2,4,6-trichloro-1,3,5-triazine

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

OECD 421, Screening reproductive/developmental toxicity: NOAEL, fertility: 1000 ppm (90 mg/kg bw/day in males and 123 mg/kg bw/day in females)

OECD 443, EOGRTS: NOAEL, fertlity: 55 mg/kg/day (600 ppm); NOAEL, maternal: 16 mg/kg/day (200 ppm)

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

29.11.2017 to 18.07.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

other: Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) Males: 10 weeks; Females: 13 weeks
- Weight at study initiation: (P) Males: 256 - 295 g; Females: 196 - 250 g
- Housing:
On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21°C
- Humidity (%): 45 to 53%.
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12 hours

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DRINKING WATER PREPARATION
- Rate of preparation of drinking water solutions (frequency): at least weekly
- Storage temperature of drinking water solutions: refrigerator

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days at maximum
- Proof of pregnancy: vaginal plug or evidence of sperm in vaginal lavage referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations analyzed in the formulations of were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of low and high dose groups were homogeneous (i.e. coefficient of variation ≤ 10%). No test item was detected in the formulation samples of the control.

Duration of treatment / exposure:

Males were exposed for 30 days, up to and including the day of scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period.
Females with offspring were exposed for 51-57 days (most females) or 62-63 days (one female of Groups 2 and 3, respectively), i.e. 14 days prior to mating (tocover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 14-16 days after delivery, up to and including the
day of scheduled necropsy.
One non-pregnant female was treated for 48 days.

Frequency of treatment:

The test item was administered to the appropriate animals by inclusion in the drinking water ad libitum for 7 days a week.

Dose / conc.:

500 ppm

Remarks:

Concentration was reduced to 333 ppm during lactation. Corresponding mean test item intake over all study phases 43 or 61 mg/kg/day in males and females, respectively.

Dose / conc.:

1 000 ppm

Remarks:

Concentration was reduced to 667 ppm during lactation. Corresponding mean test item intake over all study phases 90 or 123 mg/kg/day in males and females, respectively.

Dose / conc.:

2 000 ppm

Remarks:

Concentration was reduced to 1333 ppm during lactation. Corresponding mean test item intake over all study phases 143 or 195 mg/kg/day in males and females, respectively.

No. of animals per sex per dose:

10 females / 10 males per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels for this study were selected based on the results of a 14-day dose range finder with drinking water administration of NHDT-solution in rats. Therefore, 6 animals per dose (3M/3F) were exposed to increasing concentrations from 600, 2000 to 7000 ppm. Treatment with 7000 ppm was ceased after a few days due to animal welfare reasons, instead animals were treated at 4000 ppm for 4 more days following a recovery period of 4 days. After these 4 days, one male and two females were sacrificed in extremis. Treatment for remaining animals was stopped, consequently.

As a result from the DRF, no local corrosive effects were observed after administration of the test item via inclusion in the drinking water, and water consumption was not markedly changed by dose levels up to 2000 ppm. Therefore, it was considered feasible to conduct toxicity studies with NHDTsolution via drinking water in Wistar Han rats. As severe health effects were observed at 4000 and 7000 ppm (absence of water consumption resulting in a significant reduced food consumption, body weight loss and signs of dehydration), a two-fold lower dose level was selected as highest suitable dose level for the reproductive/developmental toxicity screening test. Thus, selected dose levels for that study were 0, 500, 1000 and 2000 ppm.

The amount of test item incorporated in the drinking water was kept at a constant level in terms of ppm, throughout the study for males and during the pre-mating and post-coitum phases for females. During the lactation phase, the concentration of the test item in the
drinking water was gradually reduced due to the considerable increase in relative water consumption for females during lactation. After termination, the actual test item intake was estimated based on the body weight and water consumption values.

Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, or spilled drinking water from the bottles.

Parental animals: Observations and examinations:

Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the administration periods up to the day prior to necropsy. Animals were weighed individually on the first day of treatment, and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. Food consumption was quantitatively measured weekly except for the matign period. Water consumption was quantitatively measured daily from start of administration onwards up to the day of necropsy at approximately the same time except for the mating period.
Measurement of total T4 was conducted for F0-males.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.

Sperm parameters (parental animals):

For the testes of all males of control and high-dose groups, and the male that failed to sire 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 into the lumen. Any cell- or stagespecificity of testicular findings were noted.

Litter observations:

Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter. Clinical observations were performed at least once daily for all pups. Live pups were weighed individually on PND 1, 4, 7 and 13. Sex was externally determined for all pups on PND 1 and 4. Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight. All male pups in each litter were examined for the number of areola/nipples on PND 13.
Measurement of total T4 was conducted for PND 14-16 pups.

Postmortem examinations (parental animals):

All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs and possible local, corrosive effects on the GI tract. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present , nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.
The organs following organs were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated: Epididymis, coagulation gland, parathyroid gland, prostate gland, seminal vesicle, thyroid gland, testes.
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
All animals: Gross lesions/masses, cervix, epididymis, ovaries, testes, thyroid gland, uterus and vagina
Males that failed to sire and females that failed to deliver pups: Cervix, coagulation gland, epididymis, ovaries, prostate, seminal vesicles, testes, uterus and vagina.
All tissues as defined were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.

Postmortem examinations (offspring):

On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was
paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter (see also section 4.10.1), and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

For each group, the following calculations were performed. Group mean values of precoital time and duration of gestation were calculated from individual values of F0-females, the remaining group values were calculated from the total number in each group.
Mating index (%): (Number of females mated / Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females / Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1 / Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): (Total number of offspring born / Total number of uterine implantation sites) x 100
Post-implantation survival index is expressed as 100% when the number of offspring exceeds the number of implantation sites recorded.

Offspring viability indices:

Live birth index (%): (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
Viability index (%): Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling)) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Findings were limited to alopecia (for one female at 1000 ppm and several females at 2000 ppm females) and piloerection (for one female at 2000 ppm). These incidental findings occurred within the range of background findings in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these findings were regarded as unrelated to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males, body weight gain was statistically significantly reduced at all dose levels (dose-dependently) throughout the treatment period. This resulted in statistically significantly lower mean body weights at 1000 ppm during the mating period, and at 2000 ppm from Day 8 of the premating period onwards. The differences in mean body weights at the end of the treatment period were 5, 7 and 12% at 500, 1000 and 2000 ppm, respectively.

In females, slight body weight loss occurred at 1000 and 2000 ppm during the pre-mating period (on average 1 and 3% of the initial weight). The resulting differences in mean body weights on Day 1 of the mating period were small and not statistically significant (3 and 5% at 1000 and 2000 ppm, respectively).

During gestation, body weight gain and mean body weights of 2000 ppm females were statistically significantly reduced from post-coitum Day 11 onwards. Mean body weight was 15% lower at the end of the post-coitum period). Mean body weights of 2000 ppm females remained statistically significantly lower than those of controls until the end of the lactation phase (about 10%), but their body weight gain during lactation was comparable to that of controls.

Gestational body weight gain at 500 and 1000 ppm was also lower than controls (statistically significant at post-coitum Days 7-17 in 1000 ppm females), but mean body weights remained close to control values. The differences in mean body weights at the end of the post-coitum period were 3 and 6% difference at 500 and 1000 ppm, respectively, reaching no statistical significance. During lactation, body weights and body weight gain at 500 and 1000 ppm were considered not to be affected by treatment. The statistically significant differences at 1000 ppm (lower mean weight at lactation Day 1 and higher weight gain at lactation Day 13) were regarded to reflect normal biological variation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males at 2000 ppm consumed about 13% less food than controls throughout the treatment period. After correction for body weight, their food consumption was about 7% lower in the premating period and close to the control values thereafter, reaching no statistical significance. Food consumption of males at 500 and 1000 ppm was considered not to be affected by treatment.

Females at 2000 ppm consumed slightly less food than controls during gestation, reaching statistical significance from post-coitum Day 7 onwards. After correction for body weight, their overall food consumption during gestation was about 5% lower than controls (not statistically significant). A tendency towards lower food intake during gestation was also noted in females at 1000 ppm, reaching statistical significance between post-coitum Days 11-17. After correction for body weight, their overall food consumption during gestation was about 8% lower than
controls (not statistically significant). Food consumption of 1000 and 2000 ppm females during the premating and lactation periods was not affected by treatment. Food consumption of females at 500 ppm was comparable to that of controls throughout the study.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Water consumption (before and after correction for body weight) of males and females at 2000 ppm was reduced throughout the study, reaching statistical significance at incidental time intervals. In males, the overall differences after correction for body weight were 26% during pre-mating and post-mating period, respectively compared to controls. In females, the overall differences after correction for body weight were 25%, 15% and 8% during the premating, post-coitum and lactation period, respectively, when compared to controls. Water consumption of males and females at 500 and 1000 ppm was considered to be unaffected by treatment. Any statistically significant changes at 500 and 1000 ppm at incidental time intervals were considered to be unrelated to treatment as these occurred in absence of a dose-related trend and did not persist over time.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serum levels of T4 in F0-males were considered not to be affected by treatment.
The statistically significantly higher mean T4 value noted at 1000 ppm (about 28%) was considered to be unrelated to treatment due to the lack of a dose-related trend (relative difference of 16% at 2000 ppm) and as all values remained within the historical control range.

Historical control data total T4 values (μg/dL) in male Wistar Han rats (Period 2015 – Jun 2017)
Mean = 4.65, P5 – P95 = 2.97 – 6.44 (n=448)

Urinalysis findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were not affected by treatment. All females had regular cycles of four days.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

One female of the 500 ppm group was not pregnant despite evidence of mating. Although the genitals of this male were reduced in size, normal histopathology was observed. No abnormalities were seen in the reproductive organs of these animals which could account for the non-pregnancy of the female.

