6-nonyl-1,3,5-triazine-2,4-diamine

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A NOAEL of 160 mg/kg bw/d for Caprinoguanamine was obtained for repeated dose toxicity as well as for reproductive/developmental toxicity from a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD guideline 422.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined Repeated Dose Toxicity Study with the Reproductive/Developmental Toxicity Screening Study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-01-26 to 2017-09-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Crl:CD(SD) rat /Charles River (UK) Ltd.
- Age at study initiation:
Males: 69 to 76 days old, Females: 83 to 90 days old.
- Weight at study initiation:
Males: 341 to 400 g, Females: 241 to 297 g.
- Housing:
polycarbonate body with a stainless steel mesh lid. used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods.
Grid bottomed polypropylene cages were used during pairing.
Number of animals per cage: Pre-pairing up to five animals of one sex; Pairing one male and one female; Males after mating up to five animals;
Gestation one female; Lactation one female + litter
- Diet (e.g. ad libitum):
SDS VRF1 Certified pelleted ad libitum
- Water (e.g. ad libitum):
ad libitum, from the public supply via polycarbonate bottles with sipper tubes.
- Acclimation period:
Males: Six days before commencement of treatment ; Females: 20 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY:
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use.
Certificates of analysis were routinely provided by the water supplier.


ENVIRONMENTAL CONDITIONS
- Temperature (°C):
20-24ºC
- Humidity (%):
40-70%
- Air changes (per hr):

- Photoperiod (hrs dark / hrs light):
12 hours light : 12 hours dark

IN-LIFE DATES: From: 2017-05-03 To: 2017-07-02

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
- VEHICLE
- Justification for use and choice of vehicle (if other than water): Standard vehicle according to guideline
- Concentration in vehicle: 8, 16, 32 mg/mL
- Amount of vehicle (if gavage): 5 mL

Details on mating procedure:

- M/F ratio per cage: 1:1 from within the same treatment groups.
- Length of cohabitation: up to two weeks.
- Proof of pregnancy: sperm in the vaginal smear.
- After successful mating each pregnant female was caged (how): individually in solid (polycarbonate) bottom cages

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.
The homogeneity and stability was confirmed for Caprinoguanamine in corn oil formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 1 day and refrigerated storage for up to 15 days.
The mean concentrations of Caprinoguanamine in test formulations analyzed for the study were within ±6% of nominal concentrations, confirming the accurate preparation of these formulations.

Duration of treatment / exposure:

Males: Two weeks before pairing up to necropsy after a minimum of five weeks of treatment (animals were killed in Week 6).
Females Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.

Frequency of treatment:

Once daily at approximately the same time each day.
Animals were not dosed if parturition was in progress at the scheduled time of administration.

Details on study schedule:

F1 animals were not mated (screening study)

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

80 mg/kg bw/day (nominal)

Dose / conc.:

160 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random):
Estrous cycles were evaluated prior to treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the study.

On Day 1 of study all animals were weighed and body weights were reviewed by Study Management before dosing commenced to ensure that variations in
body weight of animals did not exceed ±20% of the mean for each sex. The groups were adjusted to reduce inter-/intra-group variation.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.
- Cage side observations: Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: males: Before dosing on the day treatment commenced (Day 1) and weekly thereafter. On the day prior to necropsy.
females: Before dosing on the day treatment commenced (Day 1) and weekly before pairing. Days 0, 7, 14 and 20 after mating. Day 1, 4, 7 and 13 of lactation. On the day prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: not applicable, no feeding study

WATER CONSUMPTION AND COMPOUND INTAKE: not applicable, no drinking water study

Oestrous cyclicity (parental animals):

Daily for 15 days before pairing.
For four days before scheduled termination (nominally Days 11 to 14 of lactation).

Sperm parameters (parental animals):

For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the
lumen. Any cell- or stage-specificity of testicular findings was noted.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, body weight Days 1, 4, 7 and 13 post partum
Ano-genital distance Day 1 - all F1 offspring.
Nipple/areolae count Day 13 of age - male offspring.

