2,2`-[6-(phenylamino)-heteromonocycle-2,4-diyl]diphenol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adapted by the Council on 27th July 1995

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2`-[6-(phenylamino)-1,3,5-triazine-2,4-diyl]diphenol

Cas Number:

1248-66-4

Molecular formula:

C21H16N4O2

IUPAC Name:

2,2`-[6-(phenylamino)-1,3,5-triazine-2,4-diyl]diphenol

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories Ltd. Laboratory Animal Services Wölferstrasse 4 4414 Füllinsdorf / Switzerland
- Age at study initiation: (P) 11 weeks
- Weight at study initiation: (P) Males: 298 to 357 g; Females: 186 to 217 g
- Housing: Individually in Makrolon type-3 cages. During the pre-pairing period, cages with males
were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: ad libitum. Pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland).
- Water: ad libitum. Community tap-water from Füllinsdorf.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with music during the light period

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Details on exposure:

The dose formulations were prepared weekly; addition of 80% PEG 300 to the substance and homogenized; dose volume of 5 ml/kg body weight with daily adjustment to the actual body weight;

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 d
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC/UV-detection

Duration of treatment / exposure:

mailes: 14-day pre-pairing period, pairing period + 1 day; minimum 4 weeks
females: 14-day pre-pairing period, pairing period, gestation, lactation period up to day 4 post partum; approximately 7 weeks;

Frequency of treatment:

once daily orally by gavage

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50 mg/kg body weight/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
200 mg/kg body weight/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg body weight/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous short term study (RCC No. B31972) performed at Harlan Laboratories Ltd. (former RCC Ltd).
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry:
- Other:

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily;

BODY WEIGHT: Yes
- Time schedule for examinations: daily

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

Sperm parameters (parental animals):

The testes and epididymides of all parental males were weighed as pairs. In addition, adrenal glands and thymus were weighed in males.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
external examination at first Litter Check and a last litter check (day 4); clinical signs and macroscopic findings; number and sex of pups, stillbirths, live births, postnatal mortality, weight gain to day 4 post partum;

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals after pairing period
- Maternal animals: All animals on day 4 post partum

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 to the macroscopical findings.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

clinical signs considered to be incidental

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

adrenocortical changes consisting of minimal hypertrophic changes of the zona fasciculate at 200 mg/kg/day ; at 200 and 1000 mg/kg/day, erythropoiesis at minimal degree was noted in two females in each group.

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
All animals survived

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
At all dose-levels, there were no treatment-related effects;

REPRODUCTIVE Data (PARENTAL ANIMALS):
At all dose-levels, there were no treatment-related effects on precoital time, mean duration of gestation, number of corpora lutea and implantaions, post-implantation loss, pup survival or litter size from birth through to scheduled sacrifice an day 4 post partum;

ORGAN WEIGHTS (PARENTAL ANIMALS)
No test item related changes were noted in mean organ weight of thymus, adrenals, testes and epididymides in males;

In males and females at 200 mg/kg/day and in females at 1000 mg/kg/day, adrenocortical changes consisting of minimal to moderate hypertrophic changes of the zona fasciculate were noted.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: with respect to reproduction/ developement toxicity;

2

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: based on the higher locomotor activity;

3

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: based on erithropoieseis noted during the histopathology examination;

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Histopathological findings:

effects observed, non-treatment-related

Details on results (F1)

no findings regarding first litter check and last last litter check (day 4), clinical signs and macroscopic findings in all groups;

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fetal toxicity

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

NOEL (reproduction/developmental toxicity) = 1000 mg/kg bw/day

Executive summary:

The reproductive toxicity of the substance was evaluated in an experimental study performed according to the OECD Guideline 421.
Test item was administered in Milli-Q-Water as vehicle at dosages of 50, 200, and 1000 mg/kg body weight/day, and controls received the vehicle only. Test item was administered to male rats for at least 28 days and to female rats for 15 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.
All animals survived until the scheduled necropsy and no test item-related clinical signs were noted during the study. Food consumption and body weight were not affected by the treatment with the test item. At 1000 mg/kg/day locomotor activity resulted to be increased in males.
The reproduction and developmental parameters investigated did not give any indication of any
test item-related effects. The weight of thymus, adrenals, testes and epididymides were not affected by the treatment with the test item. At histopathology examination, an increased erythropoiesis was noted in females at 200 and 1000 mg/kg/day.

Based on the increased locomotor activity in males and on the increased erithropoiesis in females, the general NOEL (No Observed Effect Level) was considered to be 50 mg/kg body weight/day.
Under the conditions of this study, the NOAEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 1000 mg/kg/day.