Number of implantation sites:
Mean number of implantation sites were 11.2, 12.3, 13.3 and 10.3 for the control, 500, 1000 and 2000 ppm groups, respectively. The mean number of implantation sites at 2000 ppm was lower compared to the mean number in the other groups and the historical control mean. No statistical significance was reached.
Fertility index:
Fertility index was not affected by treatment. Except for one female at 500 ppm (no. 58), all females were pregnant. The fertility indices were 90% for the 500 ppm group and 100% for the other groups.
Gestation index:
Gestation index and duration of gestation were not affected by treatment. All pregnant females had live offspring (gestation index 100% for all groups).
Parturition/maternal care:
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Post-implantation survival index:
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 93, 95, 95% and 84% for the control, 500, 1000 and 2000 ppm groups, respectively. At 2000 ppm, the post-implantation survival index was lower than the other groups. As a similarly low post-implantation survival index occasionally occurs in historical controls and histopathology showed no test item-related changes in reproductive organs, the lower index at 2000 ppm might reflect normal biological variation. However, taken in context of the lower litter sizes and number of implantation sites of several 2000 ppm females, it cannot be ruled out that the lower post-implantation survival index at 2000 ppm was test item-related.


Historical control data for implantation sites in Wistar Han rats (Period 2015 – Jun 2017):
Mean = 12; P5 - P95 = 5 – 16 (n=486)

Historical control data for post-implantation survival index in Wistar Han rats (period 2015 – Jun 2017):
Mean = 93%; P5 - P95 = 84 – 100% (n=48 studies)

Exposure to the test item was associated with reductions in body weight (gain), food consumption and water consumption as described below:
At 2000 ppm, slight body weight loss (average of -3%) was noted for females during the premating period and body weight gain was reduced in males throughout the study and in females during the pre-mating and gestation period. The resulting lower mean body weights compared to control means were 12% in males (end of treatment) and in females 5% (end of pre-mating period), 15% (end of gestation) and 10% (end of lactation). These effects were accompanied by reductions in food and water consumption. After correction for body weight, males had a 7% reduced food consumption during the premating period and females had a 5% reduced food consumption during gestation. Water consumption, after correction for body weight, was reduced throughout the treatment period up to about 25% in both sexes. The reduced body weight gain was regarded as adverse since the decreases in mean body weights exceeded 10% and as no signs of recovery were observed during the study period.

Lower body weight gain was also noted in males and females treated at 500 and 1000 ppm. As the resulting lower mean body weights compared to control means were small (maximum of 7%), the lower weight gain at 500 and 1000 ppm was considered not to be toxicologically relevant. Except for a tendency towards lower food consumption in 1000 ppm females during gestation, food consumption at 500 and 1000 ppm and water consumption were not affected by treatment.

At 2000 ppm, the mean number of implantation sites was lower compared to controls which was caused by 4/10 females having less than 10 implantation sites (varying from 4 to 9 implantation sites). Although changes were slight and not statistically significant, a relationship to treatment with test item cannot be excluded.
No treatment-related changes were noted in the other reproductive parameters examined (i.e. mating and fertility indices, precoital time, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Key result

Dose descriptor:

NOAEC

Effect level:

1 000 ppm (analytical)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

2 000 ppm

System:

female reproductive system

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. The clinical signs observed remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Litter size:
The mean numbers of living pups at first litter check (live litter size) were 10.3, 11.7, 12.7 and 8.6 in the control, 500, 1000 and 2000 ppm groups, respectively.Mean litter size at 2000 ppm was lower compared to both the concurrent control value and the historical control mean. Statistical significance was not achieved, probably due to high standard deviations in the control and 2000 ppm groups. The small litter sizes correlated with lower numbers of implantation sites at 2000 ppm.
Live birth index:
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices were 100% for Group 3 (1000 ppm) and 99% for the other groups. At first litter check, one control pup, one pup at 500 ppm and one
pup at 2000 ppm were found dead. This incidental pup mortality was regarded as unrelated to treatment as the incidence showed no dose-related trend and remained within normal limits.
Viability index:
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not to be affected by treatment. The viability indices were 100, 100, 98 and 99% for the control, 500, 1000 and 2000 ppm groups, respectively. One pup each at 1000 ppm and at 2000 ppm were found dead on PND 2 or 3 and one pup at 1000 ppm was euthanized on PND 1 due to a wound at its hind leg. This post-natal loss was considered not to be related to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.
Lactation index:
Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. No pups died after PND 4, resulting in a lactation index of 100% for all groups.

Historical control data for live litter size in Wistar Han rats (period: 2015 – Jan 2018):
mean = 11.4; P5 - P95 = 6.0 - 15.0 (n=794).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment.
Mean pup weights (both males and females) at 500 and 2000 ppm were statistically significantly lower than control values during the entire post-natal period. Mean combined weights at 500 and 2000 ppm differed 14-11% on PND 1 and 10-9% on PND 13, respectively, when compared to concurrent controls. As the differences showed no doserelated trend and the weights of treated pups remained within the normal range, these findings were considered to reflect normal biological variation and not an effect of treatment.

Historical data for body weights (g) of pups of Wistar Han rats (period 2015 – Jan 2018):
PND 1 male pups : mean = 6.3; P5 - P95 = 5.2 - 7.5 (n = 4527)
PND 13 male pups : mean = 30.4; P5 - P95 = 25.8 - 35.1 (n = 3072)
PND 1 female pups: mean = 6.0; P5 - P95 = 5.0 - 7.2 (n = 4507)
PND 13 female pups: mean = 29.5; P5 - P95 = 25.0 - 34.3 (n = 2997)

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment.
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.
Treatment up to 2000 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the findings noted remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Developmental toxicity was observed at 2000 ppm and consisted of a reduction in live litter size, particularly in 4/10 females which had only 3-6 pups (i.e. at or below the historical control range) which was related to the lower number of implantation sites. Additionally, post-implantation survival index for the 2000 ppm group was reduced compared to controls (i.e. at the historical control range). There were no explanatory morphological findings in reproductive organs and the lower values might reflect normal biological variation. However, it cannot be ruled out that the lower post-implantation survival and litter size was related to treatment with test item.
The developmental effects occurred at a dose level associated with parental growth retardation (accompanied by reduced food and water consumption). However, the developmental toxicity could not be unequivocally attributed to maternal toxicity because parental general health was not affected and the reductions in maternal body weight and food consumption were modest during gestation.
No treatment-related changes were noted in the other developmental parameters examined (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, clinical signs, body weight, anogenital distance (PND 1), areola/nipple retention (PND 13 males), serum T4 thyroid hormone (PND 14-16) and macroscopic examination).

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

1 000 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Key result

Critical effects observed:

no

Conclusions:

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following No Observed Adverse Effect levels (NOAELs) of NHDT-solution (4,6- dichloro-1,3,5-triazin-2(1H)-one, sodium salt) were established:
Parental NOAEL: 1000 ppm, correlated to a mean test item intake level of 90 mg/kg bw/day in males and 123 mg/kg bw/day in females.
Reproduction NOAEL: 1000 ppm, correlated to a mean test item intake level of 90 mg/kg bw/day in males and 123 mg/kg bw/day in females.
Developmental NOAEL: 1000 ppm, correlated to a mean test item intake level of 90 mg/kg bw/day in males and 123 mg/kg bw/day in females.

Based on these results, suggested dose levels for the subsequent extended one generation reproductive toxicity study in rats are 0, 200, 600 and 2000 ppm.

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

In life: 13 June 2018 - 24 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3. test method: EU B.56./OECD TG 443) in rats, oral route; with the registered substance specified as follows:
- Ten weeks premating exposure duration for the parental (P0) generation:
Ten weeks premating exposure duration is required because there is no substance specific information in the dossier supporting shorter premating exposure duration as advised in the ECHA Guidance on information requirements and chemical safety assessment R.7a, chapter R.7.6 (version 4.1, October 2015).
- Dose level setting shall aim to induce some toxicity at the highest dose level:
The dose levels in this study were selected based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) with NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1h)-one, sodium salt) via drinking water administration in rats, Test Facility Study No. 519710), and in an attempt to produce graded responses to the test item. In this preliminary study, treatment at 2000 ppm (corresponding to an average test item intake of 143 mg/kg/day in males and 195 mg/kg/day in females) resulted in a slight body weight loss in females during pre-mating and a reduced body weight gain (both sexes). Mean body weights were about 10% lower than controls. These effects were accompanied by a reduced food and water consumption (about 5% and 25%, respectively, after correction for body weight and when compared to controls). Moreover, the number of implantation sites at 2000 ppm was lower, which resulted in a lower mean litter size and a lower post-implantation survival index. The effects at 500 and 1000 ppm were limited to slightly lower body weights and body weight gains (both sexes), and a slightly lower food consumption in females at 1000 ppm during gestation only.
- Cohort 1A (Reproductive toxicity)
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation:
The conditions to include the extension of Cohort 1B are currently not met. Furthermore, no triggers for the inclusion of Cohorts 2A and 2B (developmental neurotoxicity) and Cohort 3 (developmental immunotoxicity) were identified. No information on other studies justified the inclusion of Cohorts 2A and 2B and Cohort 3.
- Route of administration:
The oral route is the most appropriate route of administration for substances except gases to focus on the detection of hazardous properties on reproduction as indicated in ECHA Guidance on information requirements and chemical safety assessment (version 4.1, October 2015) R.7a, chapter R.7.6.2.3.2. The administration of the first hydrolysis product NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1h)-one, sodium salt) via inclusion in the drinking water was considered to be the most suitable route based on analytical results and the corrosive effects of cyanuric chloride.

Species:

rat

Strain:

other: Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental internal data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 7 wks
- Weight at study initiation: (P) Males: 143-185 g; Females: 111-149 g
- Housing: On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam until termination or weaning (on PND 21).
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum except before blood sampling with a maximum of approximately 24 hours.
- Water: tap water (controls) or Drinking water preparations, containing the test substance (test groups)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 47-67
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 June 2018 To:24 December 2018

Route of administration:

oral: drinking water

Details on exposure:

JUSTIFICATION OF ROUTE:
The oral route of exposure was selected as specified in the final decision on a compliance check by ECHA. Due to its corrosive properties, oral gavage was not considered an appropriate route of expsoure for cyanuric chloride (CYC). Furthermore, It was technically not feasible to prepare diets with CYC, due to its hydrolytical instability. Therefore it was decided to perform a study with exposure via drinking water with the first hydrolysis product NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1h)-one, sodium salt).

