1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: M.A.F.F. in Japan notification 12 Nousan No 8147, (November, 2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]-6-[(1R)-1-fluoroethyl]-1,3,5-triazine-2,4-diamine

EC Number:

619-749-5

Cas Number:

730979-19-8

Molecular formula:

C16H20FN5

IUPAC Name:

N-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]-6-[(1R)-1-fluoroethyl]-1,3,5-triazine-2,4-diamine

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River laboratories, St Germain-sur-l'Arbresle, France.
- Age at study initiation: adult virgin, not further specified
- Weight at study initiation: 239-299 g (females selected for study)
- Fasting period before study: not applicable
- Housing: Pregnant females were housed individually in suspended, stainless steel, wire mesh cages.
- Diet (e.g. ad libitum): Certified rodent pelleted and irradiated diet A04C-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France) was distributed ad libitum. Feed was stored in an identified room, controlled for temperature and humidity. Diet was used only until the date of expiry.
- Water (e.g. ad libitum): Water from the municipal supply was provided ad libitum with an automatic watering system.
- Acclimation period: at least 12 days prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was suspended (w/v) in an aqueous solution of methylcellulose 400 (Fluka, Mulhouse, France) at 0.5% and stored at approximately 5 °C (± 3 °C). Formulations were prepared twice (F1 and F2) during the study. The suspensions were mixed continuously before and during treatment with an electromagnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% aqueous methylcellulose 400; methylcellulose is commonly used as thickener or emulsifier in various food and cosmetic applications and toxicological studies to generate appropriate consistency.
- Concentration in vehicle: 0, 1.0, 2.5 and 20.0 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity of the suspensions was checked on the first formulation (F1) for the lowest and the highest concentrations (1 and 20 mg/mL). The mean values obtained from the homogeneity check were used as measured concentrations. In addition, the intermediate concentration of the first formulation (F1) and all concentrations of the second formulation (F2) were checked. Stability of the test substance in suspension in the vehicle at concentrations of 0.127 and 250 g/L was determined in a previous study (SA 04129) and was found to be stable for 30 days under similar conditions to those of the current study.

Homogeneity and concentration of dosing suspensions were between 92 and 100% of nominal values, which is within the in-house target range of 90 to 110% of nominal concentration. The dosing suspensions were therefore considered acceptable for use on the current study.

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: not referred to
- Verification of same strain and source of both sexes: yes, stock males of same strain from same supplier
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

GD 6 to 20

Frequency of treatment:

once daily

Duration of test:

until GD 21

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

23 pregnant females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Doses based on results obtained in a range-finding study (SA 04115), where pregnant rats received up to 400 mg/kg bw/day, from GD 6 to 20. At this dosage 1/8 females showed marked clinical signs and body weight loss. The overall mean body weight gain between GD 6-21 for this group was reduced by 29%, with mean food consumption reduced by approximately 14% between GD 16-18 and GD 18-21, compared with controls. At the limited fetal skeletal examination, a delay in ossification for several parameters was observed.
In the current study the high dose of 200 mg/kg bw/day was expected to cause slight maternal and fetal toxicity. The mid dose of 25 mg/kg bw/day provided an eight-fold factor between the high and mid dose in case 200 mg/kg bw/day should prove to be too toxic. The low dose of 10 mg/kg bw/day was expected to be a No Observed Adverse Effect Level (N.O.A.E.L.) in terms of maternal and fetal toxicity.

- Rationale for animal assignment: random

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical signs daily from GD 0 through GD 21; mortality twice daily (in the morning and in the afternoon, except at weekends and public holidays when check was carried out once daily).
- Cage side observations included: all clinical signs, mortality (dead or moribund animals)

BODY WEIGHT: Yes
- Time schedule for examinations: On GD 0, 6, 8, 10, 12, 14, 16, and 18

FOOD CONSUMPTION: Yes, for periods from GD 1-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, and 18-21
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined:
Animals killed in extremis or found dead (1 high dose female): Macroscopic examination of visceral organs; the lungs, trachea, esophagus and samples from liver were retained in 10% neutral buffered formalin for possible histological examination. The number and type of implantations and corpora lutea were noted when present.

All surviving females were killed by carbon dioxide inhalation for examination of uterine content. Each female was first subjected to macroscopic examination of the visceral organs. The liver was weighed for all pregnant females. A portion of liver was retained in 10% neutral buffered formalin for possible histological examination, and the remaining portion stored at -80 °C for possible enzyme analysis, for all females at scheduled sacrifice. The kidneys from one animal of the low-dose group and the spleen from another one were retained in 10% neutral buffered formalin for possible histological examination.

OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Sex of live fetuses, individual weight of live fetuses; if possible, sex and weight of dead fetuses were recorded and included in the study file, as well.
Uterine horn(s) without visible implantations were immersed in a 10% solution of ammonium sulfide according to the SALEWSKI method (1964) in order to visualize any sites which were not apparent.
Intra-uterine death was classified according to GLEICH and FROHBERG (1977) as:
- Early resorptions: macroscopic discrimination between fetal residues and placental material not possible.
- Late resorptions: distinct macroscopic discrimination between fetal and placental remains possible.

Dead fetuses: defined as dead conceptuses showing distinct digits on fore- and hind-paws.
Runt fetuses: defined as live fetuses weighing less than 4 g at cesarean section of the dam.

