory Index (%) = (no. of animals mated x 100) / (no. of animals paired)

Fertility Index (%) = (no. of males which induced pregnancy x 100) / (no. of males paired)


Females

Copulatory Index (%) = (no. of animals mated x 100) / (no. of animals paired)

Fertility Index (%) = (no. of pregnant females x 100) / (no. of females paired)


Males and females

Copulatory Interval = Mean number of days between pairing and mating


Females

Pre-birth loss was calculated as a percentage from the formula:
(((No. of visible implantations) - (total litter size at birth)) x 100) / (No. of visible implantations)

Pup loss at birth was calculated as a percentage from the formula:
(((Total litter size) - (live litter size)) x 100) / (Total litter size)

Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:
(((Total litter size at birth) - (live litter size at Day 4)) x 100) / (Total litter size at birth)

Sex ratios were calculated at birth and on Day 4 post partum and presented as the percentage of males per litter.

Historical control data:

not used since no effects

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: External examinations of pups at day 4 post partum

Details on embryotoxic / teratogenic effects:
External examinations of pups at day 4 post partum revealed no effects.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Clinical signs, body weight and food consumption were unaffected by treatment in both sexes.
Reproductive parameters such as fertility index, pre-coital interval and copulatory index did not show significant differences in treated groups compared to controls.
Gestation length, litter data, sex ratios and implantation losses (pre and post) were unaffected by treatment.
At macroscopic and microscopic examination, no treatment related lesions were seen.
On the basis of the results obtained in this OECD Reproduction / Developmental Toxicity Screening Test, the maximum dosage of 1000 mg/kg/day of the test item was established as the NOAEL (No Observed Adverse Effect Level) for parental animals of both sexes (general toxicity and reproduction) and for the offspring (developmental toxicitxy).

Executive summary:

Study design

The toxic effects on rats after repeated dosing, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition, development of the concepts and early lactation were investigated.

The dosage groups were as follows:

Group Number	Treatment (mg/kg/day)	Number of animals
1	0	10M+10F
2	100	10M+10F
3	300	10M+10F
4	1000	10M+10F

M = Males; F = Females

The test item was administered by oral gavage at a dose volume of 5 mL/kg.

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 32 days. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum or until the day before necropsy. The following investigations were performed on parental animals of all groups: body weight, clinical signs, food consumption, oestrous cycle, mating performance, litter data, macroscopic observations, organ weights and histopathological examination of abnormalities. Histopathological examination of testes, epididymides and ovaries was performed only on control and high dose groups.

Mortality and fate of females

One control female and one low dose female were found dead. On the basis of the macroscopic and microscopic observations, the cause of deaths is attributable to a misdosing. One low dose female was not pregnant and one mid-dose female showed unilateral implantation. The number of females with live pups on Day 4 post partum was 9 in the control group, 8 in the low dose group and 10 in each of the mid- and high dose groups.

Clinical signs

As consequence of the colour of the test item blue staining of the bedding material and tail was noted in the animals of mid- and high dose groups.

Body weight and body weight gain

No signs of toxicological significance were seen in body weight or body weight gain during the study in treated animals compared to controls.

Food consumption

No effects on food consumption were seen.

Oestrus cycle, mating performance and reproductive parameters

No treatment-related anomalies were noted in the oestrus cycle of treated females when compared to controls. Pre-coital interval, copulation plugs, copulatory and fertility index were similar between treated and control animals.

Implantation, pre-birth loss data and gestation length

No differences were observed in treated and control groups for these parameters.

Litter data and sex ratio

Litter data and sex ratios at birth and on Day 4 post partum were unaffected by treatment.

Clinical signs of pups

Pre-weaning clinical signs did not show treatment-related effects.

Necropsy findings in pups

Necropsy findings in decedent pups and pups sacrificed on Day 4 post partum did not reveal any treatment-related effect.

Terminal body weight and organ weights

Terminal body weight and organ weights were unaffected by treatment in both sexes.

Macroscopic and microscopic observations including examination of spermatogenic cycle

The lesions detected in some animals were also noted in untreated laboratory rats; they were therefore considered to be incidental and/or spontaneous in origin. No treatment-related changes were seen in testis, epididymides (including spermatogenic cycle) and ovaries evaluated in high dose males or females receiving the test item.