Thyroid Hormone Analysis:
Day 4 of age: F1 offspring, two females per litter: one for T4 (serum) and one for TSH (plasma)
Day 13 of age: F1 offspring, two males and two females per litter: two for T4 (serum) where possible one male and one female and two for TSH (plasma) where possible one male and one female

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, after week 5 investigations completed.
- Maternal animals: Day 25 after mating F0 females failing to produce a viable litter, all other females day 14 of lactation.


GROSS NECROPSY
- After a review of the history of each animal, a full macroscopic examination of the tissues was performed.
All external features and orifices were examined visually.

HISTOPATHOLOGY / ORGAN WEIGHTS
The five lowest numbered surviving males and the first five lactating females with a surviving litter per group at scheduled termination were subject to pathology procedures: The tissues indicated in Table [1] were prepared for microscopic examination and weighed, respectively.
Pathology procedures for remaining F0 males and females per group is outlined in Table 2

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring: Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia.
Thyroid glands were preserved from one male and one female in each litter.

Statistics:

A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pretreatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other comparisons the F1 approximate test was applied.
If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied.
If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead. A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pretreatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon 1945) were made. For all other comparisons the H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied.
For grip strength, motor activity, clinical pathology data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests (Fisher 1973) were performed.
For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied.
Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Reproductive indices:

Percentage mating (%)
Conception rate (%)
Fertility index (%)
Gestation index (%)

Offspring viability indices:

Post-implantation survival index (%)
Live birth index (%)
Viability index (%)
Lactation index (%)

Clinical signs:

no effects observed

Description (incidence and severity):

Parental (F0): Clinical condition, behaviour in the arena, sensory reactivity, grip strength and motor activity were unaffected by treatment.

Mortality:

no mortality observed

Description (incidence):

Parental (F0): No animals died prematurely

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Parental (F0):
Male body weight gains during the last two weeks of treatment for animals receiving the test item at all doses were lower than controls.
This resulted in lower overall weight gains (Day 1 to 38) in these groups (96, 95 and 85% of control for males receiving 40, 80 or 160 mg/kg/day, respectively).
Females receiving 160 mg/kg/day had low body weight gains, when compared to controls, throughout the gestational period and subsequently a slightly lower gain throughout lactation.
These reductions in body weight gain were considered not of sufficient magnitude to be considered adverse.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Parental (F0):
Haematological investigations did not reveal any findings that were attributed to treatment

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Parental (F0):
The biochemical examination of the blood plasma revealed slightly low plasma potassium, calcium, glucose, cholesterol and triglyceride concentrations in males receiving 40, 80 or 160 mg/kg/day.
Females receiving 40, 80 or 160 mg/kg/day had slightly higher plasma bile acid concentrations; these variations were in the absence of a dose dependent trend.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Behaviour in the arena, sensory reactivity, grip strength and motor activity were unaffected by treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Changes related to treatment with Caprinoguanamine were seen in the liver of males.
Minimal to moderate periportal hepatocellular vacuolation, was seen in most males at all doses of Caprinoguanamine, and at minimal to slight severity in two control males.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cyclicity, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by the administration of Caprinoguanamine at all dose levels.
There was no effect of treatment on the number of implantations or litter size.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages.
No cell or stage abnormalities were noted.

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproductive performance, fertility and offspring survival were unaffected by parental treatment.
There was no effect of treatment on the number of implantations, litter size, or the growth of the offspring.

Key result

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest tested dose

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

The clinical condition of the offspring, litter size, offspring survival and sex ratio were unaffected by parental treatment.

Mortality / viability:

no mortality observed

Description (incidence and severity):

Offspring survival and sex ratio were unaffected by parental treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 160 mg/kg/day group, male and female offspring body weights were marginally low on Day 1 of age, when compared with the controls; subsequent weight gain was marginally low.
There was no effect of treatment on the mean body weights of male and female offspring on Day 1 of age at 40 or 80 mg/kg/day.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the circulating levels of thyroxine (T4) in the Day 13 male and female offspring.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Ano-genital distances of both male and female offspring on Day 1 of age and nipple counts on Day 13 of age showed no adverse effects from parental treatment.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of offspring that died prior to the scheduled termination or were killed on Day 13 of age did not reveal any findings that were considered related to parental treatment at any dose level.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest tested dose

Key result

Reproductive effects observed:

no

Conclusions:

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/developmental toxicity was considered to be 160 mg/kg/day.