PREPARATION OF DOSING SOLUTIONS:
The test item was diluted with the required amount of municipal tap water. Drinking water solutions were prepared at least weekly and were stored in the refrigerator for a maximum of 8 days if not used on the same day. The preparations were removed from the refrigerator and acclimatized for at least 30 minutes at room temperature before administration. From Week 3 of treatment onwards, after stability over 8 days at room temperature was confirmed, drinking water solutions were prepared at least weekly and stored at room temperature for a maximum of 6 days before use. No adjustment was made for specific gravity of the test item. A correction factor of 12.5 was used to correct for the purity/composition of the test item.

Details on mating procedure:

- M/F ratio per cage: 1:1 within the same treatment group, avoiding sibling mating after at least 10 weeks of treatment
- Length of cohabitation: until mating, maximally 2 weeks
- Proof of pregnancy: evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug referred to as day 0 of pregnancy

Detection of mating was not confirmed in first instance for two control females, one female at 16 mg/kg/day, one female at 55 mg/kg/day and two females at 160 mg/kg/day. Evidence of mating for these animals was obtained indirectly by delivery of a litter. The mating date of these animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum. For one female at 16 mg/kg/day, evidence of mating was obtained by one implantation site recorded at necropsy; a mating date for this animal could not be estimated. Apparently, mating was overlooked in the assessment of the vaginal lavage of these animals, which explains the continuation of di-estrus during the mating in these females.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Drinking water preparation samples were collected for analysis in week 1, 11 and 22. Test substance concentration was analysed for all groups, homogeneity was analysed in low and high dose group samples. Additional drinking water solutions were prepared in Week 1 of treatment, in order to conduct stability analysis over a concentration range that covers the complete study period; those drinking water preparations were not used for administration to the animals.
Analyses were performed using a validated analytical procedure (Test Facility Study No. 519709). Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was equal or less than 10%. During the course of this study at one occasion during the treatment phase, stability of the prepared drinking water containing test item was determined for 8 days at room temperature.
Duplicate sets of each sample (approximately 500 mg) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 519709) demonstrated that the test item is stable in tap water for 2 hours at room temperature and 8 days in the refrigerator when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

P-males: 11-12 weeks, including 10 weeks prior to mating.
P-females: 16-17 weeks, including 10 weeks prior to mating
P-females which failed to deliver: 14-16 weeks
F1 Cohort 1A: 9-11 weeks
F1 Cohort 1B: 10-11 weeks
F1 Cohort 1C: 2-5 weeks
F1 Cohort Surplus: not applicable
Cohort 1C is included to obtain a sufficient amount of animals for the assessment of sexual maturation (i.e. the same number of animals that would have been available for assessment of sexual maturation in case cohorts 2 and 3 were included).

Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, via spilled water from the water bottles or when they commenced drinking from the water bottles themselves.
From weaning onwards (PND 21), F1-animals received drinking water with test item up to and including the day before scheduled necropsy (Cohort 1A) or until and including the day of necropsy (Surplus, Cohorts 1B and 1C).

Frequency of treatment:

The amount of test item incorporated in drinking water was kept at a constant level in terms of ppm, throughout one specified phase of the study period. After termination, the actual test item intake was estimated based on the body weight and water consumption values. The drinking water solutions were refreshed at least every other day at approximately the same time.

Dose / conc.:

200 ppm

Remarks:

Equal to actual mean test item intakes of 16 and 22 mg/kg bw/day for males and females, respectively; Target dose level 16 mg/kg bw/day

Dose / conc.:

600 ppm

Remarks:

Equal to actual mean test item intakes of 51 and 74 mg/kg bw/day for males and females, respectively; Target dose level 55 mg/kg bw/day

Dose / conc.:

2 000 ppm

Remarks:

Equal to actual mean test item intakes of 166 and 239 mg/kg bw/day for males and females, respectively; Target dose level 160 mg/kg bw/day

No. of animals per sex per dose:

P-animals: 25
F1-animals: 20 for each cohort (1A, 1B, 1C), 10 as surplus

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels in this study were selected based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) with NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1h)-one, sodium salt) via drinking water administration in rats (Test Facility Study No. 519710), and in an attempt to produce graded responses to the test item.
In this preliminary study, treatment at 2000 ppm (corresponding to an average test item intake of 143 mg/kg/day in males and 195 mg/kg/day in females) resulted in a slight body weight loss in females during pre-mating and a reduced body weight gain (both sexes). Mean body weights were about 10% lower than controls. These effects were accompanied by a reduced food and water consumption (about 5% and 25%, respectively, after correction for body weight and when compared to controls). Moreover, the number of implantation sites at 2000 ppm was lower, which resulted in a lower mean litter size and a lower post-implantation survival index. The effects at 500 and 1000 ppm were limited to slightly lower body weights and body weight gains (both sexes), and a slightly lower food consumption in females at 1000 ppm during gestation only.
Based on the findings of the preliminary study, the highest concentration in the main study was established at 2000 ppm, and was expected to induce some degree of toxicity, but not to compromise the study objectives. The lowest concentration, 200 ppm, was expected to represent a NOAEL.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to first administration and at least once daily from start of administration onwards, up to the day prior to necropsy. In addition, clinical observations were conducted in a standard arena once before the first administration of the test item and at weekly intervals during the treatment period

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to administration), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.
A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0-4, 4-7, 7-11, 11-14, 14-17 and 17-20 post-coitum and during lactation over Lactation Days (LD) 1-4, 4-7, 7-14 and 14-21.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was quantitatively measured at least over every two days, from start of administration onwards up to the day of necropsy, except for males and females which were housed together for mating and for females without evidence of mating. Water consumption was also measured on the days the concentration of test item in the drinking water was changed. For mated females, water consumption was measured daily during post-coitum and lactation. During lactation, water consumption was determined daily.

OTHER:
General Reproduction Data – P-Generation: From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

Oestrous cyclicity (parental animals):

Estrous stages were determined by examining the cytology of vaginal lavage samples. Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of scheduled necropsy, a vaginal lavage sample was also taken.

Sperm parameters (parental animals):

For all surviving males, the following assessments were performed:
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples. One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples. As for two animals abnormalities were noted in the left epididymis, the left side organ(s) was fixed in modified Davidson's solution, and the right side organ was used for evaluation of sperm numbers.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); surplus pups were killed and discarded (after blood collection in some pups, see below).
- On PND 4 at culling, blood was collected from two surplus pups per litter (from all litters, if possible) by decapitation, and samples were pooled per litter. If available, blood was collected from one male and one female pup per litter. If only one surplus pup per litter was available at culling, as much as possible blood was collected from this single pup. As for several litters the target volume of 0.5 mL was not reached by pooling two pups, a third surplus pup of the same litter was added.

PARAMETERS EXAMINED
The following parameters were performed for the F1 pups until weaning (PND 21):
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups,

On PND 4, the pups scheduled for culling (in case of > 8 pups per litter) were euthanized by decapitation. Sex was determined both externally and internally. Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.
Spare F1-animals which were not assigned to one of the Cohorts were sacrificed between PND 23-25 by oral administration of sodium pentobarbital (Euthasol® 20%). Animals were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex was determined (both externally and internally). Descriptions of all external abnormalities were recorded. External abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director.

GROSS EXAMINATION OF DEAD PUPS:
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally) and externally examined with emphasis on developmental morphology.
Descriptions of all external abnormalities were recorded. External abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

ASSESSMENT OF REPRODUCTIVE/DEVELOPMENTAL ENDPOINTS:
The in-life procedures, observations, and measurements listed below were performed for all F1-animals from weaning (PND 21) onwards, except for the animals of Cohort Surplus and spare F1-animals as these were terminated on PND 22-24 or 23-25:
Mortality, clinical oibservations, including arena observations, body weights, food consumption, water consumption, vaginal patency (females), balanopreputial separation (males), stage of estrus determination (females).
Cohort 1A: Estrous cycle determination (females),

Blood and urine samples
Blood of 10 selected animals/sex/group of P-animals and Cohort 1A animals was collected on the day of scheduled necropsy, for hematology, coagulation, clinicl chemistry and thyroid hormone analysis. On PND 4 at culling, blood was collected from two surplus pups per litter, and samples were pooled per litter. On PND 22-24, blood was collected from all Cohort Surplus animals (10/sex/group), if possible. These F1 culled pups and cohort surplus samples were used for thyroid hormone analysis only.
Urine was collected into a specimen vial from the 10 selected animals/sex/group of P-animals and Cohort 1A animals housed in individual metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
Cohort 2 was not included in this study.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
Cohort 3 was not included in this study.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, after successful mating or at the end of the mating period
- Maternal animals: All surviving animals at lactation day 23-25.

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

HISTOPATHOLOGY / ORGAN WEIGHTS
The organs identified in attachment 1 were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated. Representative samples of the tissues identified in attachment 1 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated.

Postmortem examinations (offspring):

SACRIFICE
- F1 animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

Cohort 1A (fasted), PND 89-95: macroscopy and organ weights (20/sex/dose), histopathology (full list for controls and high dose, gross and target tissues for low and mid dose animals)
Cohort 1B (non-fasted), PND >90: macroscopy and organ weights (19/sex/dose), no histopathology in first instance
Cohort 1C (non-fasted), after positive vaginal patency.balanopreputial separation: macroscopy (19-20/sex/dose)
Cohort Surplus (non-fasted), PND 22-24: macroscopy and organ weights (10/sex/dose)
Non-selected F1 and spare litters (non-fasted), PND 23-25: macroscopy and tissue collection

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The organs identified in attachment 1 were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated. Representative samples of the tissues identified in attachment 1 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated.

