2,4,6-tris(2,4,6-tribromophenoxy)-1,3,5-triazine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

FR-245: Waiver for Extended One-Generation Reproductive Toxicity Study (EOGRT) testing

The Registrants of the substance 2,4,6-tris (2,4,6-tribromophenoxy)-1,3,5-triazine were notified by European commission (EC letter dated 15-11-2016) regarding an Amendment of the standard information requirements in Annex IX and X of the REACH in which the OECD 443 Extended One-Generation Reproductive Toxicity Study (EOGRT) became the standard information requirement under REACH instead of the previous 2-generation reproductive toxicity study. Accordingly, the section of Reproductive Toxicity of the dossier is updated.

The registrants request to refrain from performing the (EOGRT) based on the following scientific information which was generated according to appropriate OECD guidelines and GLP. 

1.     According to the REACH Regulation Annex IX section 8.7.3, EOGRT should be performed“If the available repeated dose toxicity studies (e.g. 28-day or 90-day studies, OECD 421 or 422 screening studies) indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity.

This is equally stated in Chapter R.7a (P. 359): Endpoint Specific Guidance from ECHA Guidance on Information Requirements and Chemical Safety Assessment.

A 90- day oral repeat dose toxicity study in the rat with a 28 day recovery period was conducted according to OECD 408 testing guideline under GLP conditions (Mackay, 2009). The study included investigation of effects on some reproductive endpoints, in addition to the standard study design. The results showed no systemic toxicity effects and the No Observed Adverse Effect level (NOAEL) was determined as >1000mg/kg/day. No treatment related changes in sperm count and motility were observed. Vaginal lavages which were taken early morning during the 3 week period from all females, prior to termination of the animals, showed no treatment related changes in the oestrus cycle. In addition, there were no dose related changes in organ weight of ovaries, seminal vesicles, testis, ureter, uterus, vagina in comparison to control animals. Details of the data can be found in section 7. 5.1 (Repeated dose toxicity).

In summary, the 90-day repeated dose toxicity for this substance did not observe any adverse effect on reproductive organs or tissues.

2.     A Developmental toxicity study was conducted according to the appropriate OECD 414 guideline and following GLP standards (Kim Kang-hyun, 2013) to evaluate the potential maternal and developmental toxicity of 2,4,6-Tris(2,4,6 tribromophenoxy)-1,3,5-triazine (also designated FR-25) in rats. Doses of 250, 500 and 1000mg/kg/day test substance were administered orally during days 5-19 of gestation.No effects of the test substance treatment were observed in maternal rats in any of the parameters examined.

At cesarean section, there were no treatment-related abnormalities in such parameters as mean gravid uterine weights, the numbers of corpora lutea and implantation, or the percent implantation index, pre- and post-implantation loss in any treated groups.

As for fetuses, the number of live fetuses, percent fetal deaths, fetal body weight, placental weights, and sex ratio in treated groups were comparable to those in the control group. Any treated groups were shown no adverse effects of test substance treatment in external, visceral and skeletal examinations of live fetuses.

Based on the results of absence of toxicity (maternal and fetuses) and absence of teratogenic findings, it is concluded that the dose level of 1000 mg/kg/day of 2,4,6-Tris(2,4,6-tribromophenoxy)-1,3,5-triazine is the no-observed-adverse-effect level (NOAEL) for maternal rats and fetuses.

In summary, from the generated scientific data and according to the REACH RegulationAnnex IX it can be concluded that long term exposure to FR-245 is not likely to have adverse effect on the fertility, developmental toxicity or nor cause systemic toxicity or affect the reproductive organs. Therefore, the registrantsbelieve that performance of the EOGRT is unnecessary scientifically, and unjustified for animal welfare reasons.

Effects on developmental toxicity

Description of key information

Based on these results, it is concluded that the dose level of 1000 mg/kg/day of 2,4,6-Tris(2,4,6-tribromophenoxy)-1,3,5-triazine is the no-observed-adverse-effect level (NOAEL) for maternal rats and fetuses. The prenatal developmental toxicity of 2,4,6-Tris(2,4,6-tribromophenoxy)-1,3,5-triazine is negative under the condition of this study. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Crl:CD(SD) rats, Specific Pathogen Free (SPF)
Temperature : (min 21.5 ℃, max 23.2 ℃)
Humidity : (min 47.6 %, max 53.7 %)
Air ventilation : (10 ∼ 15) changes/hr
12 hours light/dark cycle (light during 8:00 ∼ 20:00)
(150 ~ 300) Lux