Fetal examinations:

- External examinations: Yes: All the live fetuses were killed by subcutaneous injection (0.02 mL/fetus) of Dolethal (18.22 g/100 mL, sodium pentobarbital) and subjected to an external examination.
- Soft tissue examinations: Yes: Approximately half of the live fetuses from each litter were stained in Bouin's fluid for subsequent internal examination following free-hand sectioning.
- Skeletal examinations: Yes: The remaining half of the live fetuses from each litter were skinned, eviscerated and stained with alizarin red S and alcian blue for skeletal examination of bone and cartilage. Structural deviations were classified as follows:
Malformations: A permanent structural change likely to adversely affect survival or health.
Variations: A change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health (this might include a delay in growth or morphogenesis that has otherwise followed a normal pattern of development).
- Head examinations: No

Statistics:

Mean and standard deviation for all maternal, litter and fetal parameters were calculated for each group. Statistical analyses were performed separately for all pregnant females and for all pregnant females with live fetuses.
Only for body weight descriptive statistics were done as the only exception.

For details on statistics performed please refer to "Any other information on materials and methods incl. tables".

Indices:

Maternal body weight (BW) changes for interval periods; Corrected body weight change (CBWC); Average food consumption (FC) during intervals in g/day; Pre-implantation loss per litter; Post-implantation loss per litter; Number of live fetuses per litter; Number of dead fetuses per litter; Percentage of dead fetuses per litter; Percentage of male fetuses per litter; Mean fetal body weight per litter; Mean fetal body weight per litter and per sex; Mean fetal body weight per group; Mean fetal body weight per sex per group; Percentage of fetuses affected per litter; Mean percentage of fetuses affected per group; Percentage of litters affected per group

Historical control data:

Historical control data from studies conducted in-house were provided and referred to in order to allow comparison with concurrent controls.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: not adverse

Details on maternal toxic effects:
No treatment-related mortality occurred. One animal of the high dose group had died due to gavage error as confirmed by necropsy. No treatment-related clinical signs were observed at any dose level. The pregnancy rate was unaffected by treatment (100% in 200 and 25 mg/kg bw/day and control group, 96% at 10 mg/kg bw/day). At 200 mg/kg bw/day, mean maternal body weight gain was reduced by 67% between GD 6-8, compared with the controls. Thereafter, mean maternal body weight gain was reduced by between 10 and 14% at each specified interval, resulting in an overall reduction of 14% between GD 6-21, compared with the controls. The effect was not statistically significant. There was no effect on mean maternal weight in any other group. At 200 mg/kg bw/day, mean maternal corrected body weight change (maternal body weight change independent of the uterine weight) was less pronounced (36.5 g) than in the control group (50.6 g), the effect being statistically significant (p≤0.05). There was no effect on mean maternal corrected body weight change at 25 or 10 mg/kg bw/day. At 200 mg/kg/day, mean maternal food consumption was reduced by between 4 and 10% at each specified interval compared with the controls, the effect being statistically significant between GD 6-8 (p≤0.01), GD 12-14 (p≤0.05) and GD 18-21 (p≤0.05). No effects were observed in the other groups. Mean maternal liver weights were unaffected by treatment. No treatment-related macroscopic findings were observed at autopsy.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Remarks:

maternal toxicity

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no effects observed

2

Dose descriptor:

LOEL

Remarks:

maternal toxicity

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

3

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse systemic effects observed

4

Dose descriptor:

NOEL

Remarks:

developmental toxicity

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no effects observed on development

5

Dose descriptor:

LOEL

Remarks:

developmental toxicity

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

6

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse effects on development observed

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: not adverse

Details on embryotoxic / teratogenic effects:
At 200 mg/kg bw/day, mean fetal body weight for the combined sexes was reduced by 8% and by 9% and 7% in male and female fetuses, respectively, the effect being statistically significant in each case (p≤0.01). At 10 mg/kg bw/day, mean fetal body weights for the combined and separate sexes were statistically significantly lower than the control group, as well. However, in the absence of a dose-response relationsship, this finding at 10 mg/kg bw/day was considered to be incidental.
There was no effect on other litter parameters including pre and post implantation loss, early and late resorptions, fetal death status and percentage of male fetuses.
There were no treatment-related findings at the external fetal examination. The higher incidence of runt fetuses in the treated groups did not occur in a dose-related manner and was either well-comparable to or within the in-house historical control range at the high dose level; therefore, it was not considered to be treatment-related.
The same argument is valid for the observed visceral malformations like severe renal pelvic dilatation or misshapened thymus. All other variations occurred at a higher incidence in the control group compared with the treated groups, or occurred as isolated findings and were therefore considered to have occurred by chance.
The few skeletal malformations observed were distributed across the groups including the controls, with no indication of a dose-related effect, or occurred as isolated findings. These malformations were therefore considered to have arisen spontaneously.
The incidences of the observed variations (deviations in ossification), although being higher in the treated groups than in the controls at both the fetal and litter level, either lacked a dose-response relationship or were within the in-house historical control range, or both. Therefore, these findings were considered to have occurred by chance.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Remarks:

embryotoxic / teratogenic effects

Effect level:

>= 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

CONCLUSION

There were no treatment-related mortalities or clinical signs in any dose group throughout the study. Pregnancy rate was unaffected by treatment. A dose level of 200 mg/kg/day caused slight maternal toxicity as evidenced by the reduction of mean maternal body weight gain, mean maternal corrected body weight change (maternal body weight change independent of the uterine weight), mean maternal food consumption and mean fetal body weight. Although statistically significant to some extent and very likely treatment-related, these effects were not considered adverse, as they did neither affect survival of dams or litters or their normal development nor indicate a handicap in adapting to the environment.

Therefore, a dose level of 200 mg/kg bw/day was considered as No Observed Adverse Effect Level (NOAEL) for both maternal and developmental toxicity, being the Lowest Observed Effect Level (LOEL) at the same time under the conditions of the study. A dose level of 25 mg/kg bw/day was a clear No Observed Effect Level (NOEL) in terms of both maternal and developmental toxicity.