OTHER – Cohort 1A
The following additional assessments were performed:
Sperm analysis (al males), splenic lymphocytic subpopulation anaylsis (10/sex/group; 1/sex/litter)
In addition to the procedures described above, HE stained step sections of ovaries and corpora lutea at a thickness of 5 micrometers (5 step sections in total, including the routine section) were prepared. One of the ovaries will be quantitatively evaluated for follicles (primordial and small growing follicles counted together), as well as corpora lutea initially.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Incidence: An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

Mating index males (%): (Number of males mated/Number of males paired) x 100
Mating index females (%): (Number of females mated/Number of females paired) x 100
Precoital time: (Number of days between initiation of cohabitation and confirmation of mating
Fertility index males (%): (Number of pregnant females/Number of males mated) x 100
Fertility index males (%): (Number of pregnant females/Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1/Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): (Total number of offspring born/Total number of uterine implantation sites) x 100

Offspring viability indices:

Live birth index (%): (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100
Weaning index (%): (Number of live offspring on Day 21 after littering/Number live offspring on Day 4 (after culling)) x 100
Percentage live males at weaning (%): (Number of live male pups on Day 21 after littering/ Number of live pups on Day 21 after littering) x 100
Percentage live females at weaning (%): (Number of live female pups on Day 21 after littering/ Number of live pups on Day 21 after littering) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental findings that were noted included alopecia, wounds and scabs. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered not to be signs of toxicological relevance.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights and body weight gain of treated animals were affected by treatment at 16, 55 and 160 mg/kg/day.
In males, body weights and body weight gain were statistically significantly lower than controls at 160 mg/kg/day from Week 2 of the pre-mating period onwards. Mean body weight gain was 0.79x and 0.78x of controls at the end of the pre-mating and mating period, respectively, resulting in a terminal body weight (on Day 80-85) of 0.87x of controls.
At 55 mg/kg/day, lower body weights were observed from Week 10 of pre-mating until Week 2 of mating, hereafter body weights appeared to recover for the remaining animals. At 16 mg/kg/day, lower body weights were observed from Week 9 of pre-mating until Day 1 of mating, followed by recovery to normal body weights. At necropsy, terminal body weights were lower with 0.94x and 0.93x compared with controls for treatment groups 16 and 55 mg/kg/day, respectively.
In females treated at 160 mg/kg/day, body weights were statistically significant lower than controls from Week 4 of the pre-mating period onwards. Mean body weight gain was 0.75x, 0.74x and 0.70x of controls at the end of the pre-mating, beginning of mating and end of post-coitum period, respectively, resulting in mean body weights of 0.91x, 0.91x and 0.83x of controls, respectively. Despite the still statistically significantly lower body weights at the end of the lactation period (0.92x), body weight gain increased during the lactation period for these animals, resulting in statistically significantly increased body weight gain at the end of the lactation period (2.1x).
In females treated at 55 mg/kg/day, body weights and body weight gains were lower than controls from Week 8 of treatment until Week 1 of the mating period. Mean body weight gain was 0.91x-0.89x of controls from Week 8 until Week 1 of mating, resulting in mean body weights of 0.96x (not statistically significant)-0.95x, respectively. Despite the still statistically significantly lower body weights on most days during the lactation period (0.93-0.95x), body weight gain increased during the lactation period for these animals, resulting in statistically significantly increased body weight gain at the end of the lactation period (1.5x).
Body weights and body weight gain for females at 16 mg/kg/day were considered to be unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was not affected by treatment up to 160 mg/kg/day in males and up to 55 mg/kg/day in females.
In females at 160 mg/kg/day, absolute food consumption levels were lower compared with controls during the post-coitum and lactation phase (0.86x and 0.88x, respectively). As relative food consumption levels were not affected, the lower food consumption levels were considered to be the result of the lower body weights observed for females during the post coitum period.
Any other statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment during the pre-mating and mating period.

Water consumption and compound intake (if drinking water study):

effects observed, non-treatment-related

Description (incidence and severity):

Water consumption was affected by treatment up to 160 mg/kg/day in males and females.
In males, water consumption before correction for body weights was considered to be unaffected during the pre-mating and the mating period. The incidental changes observed in the two-daily water consumption measurements were considered to have arisen as a result of naturally occurring day to day variation and to be unrelated to treatment. The water consumption corrected for body weight was increased on multiple days of treatment at 16, 55 and 160 mg/kg/day.
In females, water consumption was considered to be increased, particularly during the first 5 weeks, during the premating and the mating period at treatment with 55 and 160 mg/kg/day. During the post-coitum and the lactation period, water consumption at 160 mg/kg/day was reduced on most days. The water consumption corrected for body weight was increased on multiple days of treatment at 16, 55 and 160 mg/kg/day during the pre-mating period.
Taken into account the direction of the change and in absence of corroborative morphological alterations, the increased (relative) water consumption was considered non-adverse.
Test item concentration was adjusted during the study to account for periodic changes in water consumption and body weight. The adjustments were based on the DRF study and historical control data.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males, a decrease in mean corpuscular volume and mean corpuscular haemoglobin at 55 and 160 mg/kg/day and an increase in red blood cells at 160 mg/kg/day were noted. The increase in red blood cells may have been related to the increased incidence and severity in extramedullary hematopoiesis observed in the spleen at 160 mg/kg/day. In females, changes consisted of a decrease in lymphocytes, red blood cell distribution width and platelets at 160 mg/kg/day.
The expression of the haematological changes were mild in comparison with the controls and were in absence of degenerative changes; they were therefore considered non-adverse changes.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry changes consisted of an increase in total protein in males (55 and 160 mg/kg/day) and a decrease in creatinine in males (160 mg/kg/day) and females (at 55 and 160 mg/kg/day). The observed changes were considered slight and without morphological correlates.
In addition, an increase was noted in total T4 levels in males (55 mg/kg/day) and females (55 and 160 mg/kg/day). This increase in total T4 levels in males may have been related to the increased incidence and/or severity of follicular cell hypertrophy of the thyroid gland in males at 160 mg/kg/day, though this does not explain the increase in total T4 levels in females.
These changes were therefore considered non-adverse.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 160 mg/kg/day, an increase in pH (1.09x control) was noted in females, which was slightly above the upper limit of the historical control range.
In absence of corroborative microscopic findings, the effect on pH was considered non-adverse.
At 16 mg/kg/day, a decrease in specific gravity noted in males, was considered to have been the result of slightly increased control levels which may have been the result of the somewhat lower urine volumes and therefore unrelated to treatment.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Thyroid gland: an increased incidence and/or (mean) severity of follicular cell hypertrophy and colloid alteration was present in males treated at 160 mg/kg/day.
In males at 160 mg/kg bw/day, follicular cell hypertrophy was minimal in 12/25 animals, and slight in 7/25 (controls: 15/25 minimal, 3/25 slight), the colloid alteration was minimal in 12/25 animals, and slight in 1/25 (controls: minimal 5/25, slight 0/25).

Spleen: an increased incidence and/or (mean) severity in extramedullary hematopoiesis and pigmentation was seen in males and females at 160 mg/kg/day.
In males at 160 mg/kg bw/day, extramedullary hematopoiesis was minimal in 14/25 animals, slight in 9/25, and moderate in 1/25 (controls: 13/25 minimal, 2/25 slight, 0/25 moderate), the pigmentation was minimal in 1/25 animals, slight in 16/25, and moderate in 8/25 (controls: minimal 13/25, slight 11/25 and moderate 1/25). In females at 160 mg/kg bw/day, extramedullary hematopoiesis was minimal in 7/25 animals, slight in 1/25, and moderate in 1/25 (controls: 2/25 minimal, 0/25 slight, 0/25 moderate), the pigmentation was minimal in 1/25 animals, slight in 15/25, and moderate in 9/25 (controls: minimal 14/25, slight 11/25 and moderate 0/25).

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Sperm motility, concentration, morphology and spermatogenesis were considered not affected by treatment.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

At 160 mg/kg/day, the number of non-pregnant females versus mated females was considered increased compared with controls. The number of non-pregnant females was 0/25, 1/25, 0/24 and 5/24 in the control, 16, 55 and 160 mg/kg/day groups, respectively, resulting infertility indices of
100%, 96%, 100% and 79%, respectively. As fertility is affected by body weight, the decreased fertility index may have been the result of the decreased body weights and body weight gains of the females observed at 160 mg/kg/day. No effects on sperm parameters, estrous cycle and microscopic correlates were noted that could have explained the decreased fertility index.


No test item-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating, precoital time, number of implantations and histopathological examination of reproductive organs).
Gestation indices were 100%, 96%, 100% and 100% for the control, 16, 55 and 160 mg/kg/day groups, respectively. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 95%, 95%, 94% and 93% for the control, 16, 55 and 160 mg/kg/day groups, respectively. The live birth indices were 100%, 100%, 100% and 99% for the control, 16, 55 and 160 mg/kg/day groups, respectively. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

for details on results see overall remarks and attachments

Key result

Dose descriptor:

NOAEL

Effect level:

55 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Key result

Dose descriptor:

NOAEL

Effect level:

16 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No mortality occurred that was considered to be attributable to treatment with the test item.
One male at 55 mg/kg/day was sacrificed in extremis on Day 18 post weaning, based on abnormal posture and/or gait, tilted head, a lean appearance and slightly lower body weight. At macroscopy, a misshapen spinal cord was the only relevant finding, correlating to the observations made in-life for this animal.
One male at 160 mg/kg/day was sacrificed in extremis on Day 32 post weaning, based on overgrowth teeth, causing food intake issues and resulting in lower body weight (gain) and slight body weight loss (-5%) in the week prior to sacrifice. At macroscopy, overgrown teeth were confirmed and in addition, enlarged mandibular lymph nodes were observed.
On Day 9 post weaning, one animal (dosed at 16 mg/kg/day) appeared to be a male animal instead of a female animal and was sacrificed.
These deaths were all considered to be unrelated to treatment.

