Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to information in OECD SIDS (2002)
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
PARENTAL TEST ANIMALS
- Age at study initiation: 10 weeks old, both sexes
- Weight at study initiation: 335-364 g for males, 233-263 g for females
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
for injection
Details on exposure:
Treatment of parental animals by oral gavage administration. Test substance was not directly administered to F1 animals, as these pubs were sacrificed on day 4 post partum, i.e. day 4 of lactation.
Details on mating procedure:
- Male/female ratio per cage: 1/1
- Length of cohabitation: At the most 14 days, until proof of pregnancy was confirmed. 
- Proof of pregnancy: Formation of vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
(During cohabitation, females were checked every morning for pregnancy)

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
- Treatment period, Parental males: 49 days (14 days before mating and 35 days including 14 days for mating)
Treatment period, Parental females: 40-46 days (from 14 days prior to mating to day 3 of lactation.)
- Frequency of treatment: Daily
- Post treatment observation period: 1 day
- Duration of test, Parental males: 50 days
Duration of test, Parental females: From 14 days prior to mating to day 4 of lactation.
Duration of test, Pups: Until day 4 of lactation.

Frequency of treatment:
Daily
Details on study schedule:
- Age at mating of the mated animals in the study: 12 to 14 weeks
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
Clinical observations performed and frequency:
- Clinical signs: Twice a day (just before and after administration)
- Body weight, Males: Twice a week
Body weight, Females: Twice a week for pre-mating and mating period, 0, 7, 14, 21st day of pregnancy and 0, 4th day of lactation period.
- Food consumption, Males: Twice a week for pre-mating period and after a mating period end.
Food consumption, Females: Twice a week for pre-mating period, 2, 9, 16, 21st day of pregnancy and 4th day of lactation period.
- Hematology, Blood Chemistry, Urinalysis in parental males only. *

* Detailed in separate endpoint study record, i.e. "7.5.1 Repeated dose toxicity: oral - MHLW Repeat dose tox.: oral, rat, 2001"
Oestrous cyclicity (parental animals):
Frequency of vaginal estrus was determined from the beginning of the treatment period to the day of confirmed copulation.
Sperm parameters (parental animals):
Parameters examined in male parental animals: testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS: Not performed. The study ended on day 4 post partum.

LITTER PARAMETERS EXAMINED
- No. of pregnant females with live pups on day 0.
- Gestation index (No. of females with live pups/No. of living pregnant females x 100)
- No. of pregnant females with live pups on day 4
- Delivery index  (No. of pups born/No. of implantation sites x 100)
- No. of pups alive on day 0 of lactation,
- Live birth index (No. of live pups on day 0/No. of pups born x 100)
- Sex ratio  (Total No. of male pups/Total No. of female pups)
- No. of pups alive on day 4 of lactation
- Viability index (No. of live pups on day 4/No. of live pups on day 0 x 100)
- Clinical signs, twice a day after birth
- Body weight of live pups (on day 0 and 4).



Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes, see below
WEIGHING OF ORGANS: Yes, see below
HISTOPATHOLOGY: Yes, see below

- Terminal sacrifice, Males: Killed on the day after the treatment period.
Terminal sacrifice, Females: Killed on day 4 of lactation. Females with no delivery were killed 4 days after the delivery expected date.
Females with no copulation were killed at the end of the mating period.

- Gross pathology: All rats received a full macroscopic examination  with tissue collection.

- Organs Weights: The following organs were weighed at necropsy:
Brain, pituitary, thyroids, heart, thymus, liver, spleen, adrenals, kidneys, testes, epididymides, ovaries.

- Histopathology: The following organs were microscopically observed for the control and 1000 mg/kg bw/day groups:
Brain, pituitary gland, thyroids, heart, thymus, liver, spleen, adrenals, kidneys, testes, epididymides, ovaries,
lung, trachea, pancreas, salivary glands, esophagus, stomach, duodenum, jejunum, ileum, caecum, rectum,
colon, lymph node, bladder, uterus, vagina, parathyroids, spinal cord, sciatic nerve, eyes, Harderian glands,
mammary gland,  bone marrow, seminal vesicle, prostate.
In addition, liver and spleen (male only) were also examined for the 30, 100 and 300 mg/kg bw/day groups.