Route of administration:

oral: gavage

Vehicle:

other: 0.5% sodium carboxymethyl cellulose solution

Details on exposure:

For each dose level, the specified amount of the test substance was weighed out and suspended in 0.5% sodium carboxymethyl cellulose solution (CMC, Sigma-aldrich., Lot No. SLBB5612V). The volume of dosing solutions administered to females was 10 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses for concentration of the test substance were performed for each dose level on samples taken from upper, middle and bottom in bottle before use. At the first day before administration, dosing solutions prepared for the low-dose, middle-dose and high-dose groups were analyzed for homogeneity of the test substance.
Chemical analyses were performed at the Health Care Research Laboratory, Korea Testing and Research Institute (KTR). The test substance in samples was extracted and diluted with toluene and analyzed using a ultra fast liquid chromatograph (UFLC, UFLCXR, Shimadzu, Japan) with a column, InertsilODS-3 (GL Science Inc., USA). The analytical method to be used for the determination of the test substance in the 0.5% CMC aqueous solution was validated in a method validation study (KTR/Study No. KG-2012-446).
The analyses demonstrated that the test substance concentrations were 99.5-105.3% to the claimed values for all dose groups and that the test substance was present in the samples with 1.7-2.8% coefficients of variation (Annex 7). These suggested that the preparation was performed properly and that test substance was distributed homogeneously in the dosing solutions.

Details on mating procedure:

Normally, 1:1 (one male to one female) mating was used in this study. The females were examined next morning for the presence of vaginal plugs and sperm in vaginal smears and those showing vaginal plugs and/or sperm were considered to have copulated (Day 0 of gestation). These mating procedures were repeated for 11 days. Pregnancy was determined with implantation marks on the uterus by ammonium sulfide staining when necropsy was performed.

Duration of treatment / exposure:

The duration of dosing was 15 days (from day 5 to day 19 of gestation).

Frequency of treatment:

Once a day (from day 5 to day 19 of gestation)

Duration of test:

Study initiation date : 2013-01-22
Animal receipt date : 2013-01-24
Quarantine and acclimatization periods : 2013-01-24 ~ 2013-02-04
Mating periods : 2013-02-05 ~ 2013-02-15
Administration period of adult animals : female : 2013-02-10 ~ 2013-02-28
Necropsy date : female : 2013-02-25 ~ 2013-03-01
Experiment completion date : 2013-05-09
Final report (Draft) preparation date : 2013-06-10
Study completion date : 2013-6-13

Remarks:

Doses / Concentrations:
250 mg/kg b.w.
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
500 mg/kg b.w.
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1000 mg/kg b.w.
Basis:
nominal conc.

No. of animals per sex per dose:

0 mg/kg b.w. : 22
250 mg/kg b.w. : 19
500 mg/kg b.w. : 19
1000 mg/kg b.w. : 20

Control animals:

yes, concurrent vehicle

Details on study design:

Test groups were identified by the color of a cage lavel ad follows: control group, white; low-dose group, yellow; middle-dose group, green; and high-dose group, red.
Dose levels in this study were selected as follows based on the results of preliminary study in rats, a dose range-finding study (KTR/Study No. KG-2012-464) : 0 (vehicle only), 500 and 1000 mg/kg/day.
In the dose range-finding study, 2,4,6-Tris(2,4,6-tribromophenoxy)-1,3,5-triazine was suspended in 0.5% carboxymethyl cellulose (CMC) solution and administrated orally, via gavage, to copulated Sprague-Dawley female rats (10 per group) once per day from days 5 through 14 of gestation at dose level of 0, 500 and 1000 mg/kg/day. Mean maternal body weight on day 15 of gestation in the 1000 mg/kg/day group was significantly decreased than that in the control group. However, no adverse effect of the test substance treatment were found in 500 and 1000 mg/kg/day groups in any of the parameters (mortality, clinical signs and food consumption). Observations at necropsy on day 15 of gestation and percent of implantation rate and fetal death were comparable to those in the control group.
Based on these results, the dose level of 1000 mg/kg/day was adopted as the high dose in this study. The dose levels of 500 and 250 mg/kg/day were selected as the middle and low dosage levels, respectively.
Each test group consisted of above 21 copulation females, to which an individual animal number was given as follows:

Maternal examinations:

Clinical signs and mortality
Body weight
Body weight gain
Food consumption
Necropsy, organ weights and tissue preservation

Ovaries and uterine content:

"The ovaries and uterus contents of females on day 20 of gestation were examined. For females having uterine conceptus, the gravid uterine weight and numbers of corpora lutea and implants were determined and recorded. Percent pre-implantation loss, post-implantation loss and implantation were calculated from the following formula:
* Implantation index(%) = No. of implantation/ No. of corpus lutea × 100
* Pre-implantation loss (%) = (No. of corpora lutea - No. of implantation)/No. of corpora lutea × 100
* Post-implantation loss (%)=(No. of implantation - No. of live fetuses) / No. of implantation × 100

Fetal examinations:

Number of live fetuses and percent of fetal deaths
Sex ratio, fetal body weight and placental weight
External, visceral and skeletal malformations and variations

Statistics:

Statistical analyses were performed by comparing the treatment groups with the vehicle control group using SPSS Statistical Analysis Systems (SPSS 19 Base, SPSS Korea Data Solution. Co. Ltd.). Data from the treated groups were statistically compared with those of the control groups using the following methods. Means and standard deviations were calculated. The data was analyzed for homogeneity of variances using Levene's test. One way ANOVA analysis was performed to evaluate the significance of differences. If tests showed statistical significance, the data were analyzed by the multiple comparison procedure of Dunnett's T3 or Scheffe's (Dunnett 1955, Scheffe, 1953). The percent implantation index, pre-implantation loss, post-implantation loss and feral death were evaluated statistically using the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952). Data were presented as frequencies, and they were analyzed by χ2-test followed by the Fisher's exact test where necessary. A difference was considered statistically significant at P<0.05 or P<0.01.

Indices:

* Fetal death (%) = (No. of resorption + No. of dead fetus)/No. of implantation × 100
* Sex ratio = No. of live male fetuses / No. of live female fetuses

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Effects of the test substance treatment on maternal rats were not found in any treated groups. Dark red coloration and swell of lung and foaming materials in trachea were caused by aspiration, and eosinophilic material in alveolus of lung was observed in histopathological examination. Hepatodiaphragmatic nodule of liver in the 250 mg/kg/day group was considered to be spontaneous because of the their low incidences, and capsular fibrosis of liver was observed in histopathological examination. Gray discoloration and smooth hardness of adrenal gland were observed in the 250 mg/kg/day group was not considered to be toxicological significance, since no treatment-related change was found in histopathological examination. Hair loss was observed in the 500 mg/kg/day group (Table 6). However, no statistically significant differences were found in the incidences of these findings between the control group and any of the treated groups.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other:

Remarks on result:

other: No effects were observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
no effects

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Any treated groups were shown no adverse effects of test substance treatment in external, viceral and skeletal examinations of live fetuses.

Remarks on result:

other:

Remarks:

No effects were observed

Key result

Developmental effects observed:

no

Treatment related:

no

Conclusions:

Based on these results, it is concluded that the dose level of 1000 mg/kg/day of 2,4,6-Tris(2,4,6-tribromophenoxy)-1,3,5-triazine is the no-observed-adverse-effect level (NOAEL) for maternal rats and fetuses. The prenatal developmental toxicity of 2,4,6-Tris(2,4,6-tribromophenoxy)-1,3,5-triazine is negative under the condition of this study.

Executive summary:

No effects of the test substance treatment were observed in maternal rats in any of the parameters examined.

At cesarean section, there were no treatment-related abnormalities in such parameters as mean gravid uterine weights, the numbers of corpora lutea and implantation, or the percent implantation index, pre- and post-implantation loss in any treated groups.

As for fetuses, the number of live fetuses, percent fetal deaths, fetal body weight, placental weights, and sex ratio in treated groups were comparable to those in the control group. Any treated groups were shown no adverse effects of test substance treatment in external, visceral and skeletal examinations of live fetuses.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Appropriate OECD guidline 414 and GLP

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available (further information necessary)

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

No effects of the test substance treatment were observed in maternal rats in any of the parameters examined.

At cesarean section, there were no treatment-related abnormalities in such parameters as mean gravid uterine weights, the numbers of corpora lutea and implantation, or the percent implantation index, pre- and post-implantation loss in any treated groups.

As for fetuses, the number of live fetuses, percent fetal deaths, fetal body weight, placental weights, and sex ratio in treated groups were comparable to those in the control group. Any treated groups were shown no adverse effects of test substance treatment in external, visceral and skeletal examinations of live fetuses.

Justification for selection of Effect on developmental toxicity: via oral route:
NOAEL>1000mg/kg/day for maternal and fetus toxicity. Noteratogenic effects found.