Viability indices (number of live offspring on Day 4 before culling compared to the number of offspring on Day 1) were thus 99%, 98%, 96% and 98% for the control, 16, 55 and 160 mg/kg/day groups, respectively. The weaning indices (number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling)) were 99%, 100%, 100% and 100%% for the control, 16, 55 and 160 mg/kg/day groups, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 160 mg/kg/day, body weights of male and female pups were statistically significant decreased during the complete lactation period. The decreased body weights were likely the result of the decreased body weights observed for the dam during the post coitum period. As the body weights of pups did not recover to normal levels by the end of the lactation period, this finding was considered adverse.
Post-weaning, test item-related statistically significant decreased body weights were observed in males from 16 mg/kg/day onwards and in females from 55 mg/kg/day onwards and test item-related decreased body weight gains were observed in males at 160 mg/kg/day. The decreased body weights at the start of weaning were considered to have arisen from the slightly decreased body weights at PND 21. As body weight gains did not recover to normal levels for males at 160 mg/kg/day, the decreased body weight and body weight gain for males at 160 mg/kg/day was considered adverse.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

At 160 mg/kg/day, food intake was lower throughout the treatment period, when compared with controls. Mean food consumption in Week 10 of treatment was 0.88x and 0.78x for males and females, respectively. For males at 55 mg/kg/day, food intake was also statistically significantly lower on several occasions during the treatment period, with the mean food consumption being 0.96x of controls in Week 10.
Food consumption after correction for body weight was statistically significantly increased for males at 55 mg/kg/day (on some occasions) and for both sexes at 160 mg/kg/day.
The lower food consumption levels were considered the result of the lower body weights of these animals and was therefore considered non adverse.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, non-treatment-related

Description (incidence and severity):

Water consumption was lower during the complete treatment period for animals treated at 160 mg/kg/day, reaching statistical significance on most occasions for the males and on approximately half of the occasions for the females. Mean water consumption over the complete treatment period was 0.87x and 0.88x of controls for males and females, respectively.
The lower water consumption levels were considered the result of the lower body weights of these animals and was therefore considered non adverse.
Test item concentration was adjusted during the study to account for periodic changes in water consumption and body weight. The adjustments were based on the DRF study and historical control data.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Similar to the P-generation, non-adverse changes were noted in males of the F1-generation for hematology and clinical chemistry parameters. Test item-related changes in red blood cell distribution width and decreased mean corpuscular haemoglobin and potassium at 160 mg/kg/day, were considered non-adverse, in absence of correlating microscopic alterations and at the low magnitude of the effects.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significant increases in reticulocytes observed in males and females at 160 mg/kg/day, were possibly related to the observed extramedullary hematopoiesis and/or pigmentation in the spleen. As the observed slight alterations were in absence of degenerative changes, these effects were considered to be non-adverse.
Serum T4 levels in male and female pups culled at PND 4 were considered not to be affected by treatment. Serum T4 levels in male and female pups of Cohort Surplus at PND 22-24 were statistically significantly higher at 160 mg/kg/day. A statistically significant increase was noted in total T4 levels in males and females (at 55 and 160 mg/kg/day). Also, this increase in total T4 levels in males may have been related to the thyroid gland findings in males observed at 160 mg/kg/day, though this does not explain the increase in total T4 levels in females. As all values were considered within normal range and in absence of degenerative changes, the changes in total T4 levels were considered non-adverse.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinary parameters were unaffected by treatment.

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

The age at which balanopreputial separation (prepuce opening) occurred in males was unaffected by treatment.
The age at which vaginal patency (vaginal opening) was attained in females was unaffected by treatment up to 160 mg/kg/day. A statistically significant increase in the age of reaching vaginal patency was noted at 160 mg/kg/day (1.05x control). This increase was considered the result of the lower body weights of the animals on the day of reaching vaginal patency (0.89x control), and was therefore considered not to be a direct response to treatment.
For animal Nos. 556, 761, 772, 786 and 807, a vaginal thread was observed on the day of vaginal patency . For animal No. 772, a vaginal thread was observed at least until first estrous was reached.
For Cohort 1A, the time between acquisition of vaginal patency and the first estrus was considered to be unaffected by treatment.
Time until first estrous was on average 4, 4, 4 and 5 days for the control, 16, 55 and 160 mg/kg/day groups, respectively. First estrous was observed on average on PND 36, 34, 36 and 38, respectively. As values generally remained within the historical control range , these slight (non-statistical) differences were considered to be of no toxicological relevance. Length and regularity of the estrous cycle were not affected by treatment at 160 mg/kg/day. For all females for which estrous cycle regularity could be determined, regular cycles of 4 to 5 days were recorded between PND 75 and 88.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 160 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Test item-related statistically significant decreased axillary lymph node weights were observed in females of Cohort 1A at 160 mg/kg/day. In absence of correlating morphological alterations, this change in weights was considered non-adverse.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the thyroid gland, an increased incidence and/or (mean) severity of follicular cell hypertrophy and colloid alteration was present in males treated at 160 mg/kg/day.
Follicular cell hypertrophy was minimal in 7/20, 8/20, 9/20 and 9/20 males at 0, 16, 55, and 160 mg/kg bw/day, respectively, and slight in 2/20, 0/20, 4/20 and 6/20 males at 0, 16, 55, and 160 mg/kg bw/day. The colloid alteration was minimal in 1/20, 2/20, 2/20 and 6/20 males at 0, 16, 55, and 160 mg/kg bw/day, respectively.

In the spleen, an increased incidence and/or (mean) severity of extra medullary hematopoiesis was seen in males and females at 55 and 160 mg/kg/day. Pigmentation was increased in incidence and/or (mean) severity in males at all doses and in females at 160 mg/kg/day.
In males, extramedullary hematopoiesis was minimal in 11/20, 11/20, 12/20 and 12/20 animals at 0, 16, 55, and 160 mg/kg bw/day, respectively, and slight in 2/20, 3/20, 6/20 and 8/20 animals at 0, 16, 55, and 160 mg/kg bw/day. In females, extramedullary hematopoiesis was minimal in 8/20, 5/20, 11/20 and 11/20 animals at 0, 16, 55, and 160 mg/kg bw/day, respectively, and slight in 1/20, 1/20, 4/20 and 7/20 animals at 0, 16, 55, and 160 mg/kg bw/day,
In males, pigmentation was minimal in 12/20, 18/20, 18/20 and 15/20 animals at 0, 16, 55, and 160 mg/kg bw/day, respectively, and slight in 0/20, 1/20, 1/20 and 4/20 animals at 0, 16, 55, and 160 mg/kg bw/day. In females, pigmentation was minimal in 13/20, 13/20, 12/20 and 5/20 animals at 0, 16, 55, and 160 mg/kg bw/day, respectively, and slight in 7/20, 6/20, 8/20 and 14/20 animals at 0, 16, 55, and 160 mg/kg bw/day, Moderate pigmentation was seen in 1 female each at 16 and 160 mg/kg bw/day.

A test item-related statistically significant decrease in ovarian follicle counts (primordial and primary oocytes) was noted at 55 and 160 mg/kg/day. However, the variation in follicle counts in control females was rather high and the number of ovulating follicles were likely the same as the number of corpora lutea was not decreased by treatment with 160 mg/kg/day. In addition, no histological changes or organ weight changes of reproductive organs were noted, and therefore, there was no indication that the reproductive function was adversely affected by treatment up to 160 mg/kg/day. Furthermore, the use of ovarian follicle counts is commonly used only as a second-tier screening method as the initial reproductive toxicity will more commonly be detected by qualitative microscopic assessment or other standard methods.

Other effects:

no effects observed

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

effects observed, non-treatment-related

Description (incidence and severity):

Splenic lymphocyte subpopulation were considered to be unaffected by treatment.
Statistically significant shifts in T-cell, T-helper cell, T-cytotoxic cell and B-cell splenic lymphocyte subpopulations in males at 55 mg/kg/day when compared to splenic lymphocyte subpopulations in control males were minor in magnitude and occurred in the absence of a dose-related trend. In addition, histopathology findings in spleen of F1-animals were unrelated to lymphocytes. Therefore, these shifts were considered to represent biological variability and considered to be unrelated to treatment.

for details on results see overall remarks and attachments

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

55 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

160 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

no

for details on results see overall remarks and attachments

Conclusions:

In conclusion, based on the results of this extended one generation reproductive toxicity study (Cohort 1) performed according to OECD guideline 443 and following GLP principles, the following no-observed-adverse-effect level (NOAEL) of NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1H)-one, sodium salt) were established (the presented NOAELs are target dose levels):
General toxicity (P and F1): 16 mg/kg/day (based on body weights and body weight gain in females)
Reproduction NOAEL (P): 55 mg/kg/day (based on a lower fertility index; possibly secondary to lower body weights)
Developmental NOAEL (P and F1): 55 mg/kg/day (based on lower body weight gains in males; possibly secondary to lower maternal body weights)

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

55 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Guideline studies according to OECD TGs are available (reliable without restrictions).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Short description of key information:
Repeated dose studies:

For Cyanuric chloride no adverse effects on reproduction and development organs were observed in available repeated dose toxicity studies.
In a repeated oral toxicity study (no guideline followed) rats were treated on 5 consecutive days with Cyanuric chloride (93-0090-FKR). No systemic toxicity was found. Effects were only seen at the portal of entry based on the irritation and caustic effects of Cyanuric chloride. In a subchronic inhalation toxicity study (OECD 413) no clear treatment related effect became obvious (94-0211-DKT). In a dermal toxicity study (OECD 410) rabbits were treated repeatedly during 21 days (83-0093-FKT). Generally, effects were only seen at the portal of entry based on the irritation and caustic effects of Cyanuric chloride.

OECD 421, Screening reproductive/developmental toxicity:

In this study the potential toxic effects of NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1H)-one, sodium salt) were determined when given via drinking water for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. The dose levels in this study were selected to be 0, 500, 1000, 2000 ppm. Concerning reproductive toxicity, at 2000 ppm, the mean number of implantation sites was lower compared to controls which was caused by 4/10 females having less than 10 implantation sites (varying from 4 to 9 implantation sites). Although changes were slight and not statistically significant, a relationship to treatment with test item cannot be excluded. Therefore, the NOAEL for reproduction (fertility) was set at 1000 ppm (90 mg/kg bw/day in males and 123 mg/kg bw/day in females). In addition, treatment with the test item was associated with reductions in body weight (gain), food consumption and water consumption at 2000 ppm. The resulting lower mean body weights compared to control means were 12% in males (end of treatment) and in females 5% (end of pre-mating period), 15% (end of gestation) and 10% (end of lactation).

The reduced body weight gain was regarded as adverse since the decreases in mean body weights exceeded 10% and as no signs of recovery were observed during the study period. The NOAEL for parental generation was set at 1000 ppm ( correlated to a mean test item intake level of 90 mg/kg bw/day in males and 123 mg/kg bw/day in females).