Postmortem examinations (offspring):
Full macroscopic examination of all pups
Statistics:
Dunnett's test was used for numerical data. Chi square test was used for copulation index and fertility index analysis.
Reproductive indices:
- No. of pairs with successful copulation
- Copulation index (No. of pairs with successful copulation/No. of pairs mated x 100)
- Pairing days until copulation
- No. of pregnant females
- Fertility index (No. of pregnant animals/No. of pairs with successful copulation x 100)
- No. of corpora lutea
- No. of implantation sites
- Implantation index (No. of implantation sites/No. of corpora lutea x 100)
- No. of living pregnant females
- No. of pregnant females with parturition
- Gestation length
- For further reproductive parameters, see also the above section "Litter observations"
Offspring viability indices:
- Viability index (No. of live pups on day 4/No. of live pups on day 0 x 100).
- For further parameters indicative of the viability of the offspring, see also the above section "Litter observations"
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
only in 2 females, no effect in males
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Mortality:
- No deaths occurred in all dams during the study.

Histopathology:
- Males:  No treatment-related abnormality was observed.
- Females:  Very slight (marginally positive) extramedullary hematopoiesis in the liver was noted in two female animals of the 1000 mg/kg bw/day gro up.

Other Examinations (addressed in separate endpoint study record "7.5.1 Repeated dose toxicity: oral - MHLW Repeat dose tox.: oral, rat, 2001"):
- Hematological examinations (only for males)
- Blood chemical examinations (only for males)
- Urinary examinations (only for males)
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Sex:
male
Basis for effect level:
other: NOEL = highest dose tested. NOEL (instead of NOAEL stated by OECD SIDS) seems more appropriate, because OECD SIDS states: "No treatment related abnormality up to 1000 mg/kg bw/day in parental males."
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Sex:
female
Basis for effect level:
other: NOAEL = highest dose tested. Very slight extramedullary hematopoiesis in the liver in two parental females at 1000 mg/kg bw/day was statistically not significant, therefore not adverse.
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
No treatment-related abnormality was observed.

ADDITIONAL FETAL DATA:

No abnormality in sex ratio.

Grossly Visible Abnormalities and External Abnormalities:
Proboscis was observed in a stillbirth pup at 300 mg/kg bw/day. No treatment related external abnormality was observed among newborns.

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
dams were dosed
Sex:
male/female
Basis for effect level:
other: NOEL = highest dose tested. NOEL (instead of NOAEL stated by OECD SIDS) seems more appropriate, because OECD SIDS states: "No abnormalities in all indexes obtained from pups in each dose group."
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

There were no statistically significant differences from concurrent controls.

Conclusions:
There were no treatment related abnormalities. The no observed adverse effect level (NOAEL) for maternal toxicity and the no observed effect level (NOEL) for foetal toxicity are 1000 mg/kg bw/day.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In the available OECD 422 study in the rat, THEIC did not have any effect on reproductive and developmental endpoints at the top dose level of 1000 mg/kg/day. This is consistent with the absence of reproduction toxicity in the numerous toxicity studies with cyanuric acid structurally similar to THEIC and used for read-across regarding sub-chronic oral toxicity (Tschech 2010). In a three-generation reproduction toxicity study with cyanuric acid sodium salt in the rat, all parental and offspring animals of the highest dose group (100 mg/kg/day) were free from any effects on reproductive parameters (WHO 2004). Based on this information and the general "quasi inert" behaviour of THEIC in all toxicological and ecotoxicological studies it is not expected that the performance of an extended one generation reproductive toxicity study with THEIC would provide additional toxicological information on this substance relevant to its safe handling.

Effects on developmental toxicity

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: approx. 71 days (at Day 0 of gestation)
- Weight at study initiation: 229-287 g (at Day 0 of gestation)
- Fasting period before study: no
- Housing: 1 animal per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days before commencement of pairing

DETAILS OF FOOD AND WATER QUALITY: SDS VRF1 Certified pelleted diet; potable water from public supply

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

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amounts of test material were weighed and dissolved in water.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 10, 30, 100 mg/ml
- Amount of vehicle (if gavage): 10 ml / kg bw
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of dose formulations were analysed by HPLC with UV-detector. Mean concentrations were within an acceptable range of the nominal concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 : 1
- Length of cohabitation: until evidence of mating
- Verification of same strain and source of both sexes: yes, a colony of stud males was maintained
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from day 6 till day 19 of gestation
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels selected for investigation in this study (0, 100, 300 and 1000 mg/kg/day) were based on the results of a sub-acute repeated dose study with the reproduction / developmental toxicity screening.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations : visual inspection for ill-health or reaction to treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: days 0, 5, 12, 18, and 20