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening study according to OECD guideline 422, Caprinoguanamine (ca. 98 % a.i) was administered to 10 Sprague-Dawley rats /sex/ at dose levels of 0, 40, 80 and 160 mg/kg bw/day by gavage.

Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation.

Oral gavage administration of Caprinoguanamine for five weeks was generally well tolerated in males at dose levels of 40, 80 or 160 mg/kg/day but did result in a higher incidence and severity of periportal hepatocellular vacuolation in the liver at all dose levels when compared with the controls. For detailed information refer to repeated dose toxicity.

 

F1 Litter responses

The clinical condition of the offspring, litter size, offspring survival and sex ratio were unaffected by parental treatment.

Ano-genital distances of both male and female offspring on Day 1 of age and nipple counts on Day 13 of age showed no adverse effects from parental treatment.

 

In the 160 mg/kg/day group, male and female offspring body weights were marginally low on Day 1 of age, when compared with the controls; subsequent weight gain was marginally low. There was no effect of treatment on the mean body weights of male and female offspring on Day 1 of age at 40 or 80 mg/kg/day.

Macroscopic examination of offspring that died prior to the scheduled termination or were killed on Day 13 of age did not reveal any findings that were considered related to parental treatment at any dose level.

 

Administration of Caprinoguanamine to females for two weeks before pairing, throughout gestation and up to Day 14 of lactation was also generally well tolerated with no test item related microscopic pathology changes detected. Reproductive performance, fertility and offspring survival were unaffected by parental treatment. There was no effect of treatment on the number of implantations, litter size, or the growth of the offspring.

In the context of this study, Caprinoguanamine showed no evidence of being an endocrine disruptor.

 

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/ developmental toxicity was considered to be 160 mg/kg/day.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

160 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Data from a GLP compliant guideline studies with reliability 1.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening study according to OECD guideline 422, Caprinoguanamine (ca. 98 % a.i) was administered to 10 Sprague-Dawley rats /sex/ at dose levels of 0, 40, 80 and 160 mg/kg bw/day by gavage.

Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation.

Oral gavage administration of Caprinoguanamine for five weeks was generally well tolerated in males at dose levels of 40, 80 or 160 mg/kg/day but did result in a higher incidence and severity of periportal hepatocellular vacuolation in the liver at all dose levels when compared with the controls. For detailed information refer to repeated dose toxicity.

 

F1 Litter responses

The clinical condition of the offspring, litter size, offspring survival and sex ratio were unaffected by parental treatment.

Ano-genital distances of both male and female offspring on Day 1 of age and nipple counts on Day 13 of age showed no adverse effects from parental treatment.

 

In the 160 mg/kg/day group, male and female offspring body weights were marginally low on Day 1 of age, when compared with the controls; subsequent weight gain was marginally low. There was no effect of treatment on the mean body weights of male and female offspring on Day 1 of age at 40 or 80 mg/kg/day.

Macroscopic examination of offspring that died prior to the scheduled termination or were killed on Day 13 of age did not reveal any findings that were considered related to parental treatment at any dose level.

 

Administration of Caprinoguanamine to females for two weeks before pairing, throughout gestation and up to Day 14 of lactation was also generally well tolerated with no test item related microscopic pathology changes detected.

Reproductive performance, fertility and offspring survival were unaffected by parental treatment. There was no effect of treatment on the number of implantations, litter size, or the growth of the offspring.

In the context of this study, Caprinoguanamine showed no evidence of being an endocrine disruptor.

 

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/ developmental toxicity was considered to be 160 mg/kg/day.

 

Effects on developmental toxicity

Description of key information

A NOAEL of 160 mg/kg bw/d for Caprinoguanamine was obtained for repeated dose toxicity as well as for reproductive/developmental toxicity from a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD guideline 422.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Reproductive toxicity of Caprinoguanamine was assessed based on data froma Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening study.

The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/ developmental toxicity was considered to be 160 mg/kg/day.

Considering the available data it can be concluded, that according to GHS Regulation EC No 1272/2008 classification for reproductive toxicity is not warranted for Caprinoguanamine.