OECD 443, EOGRTS:

Pre- and postnatal effects of NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1H)-one, sodium salt) on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats were analysed in this EOGRTS. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. The target dose levels in this study were selected to be 0, 16, 55, 160 mg/kg/day which equals 0, 200, 600, 2000 ppm NHDT-solution in drinikng water. Reproductive toxicity was observed at 160 mg/kg/day. At 160 mg/kg/day, the number of non-pregnant females versus mated females was considered decreased compared with controls. As fertility is affected by body weight, the decreased fertility index may have been the result of the decreased body weights and body weight gains of the females observed at 160 mg/kg/day. No effects on sperm parameters, estrous cycle and microscopic correlates were noted that could have explained the decreased fertility index. No test item-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating, precoital time, number of implantations and histopathological examination of reproductive organs). The NOAEL for reproduction (fertility) was set at 55 mg/kg/day (600 ppm) based on lower fertility index which might be secondary to lower body weights. As reported parental toxicity was observed in females starting at 55 mg/kg/day. The statistically significant decreased body weights observed starting at 55 mg/kg/day in females did not recover to normal levels and persisted through the post coitum period and were therefore considered adverse. At 160 mg/kg in males, the statistically significant reduced body weights and body weight gains also persisted throughout the treatment period and did not recover to normal levels at 160 mg/kg/day and were therefore considered adverse. The NOAEL for general toxicity is 16 mg/kg/day based on body weights and body weight gain in females). The NOAEL for developmental toxicity is 55 mg/kg/day based on lower body weight gains in offspring F1 males which might be secondary to lower maternal body weights.

Effects on developmental toxicity

Description of key information

OECD 414, Pre-natal developmental toxicity in rats: NOAEL, developmental: 25 mg/kg/day

(adverse effects on embryo/fetal development only in combination with severe maternal toxicity: NOAEL for maternal toxicity (oral) of 25 mg/kg/day)

OECD 414, Pre-natal developmental toxicity in rabbits: NOAEL, developmental and maternal: 4000 ppm (227 mg/kg/day)

OECD 443, EOGRTS: NOAEL, developmental: 55 mg/kg/day (600 ppm); NOAEL, maternal: 16 mg/kg/day (200 ppm)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions (e.g. purity of test substance not reported)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

: food consumption and uterus weights not determined, treatment from day 6 to day 19 of gestation

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Charles River CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. , Portage, Michigan
- Age at study initiation: approximately 70 days
- Weight at study initiation: not reported
- Fasting period before study: none
- Housing: individually in suspended wire mesh cages (except during mating)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 51 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 - 24 °C
- Humidity (%): 31 - 51 %
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: mineral oil;

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Concentrations in vehicle: 5, 25 and 50 mg/mL
- weighing and suspending of test substance in vehicle (mineral oil) with a tissue homogenizer with pestel
- transfer of the mixture to a graduated mixing cylinder and addition of the respective amount of vehicle and homogenization by manual shaking of cylinder

VEHICLE
- Justification for use and choice of vehicle: none stated, but the test item is known to decompose in water
- Amount of vehicle: 1 mL/kg bw
- Purity: not reported

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male and 1 female
- Length of cohabitation: until copulation (determined by appearance of copulatory plug in females)
- Further matings after two unsuccessful attempts: not reported
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After mating females were returned to individual cages and identified by ear tag

Duration of treatment / exposure:

day 6 to day 19 of gestation

Frequency of treatment:

daily

Duration of test:

day 0 to day 20 of gestation

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: doses based on range finding study (83-0092-FKR):
block design:
The assignment to groups was based on the time point of notification of the appearance of the copulatory plug (gestation day 0).
The first mated animal was assigned to group 1 (control group), the second animal to group 2 (low dose) and so on.
Animals were assigned in this manner until the required number of females had been placed into each group

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily from day 0 to day 5 of gestation, once daily from day 6 to day 20 of gestation
- Cage side observations checked in table 2 were included.
- No difference was made in the report between cage side observations and clinical observations


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily from day 0 to day 5 of gestation, once daily from day 6 to day 20 of gestation
- Cage side observations checked in table 2 were included.
- No difference was made in the report between cage side observations and clinical observations


BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 6, 9, 12, 16 and day 20 ( day 19 values as discribed in the results section were determined on day 20)


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:
uterus
ovaries
gross examination for evident morphological changes of organs in the thoratic and the abdominal cavities

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight:No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Uteri that appear non-gravid were further examined by ammonium sulphide (10 %) staining to confirm the non-pregnant status (Kopf, R., Lorenz, D., Salewski, E.(1964). "THE EFFECT OF THALIDOMIDE ON THE FERTILITY OF RATS IN REPRODUCTION EXPERIMENTS OVER 2 GENERATIONS". Naunyn Schmiedebergs Arch Exp Pathol Pharmakol, Vol. 247, p. 121-35)

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: approximately half per litter
- Skeletal examinations: Yes: approximately half per litter
- Head examinations: Yes: all per litter (palate and eyes)
- Weighted: Yes: all per litter
- Sex determination: Yes: all per litter

Statistics:

- male to female sex rations and proportions of litters with malformations: Chi-square test criterion with Yates' correction for 2 x 2 contigency tables and/or Fisher's exact probability test as described by Sigel (see remark (III)) to judge significance of difference
- proportions of resorbed and dead fetuses and postimplantational losses : Mann Whitney U-test as described by Siegel (Siegel, S. (1956). "Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York, N. Y., U.S.A) to judge significance of difference
- mean number of corpora lutea, total implantations, live fetuses and mean body weights: one way ANOVA, Bartlett's test for homogeneity of variances and the appropriate t-test(for equal or unequal variances) as described by Steel and Torrie (Steel, R. G. D. and Torrie, J. H. (1960). "Priciples and Procedures of Statistics". McGraw-Hill, New York, N. Y., U.S.A) using Dunnett's multiple comparison tables (see remark (Dunnett, C. W. (1964). New tables for multiple comparisons with a control. Biometrics, Vol. 20, p. 482-491) to judge significance of difference

Indices:

- Group mean preimplantational loss = ((Total No. Corpora Lutea – Total No. Implantations)/ Total No. Corpora Lutea) x 100
- Group mean postimplantational loss = ((Total No. Implantations – Total No. Viable Fetusses)/ Total No. Implantations) x 100

Historical control data:

Historical data comprises data from 1579 litters, complete statistic data on:
Preimplantational loss (and complete data to calculated this value)
Postimplantational loss (and complete data to calculated this value)
Fetal sex ration
Fetal body weight
Nature and frequency of observed malformations in fetuses
Nature and frequency of observed variations in fetuses

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
- clinical signs: in the high dose group: dry brown , red or black matter around eyes, nose, mouth, face, forelimbs or anogenital area; exess salivation; matted haircoat; respiratory rales
- body weight: slightly reduced body weight gain in the high dose group, see table 3

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: Embryotoxic effects, no teratogenic effects

Details on embryotoxic / teratogenic effects:
- slight increase in mean total postimplantation loss in all treated groups compared to the control group that was considered treatment related in the high dose group and resulted in fewer viable fetuses/dam only in this group (see table 4)
- reassessment of the data revealed, that in the control group postimplatational losses ranged from 0 - 3 (mean 0.8). In the low dose group all of the gravid animals except two (no. of postimplantational losses 4 and 5) are within this range. In the mid dose group all of the gravid animals except two (no. of postimplantational losses 4 and 7) are within this range. In the high dose group all of the gravid animals except one (no. of postimplantational losses 10) are within this range. In all treatment groups only these one or two outliers per treatment group of 25 animals cause the differences.
- mean fetal body weights of the treated groups were equal or slightly higher than in the control group (see table 4)
- No meaningful differences in the incidence of fetal malformations (table 5) and developmental variations between control and treated groups

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

reduction in number of live offspring

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The study presents a NOAEL for maternal toxicity (oral) of 25 mg/kg bw/day based on clinical signs and body weight gain and a NOAEL for embryotoxic effects (oral application to the parent) of 25 mg/kg bw/day based on fewer viable fetuses/dam in the high dose group.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17.11.2017 to 18.07.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

The test item is the first hydrolysis step of cyanuric chloride.

Species:

rabbit

Strain:

New Zealand White

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

The amount of test item incorporated in drinking water was kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test item intake was estimated based on the body weight and water consumption values.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Accuracy
The concentrations analyzed in the drinking water solutions of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
No test item was detected in the control drinking water solution.

Homogeneity
The drinking water solutions of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

The test item was administered to the appropriate animals by inclusion in the drinking water ad libitum from Day 6 to the morning of Day 29 post-coitum.

Frequency of treatment:

continuously

Dose / conc.:

640 ppm (analytical)

Remarks:

Mean test item intake over the study period was 39 mg/kg.

Dose / conc.:

1 600 ppm (analytical)

Remarks:

Mean test item intake over the study period was 101 mg/kg.

Dose / conc.:

4 000 ppm (analytical)

Remarks:

Mean test item intake over the study period was 227 mg/kg.

No. of animals per sex per dose:

22 females per group

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily during administration

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily during administration

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were individually weighed on Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was quantitatively measured for Days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was quantitatively measured once daily, beginning on Day 6 post-coitum and lasting up to the day of necropsy at approximately the same time.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: All animals were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).

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: pre and post implantation loss

Fetal examinations:

- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]

Statistics:

Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant clinical sings were noted during the observation period.
A lean appearance was noted in one single female (no. 79) at 4000 ppm on the last two days of treatment. As no body weight loss was noted for this female, and as it concerns one single female only, this was not considered to be toxicologically relevant.
Reduced feces production up to a moderate degree was observed in several animals across the groups, including controls. This finding is commonly observed in rabbits of this age and strain which are housed and treated under the conditions in this study and was not considered to be toxicologically relevant as no dose-response relationship was noted.
Any other observed clinical signs, including alopecia, diarrhea, swelling, scabs, scars and erythema, occurred incidentally within the range of background findings and in absence of a dose-response relationship.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment at 4000 ppm resulted in a slight mean body weight loss of 2% over Days 6-9 postcoitum, followed by a statistically significantly reduced body weight gain up to Day 27 postcoitum. Mean body weights were reduced up to 5% compared to controls (2% on Day 29 post-coitum).
At 1600 ppm, mean body weight gains were slightly, but statistically significantly lower than controls from Day 12 post-coitum onwards. Mean body weights were reduced up to 3% compared to controls.
Body weight and body weight gain in the 640 ppm group remained in the same range as controls over the study period.