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 3, and daily from 6-20

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: full macroscopic examination of tissues
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: number of fetuses (live and dead)
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations external: Yes: all per litter
Statistics:
The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, live young, fetal, placental and litter weight data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pretreatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other comparisons t/The F1 approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The test statistic compares the mean square, NMS, for the deviations of the observed means from the maximum likelihood means, calculated under a constraint of monotonicity with the usual error mean square, EMS. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/EMS which can be compared with standard tables of the F-distribution with 1 and error degrees of freedom. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pretreatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon 1945) were made.
Indices:
Prenatal losses were separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:
Pre-implantation loss (%) = (Number of corpora lutea – Number of implantations) x 100 / Number of corpora lutea

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = (Number of implantations – Number of live fetuses) x 100 / Number of implantations

All group values and SD (as appropriate) were calculated from the individual litter values
Historical control data:
Historical control data from 7 studies performed in the last 10 months preceding the actual study with the same rat strain are provided in the report.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female receiving 300 mg/kg/day (No. 50) was killed for welfare reasons on Day 19 of gestation due to sudden and marked decline in general clinical condition. After dose administration on Day 19 of gestation, signs of salivation, piloerection, laboured respiration and dry rales were apparent. Having previously shown normal body weight gain to Day 18 of gestation, between Day 18 to 19 of gestation bodyweight loss of 12g was observed (3% total body weight). Food consumption had been unaffected. Macroscopic examination revealed that the esophagus was distended and contained clear/viscous fluid and food material; the uterus contained 18 implantation sites, all with live young. In the absence of similar events in this or higher dose groups, this isolated premature death was considered not to be associated with THEIC administration.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see figure of body weight development attached as background material.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Description (incidence and severity):
no differences in body weight gain and food consumption between dosed animals and control animals.
Water consumption and compound intake (if drinking water study):
not specified
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
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
see table on litter data attached as background material.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
see table on litter data attached as background material.
Dead fetuses:
no effects observed
Description (incidence and severity):
see table on litter data attached as background material.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
other: NOAEL = highest dose tested
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
see table on litter and fetal weights attached as background material.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
see table on litter data attached as background material.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
see table on litter data attached as background material.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
see table on litter and fetal weights attached as background material.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants showed no relationship to maternal treatment with THEIC.
At 1000 mg/kg/day there was a slight increase in the fetal and litter incidence of medially thickened/kinked ribs compared to concurrent control and outside of Historical Control Data (HCD) range. Medially thickened/kinked ribs are widely accepted as a skeletal variant which are likely repairable via post-natal skeletal remodeling; in the absence of any other fetal abnormalities this skeletal variant is not adverse in isolation, would be of no consequence to the long term survival of the fetuses and was therefore considered likely to be an incidental finding with no relationship to maternal treatment.

see table on abnormality findings attached as background material.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 300 mg/kg/day there was a slight increase in incidence of left umbilical artery compared to concurrent control but within HCD range. During prenatal development a spontaneous closure of either the left or right umbilical artery occurs, and most commonly this occurs in the left umbilical artery. After birth their function of facilitating blood flow from/to the placenta is redundant and the artery then forms the medial umbilical ligament. The retention of the left umbilical artery (rather than the right) is classified as a non-adverse variation, and since the incidence at 1000 mg/kg/day was unaffected the slight increase in incidence of left umbilical artery in this study was considered an incidental finding and of no relationship to maternal treatment.

see table on visceral abnormality findings attached as background material.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
dams were dosed
Sex:
male/female
Remarks on result:
other: NOAEL = highest dose tested
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
no effects of developmental toxicity or teratogenicity observed up to and including the highest dose level of 1000 mg/kg body weight/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

All available toxicological data on THEIC indicate a quasi-inert behavior of this substance. In an oral repeat dose toxicity study with reproduction and developmental toxicity screening in rats (OECD 422) no signs of toxicity in the parental animals and no effects on reproductive and developmental endpoints were evident up to and including the highest dose level tested, i.e. 1000 mg/kg/day. Further support to absence of effects on reproduction comes from testing results obtained with the structurally similar substance cyanuric acid. In the developmental toxicty study performed with THEIC no effects were observed up to and including the highest dose of 1000 mg/kg/day. Therefore, THEIC does not warrant any classification regarding reproductive/developmental toxicity according to European classification rules [REGULATION (EC) 1272/2008].