Corrected weight gain for gravid uterus was slightly lower in all treatment groups compared to controls. This might be due to a slightly low mean value in the current control group, when compared to the available historical control data. As no dose-response relationship was noted and as all treatment values remained in the range considered normal for rabbits of this strain and age, this was not considered to be treatment related.

Historical control data corrected body weight gain for gravid uterus (Period 2012-2017, N=495):
Corrected weight gain (gram); mean: -245; P5 to P95: -557.3 to 45.4
Corrected weight gain (%); mean: -6; P5 to P95: -13.8 to 1.3

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Over the first week of treatment (until Day 18 post-coitum), food consumption before and after correction for body weight was dose-dependently decreased. Changes in relative food consumption were statistically significant at 640 ppm over Days 12-
15 post-coitum, at 1600 ppm over Days 9-18 post-coitum and at 4000 ppm over Days 6-18 post-coitum. Differences up to about 20% in the 640 ppm group and about 40% in the 1600 and 4000 ppm groups were noted when compared to controls.
During the remainder of the treatment period, food consumption values in all treatment groups returned back to normal.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Water consumption before and after correction for body weight was statistically significantly lower at 4000 ppm from Days 7-11 and 7-9 post-coitum, respectively. In this period, absolute values were up to about 30% lower than controls. From Day 12 post-coitum onwards, water consumption values remained in the same range as controls.
The statistically significantly higher absolute water consumption at 1600 ppm on Days 23-24 post-coitum was not considered to be treatment-related as it occurred at a single occasion in absence of a dose -response relationship.

Mean test item intake over the study period was 39, 101 and 227 mg test item/kg body weight in the 640, 1600 and 4000 ppm groups, respectively.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

In the 4000 ppm group, treatment resulted in a slight body weight loss (average loss of 2%) on Day 9 post-coitum, followed by a statistically significantly reduced body weight gain up to Day 27 post-coitum. Mean body weights were reduced up to 5% compared to controls. This effect was related to a markedly reduced food consumption from Day 6 to Day 18 postcoitum. Relative food consumption differed up to about 40% when compared to controls. During the remaining of the treatment period, food consumption values returned back to normal. Moreover, water consumption was markedly reduced as well during the first four days of treatment. This was considered to be (partly) caused by the taste/smell of the test item containing drinking water, as from Day 12 post-coitum onwards, water consumption values remained in the same range as controls. These treatment-related effects were regarded to be non-adverse as the changes were marginal, and/or were transient and are likely to be (partly) due to palatability issues.
At 1600 ppm, body weights were slightly reduced during the entire treatment period. Mean body weights were reduced up to 3% compared to controls. A reduced food consumption was noted from Days 9 to 18 post-coitum, with relative differences up to about 40%. These treatment-related effects were regarded to be non-adverse as the changes were marginal, and/or were transient. At 640 ppm, treatment-related findings were limited to a reduced food consumption on Days 12-15 post-coitum. Relative food consumption was about 20% lower than controls. As values returned back to normal from Day 15 post-coitum onwards, this change was considered to be non-adverse. No treatment-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e. clinical appearance and macroscopic examination) and no maternal toxicity was observed in the 640 ppm group.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

There were 5 non-pregnant females across the groups; one each in the control, 640 and 1600 ppm groups, and two in the 4000 ppm group. All other females were pregnant and had litters with viable fetuses. As no dose-response was observed and as treatment started from implantation onwards, this non-pregnancy incidence was not considered to be test item related.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEC

Effect level:

> 4 000 ppm (analytical)

Based on:

test mat.

Remarks on result:

other: The high dose level of 4000 ppm was considered to be the highest tolerated dose, as in the dose range finder treatment at 7500 ppm, corresponding to 288 mg/kg bw/day, resulted in unacceptable toxicity.

Key result

Abnormalities:

effects observed, non-treatment-related

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Fetal body weights (both males and females) were slightly lower in all treatment groups compared to controls, especially at 640 and 1600 ppm. Mean combined (male and female) fetal body weights were 39.6, 37.2, 36.3 and 38.1 gram for the control, 640, 1600 and 4000 ppm groups, respectively. Differences compared to controls were about 6, 8 and 4%, respectively. No clear dose-response relationship was observed, and the mean values remained within the range of historical control data (at the lower end).

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment -related effects on external morphology following treatment up to 4000 ppm.
In this study there were two externally malformed fetuses . One fetus in Group 2 had carpal flexure one both sides and one fetus in Group 3 was noted with acrania. Moreover, two late resorptions from one litter of Group 3 were noted with carpal flexure on both sides as well. As these malformations occurred singly, were previously seen in historical control fetuses and occurred in the low and mid dose group only, they were considered to be chance findings.
No external variations were noted.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on skeletal morphology following treatment up to 4000 ppm.
Only one fetus from Group 4, was observed with a vertebral anomaly. This malformation was noted previously in historical controls and as it occurred singly, it was considered to be a chance finding and not related to treatment. Skeletal variations that were noted in this study occurred at low incidences, and/or at frequencies that were within the range of available historical control data. Therefore, these variations were considered not to be test item-related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on visceral morphology following treatment up to 4000 ppm.
Visceral examination revealed 8 fetuses across the groups noted with malformations: The fetus of Group 3, noted with acrania, also had multiple visceral abnormalities (i.e.one eye present inside and situs inversus). There were two fetuses with a malpositioned
testis, one from the control group and one from Group 4. Tetralogy of Fallot was observed in four fetuses, one control fetus, one fetus from Group 2, and two fetuses from Group 4. One more malformed fetus of Group 4, was noted with cardiovascular malformations including atrial septum defect, atretic pulmonary trunk and dilated aortic arch. As there was no dose response-relationship and/or as they occurred incidentally and as all malformations were noted previously in historical controls, these malformations did not indicate a treatment relationship.
All the visceral variations noted were not considered treatment related as they occurred infrequently, in absence of a dose-response relationship and/or occurred at frequencies that were within the range of available historical control data.

Details on embryotoxic / teratogenic effects:

Fetal body weights were slightly lower in all treatment groups compared to the concurrent control group (up to 8%). A comparable effect was observed in all treatment groups of the dose range finder at dose levels of 1800, 3500 and 7500 ppm, in which differences of 5-11% were noted, compared to controls. Although no clear dose-response relationship was observed in both studies, a relationship to treatment with test item cannot be excluded. As the changes were marginal, as all values remained within the range of historical control data (at the lower end), and as ossification parameters were unaffected, indicating no growth retardation effects, this was considered to be non-adverse. No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, sex ratio, external, visceral and skeletal malformations and developmental variations).

Key result

Dose descriptor:

NOAEC

Effect level:

> 4 000 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: The high dose level of 4000 ppm was considered to be the highest tolerated dose, as in the dose range finder treatment at 7500 ppm, corresponding to 288 mg/kg bw/day, resulted in unacceptable toxicity.

Key result

Abnormalities:

effects observed, non-treatment-related

Key result

Developmental effects observed:

no

for details on results see overall remarks and attachments

Conclusions:

Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for NHDT-solution (4,6- dichloro-1,3,5-triazin-2(1H)-one, sodium salt) was established as being at least 4000 ppm, corresponding to an average actual test item intake of 227 mg/kg bw/day. The high dose level of 4000 ppm was considered to be the highest tolerated dose, as in the dose range finder treatment at 7500 ppm, corresponding to 288 mg/kg bw/day, resulted in unacceptable toxicity.

Executive summary:

The objectives of this study were to determine the potential of NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1H)-one, sodium salt) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally via drinking waterto time-mated female New Zealand White rabbits from Day 6 to 29post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels in this study were selected to be 0, 640, 1600, 4000 ppm, based on the results of the dose range finder. Chemical analyses of drinking water preparations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and endpoints were evaluated in this study for the F₀-generation: mortality/moribundity, clinical signs, body weights, food consumption, water consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents.  In addition, the following parameters were determined for the F₁-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations. Formulation analyses confirmed that drinking water preparations containing test item were prepared accurately and homogenously.

Treatment at 4000 ppm resulted in a slight body weight loss (average of 2%) on Day 9 post-coitum followed by a reduced body weight gain up to Day 27 post-coitum. This effect was related to a markedly reduced food consumption from Day 6 to Day 18 post-coitum (relative difference of 40%). During the remaining of the treatment period, food consumption values returned back to normal. Moreover, water consumption was markedly reduced as well during the first four days of treatment. This was considered to be (partly) caused by the taste/smell of the test item containing drinking water, as from Day 12 post-coitum onwards, water consumption values remained in the same range as controls. These treatment-related effects were regarded to be non-adverse as the changes were marginal, and/or were transient and are likely to be (partly) due to palatability issues.

At 1600 ppm, slightly reduced body weight gain over the entire treatment period and a reduced food consumption of 40% up to Day 18 post-coitum were noted. These treatment-related effects were regarded to be non-adverse as the changes were marginal, and/or were transient. The reduced food consumption of 20% over Days 12-15 post-coitum at 640 ppm was considered to be non-adverse as it recovered during the treatment period.

Fetal body weights were slightly lower in all treatment groups compared to the concurrent control group (up to 8%). A comparable effect was observed in all treatment groups of the dose range finder. Although no clear dose-response relationship was observed, a relationship to treatment with test item cannot be excluded. As the changes were marginal, as all values remained within the range of historical control data (at the lower end), and as ossification parameters were unaffected, indicating no growth retardation effects, this was considered to be non-adverse. No treatment-related malformations or variations were observed.

In conclusion, based on the results in this prenatal developmental toxicity study a maternal and developmental No Observed Adverse Effect Level (NOAEL) for NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1H)-one, sodium salt) of at least 4000 ppm was established, corresponding to an average actual test item intake of 227 mg/kg bw/day.

The high dose level of 4000 ppm was considered to be the highest tolerated dose, as in the dose range finder treatment at 7500 ppm, corresponding to 288 mg/kg bw/day, resulted in unacceptable toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline studies according to OECD TGs are available (reliable without restrictions).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Short description of key information:

OECD 414, Pre-natal developmental toxicity in rats:

In a key teratology study (83-0091-FGT) pregnant rats consecutively assigned to one control and three treatment groups of 25 animals each were used to determine the teratogenic potential of Cyanuric chloride. Dosage level of 5, 25 and 50 mg/kg/day were administered orally by gavage as a single daily dose on days 6 through 19 of gestation at a constant volume of 1.0 mL/kg. The control animals received the vehicle only, mineral oil, on a comparable regimen. Cesarean sections were performed on all surviving females on gestation day 20 and the fetuses were removed for teratologic evaluation. Survival in the control and in the 5 and 50 mg/kg/day dosage groups was 100% during the course of this study. One rat in the 25 mg/kg/day dosage group died due to an intubation error. Compound-related antemortem observations noted in the 50mg/kg/day dosage group included dry matter around the face, forelimbs and angenital area, matted haircoat, excess salivation and respiratory rales. Necropsy findings in all treated groups were comparable to those of the control group. A slight decrease in group mean maternal body weight gain values during the treatment (day 6 to 19) and gestation (day 0 to 20) periods occured in the 50 mg/kg/day group when compared to the control group values and was considered treatment-related. The values for this parameter over the same time periods for the 5 and 25 mg/kg/day dosage groups were comparable to the control group values. There were slight increases in postimplantation loss/dam in all treated groups, but only in the high dose group this was considered significant and resulted in a lower value for viable fetuses/dam when compared to that of the control group. The values for viable fetuses/dam of the 5 and 25 mg/kg bw/day dosage groups were comparable to the control values. Mean corpora lutea, total implantations, fetal body weight and fetal sex ratio values for all treated groups were comparable to those of the control group. There were no biologically meaningful differences in the incidence of malformations and developmental variations in the treated groups when compared to the control group. Based on these results, the test substance was not considered teratogenic in rats when administered orally by gavage at dosage levels of 50 mg/kg/day or less. The NOAEL of 25 mg/kg bw for maternal toxicity and of 25 mg/kg bw for developmental toxicity was deduced under the conditions of the study.

 

OECD 414, Pre-natal developmental toxicity in rabbits:

One pre-natal developmental toxicity study according to OECD 414 is available to determine the potential of NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1H)-one, sodium salt) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally via drinking water to time-mated female rabbits from Day 6 to 29 post-coitum, inclusive. The dose levels in this study were selected to be 0, 640, 1600, 4000 ppm. During the study, reduced body weights, body weight gains, food and water consumptions were reported. However, during the remaining of the treatment period these treatment-related effects were regarded to be non-adverse as the changes were marginal, and/or were transient and are likely to be (partly) due to palatability issues. Fetal body weights were slightly lower in all treatment groups compared to the concurrent control group (up to 8%). A comparable effect was observed in all treatment groups of the dose range finder. Although no clear dose-response relationship was observed, a relationship to treatment with test item cannot be excluded. As the changes were marginal, as all values remained within the range of historical control data (at the lower end), and as ossification parameters were unaffected, indicating no growth retardation effects, this was considered to be non-adverse. In conclusion, based on the results in this prenatal developmental toxicity study a maternal and developmental No Observed Adverse Effect Level (NOAEL) for NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1H)-one, sodium salt) of at least 4000 ppm was established, corresponding to an average actual test item intake of 227 mg/kg bw/day.

 

OECD 421, Screening reproductive/developmental toxicity:

In this study the potential toxic effects of NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1H)-one, sodium salt) were determined when given via drinking water for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. The dose levels in this study were selected to be 0, 500, 1000, 2000 ppm. Developmental toxicity was observed at 2000 ppm and consisted of a reduction in live litter size, particularly in 4/10 females which had only 3-6 pups (i.e. at or below the historical control range), which was related to the lower number of implantation sites. Additionally, post-implantation survival index for the 2000 ppm group was reduced compared to controls (i.e. at the historical control range). There were no explanatory morphological findings in reproductive organs and the lower values might reflect normal biological variation. However, it cannot be ruled out that the lower post-implantation survival and litter size was related to treatment with test item. The developmental effects could not be unequivocally attributed to maternal toxicity due to the modest severity of the parental effects at 2000 ppm. The NOAEL for developmental toxicity was set at 1000 ppm (correlated to a mean test item intake level of 90 mg/kg bw/day in males and 123 mg/kg bw/day in females).

Concerning reproductive toxicity, at 2000 ppm lower mean number of implantation sites were reported and the NOAEL for reproduction (fertility) was set at 1000 ppm (90 mg/kg bw/day in males and 123 mg/kg bw/day in females). In addition, treatment with the test item was associated with reductions in body weight (gain), food consumption and water consumption at 2000 ppm. The resulting lower mean body weights compared to control means were 12% in males (end of treatment) and in females 5% (end of pre-mating period), 15% (end of gestation) and 10% (end of lactation). The reduced body weight gain was regarded as adverse since the decreases in mean body weights exceeded 10% and as no signs of recovery were observed during the study period. The NOAEL for parental generation was set at 1000 ppm (correlated to a mean test item intake level of 90 mg/kg bw/day in males and 123 mg/kg bw/day in females).

 

OECD 443, EOGRTS:

Pre- and postnatal effects of NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1H)-one, sodium salt) on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats were analysed in this EOGRTS. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. The target dose levels in this study were selected to be 0, 16, 55, 160 mg/kg/day which equals 0, 200, 600, 2000 ppm NHDT-solution in drinking water.

Reproductive toxicity was observed at 160 mg/kg/day. At 160 mg/kg/day, the number of non-pregnant females versus mated females was considered decreased compared with controls. As fertility is affected by body weight, the decreased fertility index may have been the result of the decreased body weights and body weight gains of the females observed at 160 mg/kg/day. Thus, the NOAEL for reproduction was 55 mg/kg/day. As reported parental toxicity was observed in females starting at 55 mg/kg/day. The statistically significant decreased body weights observed starting at 55 mg/kg/day in females did not recover to normal levels and persisted through the post coitum period and were therefore considered adverse. The NOAEL for general toxicity is 16 mg/kg/day based on body weights and body weight gain in females.

Developmental toxicity was observed at 160 mg/kg/day.

At 160 mg/kg/day, body weights of male and female pups were statistically significant decreased during the complete lactation period. The decreased body weights were likely the result of the decreased body weights observed for the dam during the post coitum period. As the body weights of pups did not recover to normal levels by the end of the lactation period, this finding was considered adverse. Test item-related statistically significant increases in total T4 were observed in both males and females at 160 mg/kg/day between PND 22-24. As values remained within the historical control range and in absence of microscopic examinations, these increases in total T4 levels were considered non-adverse. No test item-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, live birth index, viability and lactation indices,

duration of gestation, parturition, sex ratio, litter size, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, organ weights and macroscopic examination). Developmental results for F1 generation at post-weaning showed test item-related statistically significant decreased body weights were observed in males from 16 mg/kg/day and in females from 55 mg/kg/day and test item-related decreased body weight gains in males at 160 mg/kg/day. The decreased body weights at the start of weaning were considered to have arisen from the slightly decreased body weights at PND 21. As body weight gains did not recover to normal levels for males at 160 mg/kg/day, the decreased body weight and body weight gain for males at 160 mg/kg/day was considered adverse. The decrease in body weight gain may be related to the lower maternal body weight and is possibly a secondary effect. Other effects such as changes in hematology, increased total T4 levels, decreased axillary lymph node weights were reported to be slight and therefore considered non-adverse in absence of degenerative changes or changes were within historical control ranges. A test item-related statistically significant decrease in ovarian follicle counts (primordial and primary oocytes) was noted at 55 and 160 mg/kg/day. However, the variation in follicle counts in control females was rather high and the number of ovulating follicles were likely the same as the number of corpora lutea was not decreased by treatment with 160 mg/kg/day. In addition, no histological changes or organ weight changes of reproductive organs were noted, and therefore, there was no indication that the reproductive function was adversely affected by treatment up to 160 mg/kg/day. Furthermore, the use of ovarian follicle counts is commonly used only as a second-tier screening method as the initial reproductive toxicity will more commonly be detected by qualitative microscopic assessment or other standard methods. No test item-related changes were noted in any of the other developmental parameters investigated in this study (i.e. mortality, clinical signs, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrous, time between vaginal opening and first estrous length and regularity of the estrous cycle, coagulation, and sperm parameters).

The NOAEL for developmental toxicity is 55 mg/kg/day based on lower body weight gains in offspring F1 males which might be secondary to lower maternal body weights.

 

other studies:

In an exploratory range-finding teratology study in rats (83-0092-FGT) with Cyanuric chloride a NOAEL for maternal toxicity of 30 mg/kg bw/day based on overall effects and a LOAEL for maternal toxicity of 40 mg/kg bw/day based on body weight was deduced under the test conditions of the study.

A dose range finder in rabbits (2017 -0016 -DGR) was conducted to select dose levels for the Prenatal Developmental Toxicity Study with NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1H)-one, sodium salt) in drinking water in rabbits. A NOAEC for maternal toxicity of 3500 ppm (160 mg/kg bw/day) based on water consumption, body weight. Fetal body weights were decreased in all treatment groups (not dose-dependently), compared to the control group. The mean values of the treatment groups remained within the range of historical control data (at the lower end), and therefore, these changes were considered to be non-adverse. A NOAEC for fetuses was set at 7500 ppm (220 mg/kg bw/day).

Justification for classification or non-classification

The observed effects of Cyanuric chloride or its first hydrolysis product NHDT-solution (4,6 -dichloro-1,3,5 -triazin-2(1H)-one, sodium salt) on development, sexual function and fertility are considered to be a non-specific consequence of maternal toxicity. The reported effects can be clearly linked to maternal toxicity and were only present in the absence of histopathological changes/degenerations. Thus classification for reproductive/developmental toxicity is not warranted. In addition, no effects on or via lactation were identified.

Based on the results of all available studies adressing the toxicity to reproduction Cyanuric chloride is not classified according to CLP Regulation (EC) No 1272/2008.